Note: Descriptions are shown in the official language in which they were submitted.
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EXONS 4 AND 7 ENCODE SEPARATE TRANSACTIVATING AND
CHROMATIN LOCALIZING DOMAINS IN ESX
CROSS-REFERENCE TO RELATED APPLICATIONS
This claims benefit under 35 U.S.C. ~ 119 of provisional patent application
USSN 60/089,409, filed on June 16, 1998 and is a continuation-in-part of USSN
08/978,217,
filed on November 25, 1997, which claims benefit under 35 U.S.C. ~ 119 of
provisional
patent application USSN 60/031,504, filed on November 27, 1996, all of which
are herein
incorporated by reference, in their entirety, for all purposes.
FIELD OF THE INVENTION
This invention pertains to the field of oncology. In particular, this
invention
pertains to the discovery of domains of a transcription factor gene that
provide novel targets
for modulators of that gene and that are implicated in the etiology of human
epithelial
cancers, including breast cancer, and other malignancies including gastric,
ovarian, and lung
adenocarcinomas.
BACKGROUND OF THE INVENTION
Many cancers are believed to result from a series of genetic alterations
leading to progressive disordering of normal cellular growth mechanisms
(Howell (1976)
Science 194: 23, Foulds (1958) J. Chronic Dis. 8: 2). The deletion or
multiplication of
copies of whole chromosomes or chromosomal segments, or specific regions of
the genome
are common (see, e.g., Smith et al. (1991) Breast CancerRes. Treat., 18:
Suppl. l: 5-14; van
de Vijer & Nusse (1991) Biochim. Biophys. Acta. 1072: 33-50; Sato et al.
(1990) Cancer.
Res., 50: 7184-7189). In particular, the amplification and deletion of DNA
sequences
containing proto-oncogenes and tumor-suppressor genes, respectively, are
frequently
characteristic of tumorigenesis. Dutrillaux, et al. (1990) Cancer Genet.
Cytogenet., 49: 203-
217. As an example, overexpression of the HER2/neu (c-erbB-2) proto-oncogene
product is
found in approximately 20-30% of primary breast cancers and in a similar
fraction of human
gastric, ovarian, and lung carcinomas. For many of these malignancies, this
overexpressed
membrane growth factor receptor (pl8SHE~) is associated with HER2 gene
amplification,
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more aggressive tumor growth, and reduced patient survival. Maguire & Greene (
1989)
Semin. Oncol.~ 16: 148-155; Singleton & Strickler (1992) Pathol. Anna. 1: 165-
190; Tripathy
& Benz (1993} in Oncogenes and Tumor Suppressor Genes in Human Malignancies
(Benz
and Liu, eds.) pp. 15-60, Kluwer, Boston. In approximately 10-20% of HER2-
overexpressing breast tumors, some gastric, and virtually all HER2-positive
lung cancers,
HER2 mRNA and protein overexpression occur in the absence of increased gene
copy
number, suggesting that HER2 transcriptional dysregulation may be a
fundamental defect of
clinical significance in these malignancies. Bergen et al. ( 1988) Cancer Res.
48: 1238-1243;
Kameda et al. (1990) Cancer Res. 50: 8002-8009; Kern et al. (1990) Cancer Res.
50: 5184-
5191; King et al. (1989) CancerRes. 49: 4185-4191; Slamon et al. (1989)
Science 244: 707-
712; Tandon et al. (1989} J. Clin. Oncol. 7: 1120-1128. It has been speculated
that a
primary defect leading to dysregulated HER2 transcription might also
predispose to the in
vivo development of gene amplification and stable acquisition of a more
malignant tumor
cell phenotype. Kameda et al., supra.; King et al., supra.; Hynes et al.
(1989) .J. Biol. Chem.
39: 167-173; Kraus et al. (1987) EMBD,T. 6: 605-610; Pasleau et al. (1993)
Oncogene 8:
849-854.
Recently, a previously unrecognized response element similar to those
recognized by the ets transcriptional regulator family was identified within
both the human
HER2 and marine neu promoters. Scott et al. (1994} J. Biol. Chem. 269: 19848-
19858. The
ets multigene family of transcriptional regulators includes more than thirty
known members
that are involved in early embryonic development and late tissue maturation,
directing stage-
specific and tissue-restricted programs of gene expression. The ETS
transcription factors,
which are recognizable primarily by their 85 amino acid ETS DNA-binding
domain, are
dispersed across all metazoan lineages into distinct subfamilies. Ets genes
can produce
malignancies in humans and other vertebrates when overexpressed or rearranged
into
chimeras retaining the ETS domain.
Members of the Ets family of transcription factors have been shown to play
important roles in regulating gene programs critical for normal organismal
development and
cellular differentiation, while fusion proteins involving Ets family members
arising from
chromosomal translocations are thought to account for a significant fraction
of all human
leukemias and lymphomas as well as virtually all Ewings sarcomas and Primitive
Neuro-
Ectodermal Tumors (PNET), otherwise known as small round cell bone and soft
tissue
sarcomas of childhood (reviewed in Crepieux et al. (1994} Critical Reviews in
Oncogenesis,
S: 615-6381; Dittmer and Nordheim (1998) Biochim. Biophys. Acta, 1377: F1-11;
Hromas
-2-
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and Klemsz (1994) Int. J. Hematol., 59: 257-265; Sharrocks et al. (1997) Int.
J. Biochem.
Cell Biol., 29: 1371-1387; Wasylyk and Nordheim (1997) Transcription Factors
in
Eukaryotes. Papavassiliou AG (ed) Springer-Verlag: Heidelberg, Germany, pp.
253-286).
Searching for Ets factors potentially involved in human mammary gland
development and malignancy, we recently cloned and characterized a novel Ets
factor, ESX
Epithelial-restricted with Serine boX; HUGO/GDB:6837498), which was found to
be
transcriptionally upregulated in a subset of early breast tumors and breast
cancer cell lines
where it was postulated to be a candidate transactivator of the Ets responsive
proto-
oncogene, ErbB2 (USSN 08/978,217, Chang et al. (1997) Oncogene, 14: 1617-
1622). Four
other groups have since published on the potential biological and
developmental importance
of this epithelium-specific Ets factor (variably named ESE-1, ELF-3, Jen, or
ERT) in non-
mammary epithelial systems (Andreoli et al. (1997) Nucleic Acids Res., 25,
4287-4295I
Choi et al. (1998) J. Biol. Chem., 273: 110-1171; Oettgen et al. (1997) Mol.
Cell. Biol., 17:
4419-4433; Tymms et'al. (1997) Oncogene, 15, 2449-2462). While its expression
profile
suggests that ESX is associated with development of both simple and stratified
epithelium
(Andreoii et al., supra.; Choi et al. supra.; Chang et al., supra.; Oettge~n
et al. (1997) supra.,
Tymms et al. supra.), detailed studies were first performed in the latter and
these showed
that ESX is unique among transcription factors generally, and Ets factors
specifically, for its
restricted expression in terminally differentiated epidermal cells ((Andreoli
et al., supra.;
Choi et al. supra.; ). In stratified epithelium, ESX is thought to
transactivate such genes as
the transforming growth factor-~i type II receptor (TGF-~3RI1], Endo-Alkeratin-
8, and several
markers of epidermal cell differentiation including transglutaminase 3,
SPRR2A, and
profilaggrin (Andreoli et al., supra.; Choi et al. supra.; Oettgen et al.
(1997) supra.; Tymms
et al. supra.).
Because most, if not a11, cancers involve dysregulation of gene expression, a
need exists for information as to transcription factors and other regulatory
moieties that are
involved in mediating the dysregulation. More particularly, knowledge of
particular
domains or subunits within transcription factors that provide good targets for
the
development of modulators of transcription factor activity is is helpful in
developing
methods and compositions for use in diagnosing and treating cancers. The
present invention
fulfills this and other needs.
StfMMARY OF THE INVENTION
This invention pertains to discoveries regarding the structural and fimctional
organization of ESX epithelial-restricted with serine bola member of the ETS
family of
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genes. In particular, this invention is premised, in part, on the discovery
that axons 4 and 7
encode separate transactivating and chromatin localizing domains in ESX.
In one embodiment, this invention provides methods of screening for a
modulator of ESX activity. The methods involve providing a target such as a
nucleic acid
encoding a polypeptide of ESX axon 7 (or axon 4), and/or polypeptide
comprising ESX axon
7 (or axon 4); contacting the nucleic acid or polypeptide with the test agent;
and detecting
binding of the test agent to the nucleic acid or polypeptide wherein a test
agent that binds to
said nucleic acid or polypeptide modulates ESX activity. In one preferred
embodiment, the
target is a nucleic acid encoding a polypeptide of ESX axon 7, while in
another preferred
embodiment the polypeptide comprises ESX axon 7. In preferred embodiments, the
polypeptide is not a full-length ESX polypeptide. The nucleic acid or
polypeptide can be
labeled with a detectable label (e.g., radioisotope, enzyme, fluorescent
molecule, quantum
dot, chemiluminescent molecule, bioluminescent molecules, colloidal metals,
etc.) and
preferred detectable labels are fluorescent labels. Preferred detection
methods include
immunoassays, particularly immunoassays utilizing an antibody that
specifically binds to a
polypeptide of ESX axon 7.
This invention also provides methods of activating transcription of a gene or
a
cDNA. The methods involve contacting the "target" gene or cDNA with a
construct
comprising a DNA binding domain attached to a polypeptide comprising axon 4 of
ESX. In
a preferred embodiment, the polypeptide is not a full-length ESX polypeptide.
In one
embodiment, the polypeptide comprising axon 4 of ESX contains one or more
mutations of
amino acids at positions 155, 154, 152, 150, 145, 135, 131, and 132. The
polypeptide can
also comprise conservative substitutions in the ESX exon(s). Alternatively, or
in addition,
the polypeptide can comprise an axon 4 of ESX having a carboxyl terminal
deletion of axon
4 of up to 27 amino acids and/or an amino terminal deletion of axon 4 of up to
a 5 amino
acids. In one particularly preferred embodiment, the ESX axon 4 region
contains both an
amino terminal and a carboxyl terminal deletion of axon 4 leaving the axon 4
amino acids
134 through 147. The transactivation is most effective against target
genes/cDNAs under
the control of a promoter (preferably an epithelial gene promoter) having an
endogenous or
engineered Ets response element. The gene or cDNA can be an endogenous gene or
cDNA
or present in a transfected (e.g., transiently transfected) vector. A
preferred DNA binding
domain is a GAL4 DNA binding domain.
This invention also provides constructs comprising a nucleic acid (e.g. DNA)
binding domain attached to a polypeptide comprising axon 4 or axon 7 of ESX.
Also
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provided are nucleic acids encoding such constructs. Preferred constructs (and
their
corresponding nucleic acids) are constructs comprising a DNA binding domain
attached to a
polypeptide comprising exon 4 of ESX. Any of the exon 4 polypeptides are
described herein
are suitable. The nucleic acid binding domain is either chemically conjugated
to the ESX
polypeptide or they are expressed as a fusion protein.
In still another embodiment, this invention provides an affinity matrix
comprising a solid support attached to a polypeptide comprising exon 4 or exon
7 of ESX as
described herein. The polypeptide is preferably not a full-length ESX.
Preferred solid
supports include glass, plastic, metal, ceramic, gels and aerogels.
Also provided are kits for performing any of the methods described herein.
Preferred kits comprising a container containing one or more of the constructs
selected from
the group consisting of a polypeptide that is not a full-length ESX and that
comprises ESX
exon 4 or ESX exon 7, a DNA binding domain attached to a polypeptide
compzising exon 4
or exon 7 of ESX, a nucleic acid encoding a polypeptide that is not a full-
length ESX and
that comprises ESX exon 4 or ESX exon 7, and a nucleic acid encoding a DNA
binding
domain attached to a polypeptide comprising exon 4 or exon 7 of ESX.
This invention also provides methods of detecting dysregulation of an ESX
gene (e.g., in an organism). The methods involve detecting the degree of
acetylation of ESX
in a biological sample; and comparing said degree of acetylation of ESX in the
biological
sample with the degree of acetylation in a control sample from a normal
healthy tissue,
wherein a difference in the degree of acetylation of ESX in said biological
sample with the
degree of acetylation in said control sample indicates dysregulation of an ESX
gene. In
preferred embodiments, the difference is a statistically significant
difference (e.g. at least a
1.5 fold difference, more preferably at least a two-fold difference, and most
preferably at
least a 5-fold or even at least a 10-fold difference). The detecting can be by
use of an
antibody that specifically binds to an acetylated ESX and not to an
unacetylated ESX or vice
versa. In one embodiment, the statistically significant difference is
indicative of an
epithelial cancer (e.g., human breast cancer). In one embodiment the healthy
tissue
comprises normal human mammary epithelial cells. In one embodiment the
statistically
significant difference is indicative of an unfavorable prognosis. The method
can fiuther
involve selecting an appropriate treatment regime.
Also provided are methods of inhibiting growth or proliferation of neoplastic
cells. The methods involve administering to said cells an effective amount of
an agent that
inhibits activity of exon 4 or exon 7. The neoplastic cells can comprise a
cancer in an
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organism. The method can involve transfecting cells of the mammal with a
vector
expressing an ~antisense ESX nucleic acid that specifically binds to a nucleic
acid of exon 4
or exon 7. Various agents include, but are not limited to an exon 4 mutein, an
exon 7
mutein.
This invention also provides methods of depressing transcription of a gene or
a cDNA. The methods involve contacting the gene or cDNA with a recombinantly
expressed
polypeptide comprising an exon 4 of ESX where the polypeptide is not a full-
length ESX
and lacks a DNA binding domain.
This invention also provides probes for detection or localization of proteins
that bind to ESX. These probes comprise a detectable label attached to a
polypeptide
comprising ESX exon 4 wherein the polypeptide is not a full length ESX
polypeptide.
Probes are also provided for detection or localization of proteins or nucleic
acids that bind to
ESX. These probes comprise a detectable label attached to a polypeptide
comprising ESX
exon 7 wherein said polypeptide is not a full length ESX polypeptide. The
probes can be
isolated, recombinantly expressed, or chemically synthesized. Preferred
detectable labels
include radioisotopes, enzymes, fluorescent molecules, chemiluminescent
molecules,
quantum dots, bioluminescent molecules, and colloidal metals.
Definitions
The term "antibody" refers to a polypeptide substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof which
specifically
bind and recognize an analyte (antigen). The recognized immunoglobulin genes
include the
kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as
well as the
myriad immunoglobulin variable region genes. Light chains are classified as
either kappa or
lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An
exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each
tetramer is
composed of two identical pairs of polypeptide chains, each pair having one
"light" (about
25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain
defines a
variable region of about 100 to 110 or more amino acids primarily responsible
for antigen
recognition. The terms variable light chain (VL) and variable heavy chain (VH)
refer to these
light and heavy chains respectively.
Antibodies exist e.g., as intact immunoglobulins or as a number of well-
characterized fragments produced by digestion with various peptidases. Thus,
for example,
pepsin digests an antibody below the disulfide linkages in the hinge region to
produce
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F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a
disulfide bond.
The F(ab)'Z may be reduced under mild conditions to break the disulfide
Linkage in the hinge
region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab'
monomer is
essentially an Fab with part of the hinge region (see, Fundamental Immunology,
Third
Edition, W.E. Paul, ed., LZaven Press, N.Y. 1993). While various antibody
fi~agments are
defined in teens of the digestion of an intact antibody, one of skill will
appreciate that such
fragments may be synthesized de novo either chemically or by utilizing
recombinant DNA
methodology. Thus, the term antibody, as used herein, also includes antibody
fragments
either produced by the modification of whole antibodies or those synthesized
de novo using
recombinant DNA methodologies (e.g., single chain Fv).
An "anti-ESX antibody" is an antibody or antibody fragment that specifically
binds a poLypeptide encoded by the ESX gene, cDNA, or a subsequence thereof.
A "chimeric antibody" is an antibody molecule in which (a) the constant
region, or a portion thereof, is altered, replaced or exchanged so that the
antigen binding site
(variable region) is linked to a constant region of a different or altered
class, effector
function and/or species, or an entirely different molecule which confers new
properties to the
chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.;
or (b) the
variable.region, or a portion thereof, is altered, replaced or exchanged with
a variable region
having a different or altered antigen specificity.
The term "immunoassay" is an assay that utilizes an antibody to specifically
bind an analyte. The immunoassay is characterized by the use of specific
binding properties
of a particular antibody to isolate, target, and/or quantify the analyte.
The terms "isolated" "purified" or "biologically pure" refer to material which
is substantially or essentially free from components which normally accompany
it as found
in its native state.
The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide
polymer in either single- or double-stranded form, and unless otherwise
Limited,
encompasses known analogs of natural nucleotides that can function in a
similar manner as
naturally occurring nucleotides.
The terms "polypeptide", "peptide" and "protein" are used interchangeably
herein to refer to a polymer of amino acid residues. The terms apply to amino
acid polymers
in which one or more amino acid residue is an artificial chemical analogue of
a
corresponding naturally occurring amino acid, as well as to naturally
occurring amino acid
polymers.
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A "label" is a composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, or chemical means. For example, useful labels
include 32P,
fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in
an ELISA);
biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal
antibodies are
S available (e.g., the peptide of SEQ ID NO 2 can be made detectable, e.g., by
incorporating a
radio-label into the peptide, and used to detect antibodies specifically
reactive with the
peptide).
As used herein a "nucleic acid probe" is defined as a nucleic acid capable of
binding to a target nucleic acid of complementary sequence through one or more
types of
chemical bonds, usually through complementary base pairing, usually through
hydrogen
bond formation. As used herein, a probe may include natural (i. e. A, G, C, or
T) or modified
bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may
be joined by a
linkage other than a phosphodiester bond, so long as it does not interfere
with hybridization.
Thus, for example, probes may be peptide nucleic acids in which the
constituent bases ase
joined by peptide bonds rather than phosphodiester linkages. It will be
understood by one of
skill in the art that probes may bind target sequences lacking complete
complementarity with
the probe sequence depending upon the stringency of the hybridization
conditions. The
probes are preferably directly labeled as with isotopes, chromophores,
lumiphores,
chromogens, or indirectly labeled such as with biotin to which a streptavidin
complex may
later bind. By assaying for the presence or absence of the probe, one can
detect the presence
or absence of the select sequence or subsequence.
A "labeled nucleic acid probe" is a nucleic acid probe that is bound, either
covalently, through a linker, or through ionic, van der Waals or hydrogen
bonds to a label
such that the presence of the probe may be detected by detecting the presence
of the label
bound to the probe.
The term "target nucleic acid" refers to a nucleic acid (often derived from a
biological sample), to which a nucleic acid probe is designed to specifically
hybridize. It is
either the presence or absence of the target nucleic acid that is to be
detected, or the amount
of the target nucleic acid that is to be quantified. The target nucleic acid
has a sequence that
is complementary to the nucleic acid sequence of the corresponding probe
directed to the
target. The term target nucleic acid may refer to the specific subsequence of
a larger nucleic
acid to which the probe is directed or to the overall sequence (e.g., gene or
mRNA) whose
expression level it is desired to detect. The difference in usage will be
apparent from
context.
_g_
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"Subsequence" refers to a sequence of nucleic acids or amino acids that
comprise a part of a longer sequence of nucleic acids or amino acids (e.g.,
polypeptide)
respectively.
The term "recombinant" when used with reference to a cell, or nucleic acid,
or vector, indicates that the cell, or nucleic acid, or vector, has been
modified by the
introduction of a heterologous nucleic acid or the alteration of a native
nucleic acid, or that
the cell is derived from a cell so modified. Thus, for example, recombinant
cells express
genes that are not found within the native (non-recombinant) form of the cell
or express
native genes that are otherwise abnormally expressed, under expressed or not
expressed at
all.
The term "identical" in the context of two nucleic acids or polypeptide
sequences refers to the residues in the two sequences which are the same when
aligned for
maximum correspondence. Optimal alignment of sequences for comparison can be
conducted, e.g., by the.local homology algorithm of Smith and Waterman (1981)
Adv. Appl.
1 S Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch
(1970) J.
Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (
/ 988) Proc.
Nat'l. Acad Sci. USA 85: 2444, by computerized implementations of these
algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, W>7, or by inspection.
An additional algorithm that is suitable for determining sequence similarity
is
the BLAST algorithm, which is described in Altschul et al. (i990) J. Mol.
Biol. 215: 403-
410. Software for performing BLAST analyses is publicly available through the
National
Center for Biotechnology Information (http:!/www.ncbi.alm.nih.gov~. This
algorithm
involves first identifying high scoring sequence pairs (HSPs) by identifying
short words of
length W in the query sequence that either match or satisfy some positive-
valued threshold
score T when aligned with a word of the same length in a database sequence. T
is referred to
as the neighborhood word score threshold (Altschul et al, supra.). These
initial
neighborhood word hits act as seeds for initiating searches to find longer
HSPs containing
them. The word hits are extended in both directions along each sequence for as
far as the
cumulative alignment score can be increased. Extension of the word hits in
each direction
are halted when: the cumulative alignment score falls off by the quantity X
from its
maximum achieved value; the cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue alignments; or the end of
either
sequence is reached. The BLAST algorithm parameters W, T and X determine the
~35 sensitivity and speed of the alignment. The BLAST program uses as defaults
a word length
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(W) of 11, the BLOSLTM62 scoring matrix (see Henikoff and Henikoff ( 1992)
Proc. Natl.
Acad Sci. USA, 89: 10915-10919) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4,
and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity
between two sequences; see, e.g., Karlin and Altschul (1993) Proc. Nat'1. Acad
Sci. USA
90: 5873-5787. One measure of similarity provided by the BLAST algorithm is
the smallest
sum probability (P(N)), which provides an indication of the probability by
which a match
between two nucleotide or amino acid sequences would occur by chance. For
example, a
nucleic acid is considered similar to an ESX nucleic acid if the smallest sum
probability in a
comparison of the test nucleic acid to an ESX nucleic acid is less than about
0.1, more
preferably less than about 0.01, and most preferably less than about 0.001.
Where the test
nucleic acid encodes an ESX polypeptide, it is considered similar to a
specified ESX nucleic
acid if the comparison results in a smallest sum probability of less than
about 0.5, and more
preferably less than about 0.2.
The terns "substantial identity" or "substantial similarity" in the context of
a
polypeptide indicates that a polypeptides comprises a sequence with at least
70% sequence
identity to a reference sequence, or preferably 80%, or more preferably 85%
sequence
identity to the reference sequence, or most preferably 90% identity over a
comparison
window of about 10-20 amino acid residues. An indication that two polypeptide
sequences
are substantially identical is that one peptide is immunologically reactive
with antibodies
raised against the second peptide. Thus, a polypeptide is substantially
identical to a second
polypeptide, for example, where the two peptides differ only by a conservative
substitution.
An indication that two nucleic acid sequences are substantially identical is
that the polypeptide which the first nucleic acid encodes is immunologically
cross reactive
with the polypeptide encoded by the second nucleic acid.
Another indication that two nucleic acid sequences are substantially identical
is that the two molecules hybridize to each other under stringent conditions.
"Bind(s) substantially" refers to complementary hybridization between a
probe nucleic acid and a target nucleic acid and embraces minor mismatches
that can be
accommodated by reducing the stringency of the hybridization media to achieve
the desired
detection of the target polynucleotide sequence.
The phrase "hybridizing specifically to", refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence under
stringent conditions
when that sequence is present in a complex mixture (e.g., total cellular) DNA
or RNA. The
term "stringent conditions" refers to conditions under which a probe will
hybridize to its
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target subsequence, but to no other sequences. Stringent conditions are
sequence-dependent
and will be different in different circumstances. Longer sequences hybridize
specifically at
higher temperatures. Generally, stringent conditions are selected to be about
S°C lower than
the thermal melting point (Tm) for the specific sequence at a defined ionic
strength and pH.
The Tm is the temperature (under defined ionic strength, pH, and nucleic acid
concentration)
at which 50% of the probes complementary to the target sequence hybridize to
the target
sequence at equilibrium. (As the target sequences are generally present in
excess, at Tm,
50% of the probes are occupied at equilibrium). Typically, stringent
conditions will be those
in which the salt concentration is less than about 1.0 M sodium ion, typically
about 0.01 to
1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at
least about 30°C for short probes (e.g., 10 to SO nucleotides) and at
least about 60EC for
long probes (e.g., greater than 50 nucleotides). Stringent conditions may also
be achieved
with the addition of destabilizing agents such as formamide.
The phrases "specifically binds to a protein" or "specifically immunoreactive
with", when referring to an antibody refers to a binding reaction which is
determinative of
the presence of the protein in the presence of a heterogeneous population of
proteins and
other biologics. Thus, under designated immunoassay conditions, the specified
antibodies
bind preferentially to a particular protein and do not bind in a significant
amount to other
proteins present in the sample. Specific binding to a protein under such
conditions requires
an antibody that is selected for its specificity for a particular protein. A
variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are routinely
used to
select monoclonal antibodies specifically immunoreactive with a protein. See
Harlow and
Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New
York, for a description of immunoassay formats and conditions that can be used
to determine
specific immunoreactivity. For determination of specific binding of an anti-
ESX antibody,
an immunoprecipitation assay is preferred. Under appropriate conditions, an
antibody that
specifically binds to an ESX polypeptide will immunoprecipitate ESX, but not
other ETS
transcription factors.
A "conservative substitution", when describing a protein refers to a change in
the amino acid composition of the protein that does not substantially alter
the protein's
activity. Thus, "conservatively modified variations" of a particular amino
acid sequence
refers to amino acid substitutions of those amino acids that are not critical
for protein activity
or substitution of amino acids with other amino acids having similar
properties (e.g., acidic,
basic, positively or negatively charged, polar or non-polar, etc.) such that
the substitutions of
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even critical amino acids do not substantially alter activity. Conservative
substitution tables
providing functionally similar amino acids are well known in the art. The
following six
groups each contain amino acids that are conservative substitutions for one
another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (17, Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (V~.
See also, Creighton (1984) Proteins W.H. Freeman and Company. One of
skill in the art will appreciate that the above-identified substitutions are
not the only possible
conservative substitutions. For example, one may regard all charged amino
acids as
conservative substitutions for each other whether they are positive or
negative (see, e.g.,
Figures 2B, 2C, and 2D). In addition, individual substitutions, deletions or
additions which
alter, add or delete a single amino acid or a small percentage of amino acids
in an encoded
sequence are also "conservatively modified variations".
The terms human "esx " or human "ESX gene or cDNA" are used
interchangeably to refer to the human esx gene, which is a transcription
factor gene that is
also involved in the etiology of cancers, for example, epithelial cancers. The
esx gene is
determined to be a member of the ETS gene family by significant homology
between the
ESX DNA binding domain and the DNA binding domain of other members of the ETS
family. ESX, however, is distinct from previously known ETS genes because of 5
non-
conservative substitutions in the ETS consensus sequence. Nevertheless, ESX is
still
recognized to belong to the ETS family because ESX contains 27 identical amino
acid
residues among the 38 recognized consensus residues making up the ETS DNA
binding
domain (i.e., greater than 50% *sequence identity, more preferably greater
than 60%
sequence identity and most preferably greater than 70% sequence identity in
the ETS
consensus sequence). Similarly the terms mouse or marine ESX genes or cDNAs
refer to
the mouse or marine ESX genes or cDNAs respectively.
A "gene product", as used herein, refers to a nucleic acid whose presence,
absence, quaatity, or nucleic acid sequence is indicative of a presence,
absence, quantity, or
nucleic acid composition of the gene. Gene products thus include, but are not
limited to, an
mRNA transcript, a cDNA reverse transcribed from an mRNA, an RNA transcribed
from
that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the
amplified DNA
or subsequences of any of these nucleic acids. Polypeptides expressed by the
gene or
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subsequences thereof are also gene products. The particular type of gene
product will be
evident from the context of the usage of the term.
An "abnormal esx gene or cDNA" refers to an esx gene or cDNA that
encodes an increased or decreased amount of ESX polypeptide, a non-functional
ESX
polypeptide, or an ESX polypeptide of substantially reduced functionality.
Animal cells
having non-functional, or reduced functionality, ESX polypeptides are
characterized by a
decrease in ESX-mediated transcriptional regulation. In a cancer cell, this
relaxation of
ESX-mediated regulation can result in a decrease in neoplastic cell
proliferation. Similarly,
"abnormal ESX gene product" refers to a nucleic acid encoding a non-functional
or reduced
functionality ESX polypeptide or the non-functional or reduced functionality
ESX
polypeptide itself. Abnormal esx genes or gene products include, for example,
esx genes or
subsequences altered by mutations (e.g. insertions, deletions, point
mutations, etc.), splicing
errors, premature termination codons, missing initiators, etc. Abnormal ESX
polypeptides
include polypegtides expressed by abnormal esx genes or nucleic acid gene
products or
subsequences thereof. Abnormal expression of esx genes includes
underexpression (as
compared to the "normal" healthy population) of ESX, B.g., through partial or
complete
inactivation, haploinsufficiency, etc.
The terms "rodent" and "rodents" refer to all members of the phylogenetic
order Rodentia including any and all progeny of all future generations derived
therefrom.
The term "marine" refers to any and all members of the family Muridae,
including rats and mice.
A "therapeutic lead compound" refers to a compound that has a particular
characteristic activity, e.g., an activity that is therapeutically useful. ~
While the compound
itself may not be suitable a therapeutic the compound provides a basis or
starting point for
the creation and/or screening of analogues for similar desired activity (e.g.,
for ESX
modulatory activity).
The term "test agent" (used interchangeably herein with "candidate agent"
and "test compound" and "test composition") refers to an element molecule or
composition
whose effect e.g., on ESX activity it is desired to assay. The "test
composition" can be any
molecule or mixture of molecules, optionally in a suitable carrier.
A "polypeptide comprising exon X of ESX" (where X is the exon number)
refers to a polypeptide encoded by exon X of ESX. In some instances,
particularly where
the exon is present in a construct that is not a full-length ESX, the exon can
include deletions
and/or mutations that transactivation activity of this domain.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleotide (SEQ ID NO:1 ) and deduced amino acid (SEQ
ID N0:2)sequences of a human ESX cDNA.
Figures 2A through 2E show the amino acid sequence of the human ESX
S polypeptide and the domain homologies of the ESX polypeptide as compared to
other
members of the ETS transcription factor family. Figure 2A shows the amino acid
sequence
corresponding to the longest open reading frame in the human ESX cDNA (SEQ ID
N0:2).
Highlighted regions (boxed, bold font) are homologous to domains of other ETS
transcription factors; these include the A-region/Pointed domain (amino acids
64-103), the
serine-rich box (amino acids 188-238), and the ETS DNA binding domain (amino
acids 274-
354). Four regions that are not homologous to other Ets transcription factor
domains are
unboxed. Figure 2B presents a comparison of the A-region/Pointed domain of ESX
(SEQ
117 N0:17) to that encoded by the human ETS-1 gene (SEQ D7 N0:18). Consensus
residues
most highly conserved-among Ets family members are shown (Lautenberger et al.
(1992)
Oncogene 7: 1713-1719. Conservative substitutions are indicated by (+). Figure
2C shows
the similarity between the ESX serine box (SEQ ID N0:19) and that of SOX4 (SEQ
ID
N0:20)..,A portion of the ESX serine box (SEQ ID N0:21) is shown in a helical
wheel
model to demonstrate clustering of serine residues opposite a hydrophobic
helical face
(boxed residues). Figure 2D shows the amino acid identity and similarity
within the ETS
DNA binding domain of the two related subfamily members, ESX (SEQ ID N0:22)
and Elf
1 (SEQ 11? N0:23). Consensus residues in this domain are the most highly
conserved
among all Ets family members (Janknecht and Nordheim (1993) Biochem. Biophys.
Acta.
1155: 346-356). Conservative (~) and non-conservative (*) substitutions found
in ESX
relative to the consensus residues (SEQ ID NOs:24-29) and their locations
within lmown
structural components of the ETS domain are shown (Wemer et al. (1995) Cell
83: 761-771;
Kodandapani et al. (I996) Nature 380: 457-460). Figure 2E illustrates the
human ESX
protein sequence (SEQ ID N0:2) showing the residues encoded by exon 4 (bold),
the
residues conserved in all Topo-I proteins (~) the Topo-I homologous fragment
(.~) and the
Lysine~45 critical for transactivation (circled and bolded K).
Figure 3 Illustrates the marine ESX (mES~ genomic organization and gene
product.
Figure 4 shows the human ESX (hES~ (cDNA=SEQ ID NO:1, amino acid =
SEQ ID N0:2) exon/intron junctions. The bold sequences contain the
"tranactivating
domain" as mapped by GAL4 fusion studies.
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Figure 5 shows the mouse ESX (mES~ (SEQ m NO: I6) and human ESX
(hESX) (SEQ ID N0:2) primary structure and domain homologies.
Figure 6 shows the conserved elements in the mouse ESX (mES~ (SEQ ID
N0:30) and human ESX(hES.i~ (SEQ ID N0:2) proximal promoter.
Figure 7 illustrates the mouse ESX (mESX) and human ESX (hES~ genomic
DNA structure.
Figures 8A through 8D show the results of DNA binding and transactivation
by recombinant ESX gene product, as well as chromosomal localization and copy
number of
the ESX gene. Figure 8A shows specific DNA-binding of full-length (42 kDa)
recombinantly expressed ESX to an oligonucleotide sequence (TAS) containing
the Ets
responsive element (GGAA) from the HERZ/neu promoter. Five different competing
unlabeled (cold) oligonucleotides containing specific mutations in the wild-
type (WT) TAS
(SEQ ID N0:32) sequence, ml-m5 (SEQ ID NOs:33-37), were added at 50-fold molar
excess; gel lanes containing the excess cold competitors are labeled. Figure
8B shows a
DNase-I hypersensitivity site and footprint produced by ESX on the antisense
strand of an
Ets response element in the HER2Ineu promoter. The antisense strand sequence
(SEQ m
N0:38) as shown (~40 by to ~26 by upstream of major. transcriptional start
site in. HER2/neu
promoter) is marked with asterisk at the hypersensitivity site within Ets
response element
(GGAA on sense strand). Figures 8C and 8D show the induction of CAT activity
from two
different ETS-responsive reporter constructs (p3TA5-BLCATS, pHER2-CAT) in COS
cells
cotransfected with an ESX expression plasmid (pcDNAI-ESX). Mutant reporter
plasmids
(p3TA5P-BLCATS, pHER2m-CAT) are identical to their normal counterparts except
for
alterations in the Ets response element within the TAS sequence (GGAA to GAGA
and
GGAA to TTAA, respectively).
Figure 9 illustrates mapping of the hESX activation domain. The varying
hESX deletion constructs and their transactivation activity is shown.
Figure 10 shows a comparison of exon-encoded mouse (m) and human (h)
ESX amino acid sequences. The 371 amino acid sequences encoded by genomic
exons 2-9
were determined by comparing cloned mouse and human cDNA sequences, with the 7
exon
boundaries mapped (arrows) after comparison between mouse and human genomic
sequences, as described in Methods. Amino acid identities (vertical lines) and
similarities (:,
.) are as indicated.
Figure 11 illustrates nucleotide sequences and consensus response elements
conserved between mouse and human ESX promoters. Comparison of aligned mouse
and
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human genomic sequences revealed 83% nucleotide identity (vertical lines)
between the 0.4
kb of upstream 'sequences shown above. Sequence numbering is relative to a
putative
transcriptional start site (+1) within a conserved pyrimidine-rich type Inr
(box with arrow)
located ~75 by downstream from a conserved CCAAT sequence (box); this putative
site was
also identified as the most 5'-terminal nucleotide from a hESX cDNA clone and
agrees with
a previously determined hESX transcriptional start site (Oettgen et al. (1997)
Mol. Cell.
Biol. 17: 4419-4433). The locations of conserved consensus response elements
for Ets, AP-
2, SP1/GC box, USF, Oct, and NF-xB are indicated by the horizontal bars; of
the pair of Ets
elements, the 5' element (GGAA) appears displaced by 3 nucleotides while the
3' element
(TTCC) is positionally conserved in both promoters. As described in Methods,
this 0.4 kb of
mESX promoter sequence was cloned into pGL2-Basic to produce the mESX-luc
reporter
construct described in Example 6.
Figures 12A and 12B show the organization and features of the_11 kb Hind
III ESX genomic fragment and comparison of Ets domain protein homologies
showing
exon-intron junctions. Figure 12A shows regions of identity between the 11 kb
Hind III
ESX genomic fragment and the three contigs THC13038 (identical also to regions
of UEV-
1, assesssion no. U49278), THC209687 and THC203540 are identified by the
respective
bracketed region. Coding ESX exons are shown as shaded boxes while noncoding
5' and 3'
exonic sequences are shown as open boxes. ESX transcription start, translation
irritation and
translation termination are indicated by an arrow, ATG and TGA respectively.
Polyadenylation signal for the 2.2 kb ESX transcript indicated by
AATAAA(2.2kb) with
presumptive polyadenylation signals for the 4.1 kb ESX transcript in THC203540
noted by
ATTAAA(4.lkb) and GATAAA(4.lkb). Presumptive UEV-1 polyadenylation signal in
THC213038 (also in corresponding genomic sequence) noted by AATAAA(tJEV). The
350
by of ESX promoter with >80% homology to that of mouse is shown as a darkly
stippled
box while the 1150 by region with <50% homology is shown as a lightly stippled
box.
Regions homologous to Alu sequences and containing CpG islands are indicated.
Figure
12B: Protein sequence alignment of the Ets DNA-binding domain from Ets family
members
whose genomic structure has been determined. The armw (if applicable)
indicates the
positions) of exon-intron junctions. Where exon-intron information for a
particular Ets
factor exists in different species (m=mouse, r=rat, c=chicken, d Drosophila)
only the human
prototype is shown. Numbers flanking the protein sequences reference their
location within
the full-lengh protein. Percent protein homology to ESX, ERF and ETS-1 were
determined
as percentage of identical amino acids within the corresponding Ets domains.
References for
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WO 99/65929 PCT/US99/13277
the genomic structures are: human/mouse ERF (de Castro et al. ( 1997)
Genomics, 42: 227-
235; Liu et al.~(1997) Oncogene, 14: 1445-1451), human/mouselrat/chicken ETS1
(Bellacosa et al. (1994) .l. Yirol., 68: 2320-2330; Jorcyk et al. (1991)
Oncogene, 6: 523-
532; Watson et al. (1988) Virology, 164: 99-105),
human/mouse/chicken/drosophilia ETS2
(Pribyl et al. (1988) Dev. Biol., 127: 45-53; Watson et al. (1990) Oncogene 5:
1521-1527),
drosphilia PNT (Klambt C. (1993) Development, 117: 163-176), human ERM (Monte
et al.
(1996) Genomics, 35: 236-240), human TEL (Baens et al. (1996) Genome Res., 6:
404-
413), mouse ELK/ (Grevin et al. (1996) Gene, 174: 185-188), mouse PU.1 (Moreau-
Gachelin (1989) Oncogene, 4: 1449-1456) and Spi-B (Chen et al. (1998) Gene,
207: 209-
218).
Figure 13 shows that the transactivating capacity of ESX localizes to exon 4.
Top panel presents reporter (luciferase) activity induced by a series of C-
terminally deleted
ESX constructs fused to the GAL4 DNA-binding domain (G) and shown relative to
G-
VP16(413/490) induced activity which served as a positive control. Bracketed
numbers
specify positions of the flanking amino acids in the ESX or VP16 fusion
constructs. Middle
panel presents the activity induced by various fragments of ESX fused to the
GAL4 DNA-
binding domain and shown relative to G-VP16(413/490). Activity of an ESX
construct
harboring the double mutations, S131R/S132A, and its normal counterpart, G-
ESX(1/156),
are displayed in the bottom two rows of this panel. Schematic at bottom of
figure depicts
approximate domain positioning within ESX including the Pointed domain, SOX
box, A/T
hook, and Ets domain shown above boundary lines specifying the location of the
8 coding
exons (2-9) in ESX.
Figures 14A and 14B show the results of a mutational analysis of exon 4.
Figure 14A shows reporter (luciferase) activity induced by exon 4 constructs
containing
single or double amino acid substitutions fused to the GAL4 DNA binding domain
(G) and
expressed as a percentage of induced activity by the unmodified exon 4 G-
ESX(129-159)
construct. For each exon 4 mutational construct, the positions) of the
substituted amino
acids) relative to unmodified exon 4 amino acids shown on the first row are
indicated (A =
alanine, P = proline and Q = glutamine). The bottom four rows represent
truncated exon 4
constructs fused to the GAL4 DNA-binding domain (G). Positions of the terminal
amino
acids of the truncated ESX constructs are shown in brackets. Figure 14B show
mutational
analysis of the FXX~~ motif within exon 4 (F = phenylalanine, X = any amino
acid, ~~ _
hydrophobic amino acid). Top panel aligns the FXX~~ motif within ESX, VP16,
p65NF-xB
and p53 proteins. The reporter (luciferase) activity induced by exon 4
mutations within the
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FXX~~ motif are expressed as a percentage of induced activity by the
unmodified exon 4 G-
ESX(129-159) construct. The positions) of alanine substitutions) in the
various exon 4
constructs are shown. Positive and negative controls consisted of the double
VP16 mutant
(F479A~I,483A) and the unmodified VP16 fusion construct G-VPI6(413-490), and
their
activities are shown in the bottom two rows.
Figures 1 SA and 1 SB show evidence for a-helical secondary structure within
ESX exon 4 domain. Figure 15A shows a helical wheel projection of the 13 amino
acids (aa
134-146) from the acidic core transactivating domain of exon 4 demonstrates
their
amphipathic distribution. Figure 15B shows CD spectra of a 25 amino acid exon
4 peptide
(aa 131-155) recorded at six different methanol:water concentrations. Insert
gives the
percent a-helical content of the peptide at each methanol concentration.
Figures I6A and 16B shows cell line dependent squelching of heterologous
promoters by ESX exon 4. Figure 16A: In transiently transfected SKBr3 cells,
'expression off
a GAL4(DBD)-exon 4'fusion construct, G-ESX(129-159), is shown to suppress the
activity
of two GAL4(DBD)-independent luciferase reporters, the SV40 early promoter and
a
synthetic promoter containing three tandem copies of the erbB2/IiER2
promoter's Ets w
response.element (Ets triple repeat}. The level of G-ESX(129-159) induced
squelching is
comparable to that induced by expression of the positive control construct
containing the
VPI6 transactivation domain, G-VP16(413-490). Activity is presented relative
to luciferase
activity following transfection of the GAL4(DBD) negative control expression
construct (G).
Similar squelching results were obtained in transfected COS-7 cells (data not
shown). Figure
16B: Transient transfection into either COS-7 or SKBr3 cells using a full-
length ESX
construct, pcDNAl-ESX, produced >4 fold upregulation of the Ets responsive
reporter (Ets
triple repeat) in the COS-7 cells but squelching (to <0.25 relative activity)
of this same
reporter in SKBr3 cells. Transfection of these cells with similar pcDNAl-ESX
expression
constructs either deleted of its exon 4 domain, pcDNAI-ESX~(129-I59), or
bearing double
mutations in this domain, pcDNAI-ESX(D134A/E135A), produced no significant
difference
in reporter activity relative to cells transfected with pcDNAl (empty vector)
alone.
Figure 17 schematically illustrates how the axon 4 domain is capable of
multiple protein-protein interactions. Given the link between ESX
overexpression and
erbB2 amplification/overexpression, and the ability of ESX to bind and
transactivate the
erb'B2 promoter, it is iikely that this domain predisposes this oncogene to
both
overexpression and amplification (unscheduled DNA replication) as illustrated
in the figure.
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DETAILED DESCRIPTION
This invention pertains to the discovery of a transcription factor associated
with the etiology of cancers, including epithelial cancers. This transcription
factor, referred
to as ESX (for epithelial-restricted with serine box), is located at
chromosome 1q32 in a
region known to be amplified in 50% of early breast cancers. ESX is heregulin-
inducible
and overexpressed in HER2/neu activated breast cancer cells. Tissue
hybridization suggests
that ESX becomes overexpressed at an early stage of human breast cancer
development
known as ductal carcinoma in situ (DCIS).
More particularly, this invention pertains to the discovery that ESX exons 4
and 7 encode separate transactivating and chromatin localizing domains in ESX.
Comparison of cloned marine and human cDNAs and genomic sequences (including
intron-
exon mapping), along with creation of GAL4 DBD and GST fusion proteins with
full-length
or partial ESX sequences have revealed that ESX contains the following unique
structural
and functional domains in addition to its defining 85 amino acid (C-terminal)
DNA-binding
domain.
The first domain characterized herein is a 33 amino acid transactivating
domain ~exon 4-encoded), with transactivating potency comparable to VP16 when
fused to a
GAIL-DBD (in a mammalian cell 2-hybrid-type experiment), and when fused to GST
in a
"pull-down" assay is able to bind specifically to both TATAA Binding Protein
(TBP) and
and the major subunit of Replication Protein A (RPA). It is believed that the
ability of exon
4 to potentially induce DNA replication by recruiting RPA (binding to the
major subunit but
not neither of the 2 minor RPA subunits), as has been shown for a few other
transcription
factors, has not yet been reported for any Ets factor. Since different
specific exon 4 residues
are critical for each of these binding functions (as described in Example 7),
they can be
separated to generate reagents with one or the other functions or to design
methods of
inhibiting either or both of these functions. This domain also has 50%
homology to the PDZ
domain in the Notch-interacting Dishevelled (Dsh) gene product and, in another
region,
significant homology to the highly conserved core domain of topoisomerase-I
(Topo I).
Using purified recombinant ESX we have been able to show that the full-length
ESX protein
has Topo I-like supercoil-relaxing activity not present in other Ets factors.
Thus, it is likely
that this exon 4 domain is designed for multiple protein-protein interactions,
specifying
critical tissue- and development-specific gene regulatory functions. Given the
link between
ESX overexpression and erbB2 amplification/overexpression, and the ability of
ESX to bind
and transactivate the erbB2 promoter, it is Likely that this domain
predisposes this oncogene
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to both overexpression and amplif cation (unscheduled DNA replication) in a
fashion
schematically represented in the diagram below (Figure 17). With this
bifunctional property,
in vitro systems employing ESX or portions thereof can be developed that both
transcribe
and replicate from the same DNA template.
The second domain characterized herein is a typical bipartite nuclear
localization signal, (Example 6), located in a domain (exon 7-encoded) also
having strong
homology to the A/T hook domain of HMG-Y. This sequence-nonspecific DNA-
binding
motif recognizes and stabilizes architecturally irregular DNA structures like
the H-DNA
form thought to be present within the GGA mirror-repeat and nuclear matrix
binding region
of the erbB2 proximal promoter (adjacent to the ESX binding EBS). This domain
has now
been shown to result in the nuclear sublocalizing predilection of ESX for the
matrix-
chromatin fi~action, unlike other transcription factors (e.g. AP-2) and other
related Ets factors
(e.g. Elf 1). Clustered lysine (K) residues in this domain are homologous in
number and
position to the functionally important acetylation sites known to be present
in the A/T hook
1 S domain of HMG-Y and in the DNA-binding domain of p53; and our studies
using the active
histone acetyl transferase (HAT) domain from the nuclear co-regulatory factor,
pCAF, have
now shown that exon 7 of ESX not only binds pCAF/HAT but is also acetylated by
pCAF/HAT to a degree exceeding that of p53 but not quite as completely as
histones.
Without being bound to a particular theory, it is predicted that acetylation
of ESX exon 7
results in its altered function (e.g. DNA-binding, perhaps also its nuclear
sublocalization and
its protein-protein interactions) and defines at least two populations of
intracellular ESX,
acetylated vs. non-acetylated ESX, for which specific antibodies and other
reagents can be
designed. Since the A/T hook domain firm HMG-Y has been shown to be
chimerially fused
by chromosomal translocation in human tumors, this same domain of ESX could be
genetically rearranged and involved in human tumorigenesis.
I. Uses of the ESX exons 4 and 7.
As indicated above, the ESX gene of this invention is a transcription factor
gene. Defects in the expression of this gene are associated the onset of
various cancers (e.g.,
cancers of the ovary, bladder, head and neck, and colon, etc.), particularly
with epithelial
cancers, including breast cancer among others.
It was a discovery of this invention that exons 4 is a strong transactivator
and
that exon 7 is bipartite nuclear localization signal (Example 7) that is a
sequence-nonspecific
DNA-binding motif that recognizes and stabilizes architecturally irregular DNA
structures.
In addition, exon 7 is subject to acetylation and thus appears to be
intimately involved in
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transcription regulation. ESX exons 4 and 7 (the nucleic acid or the encoded
protein) are
thus good targets for agents that are capable of modulating (upregulating or
downregulating)
ESX activity. Thus, in one embodiment, this invention provides methods of
screening for
potential regulators of ESX activity by screening for agents capable of
specificallly binding
to the ESX exon 4 or 7 nucleic acid or expressed protein.
In addition, because ESX exon 7 is subject to acetyation, a measure of the
relative acetylation (e.g. ratio of acetylated to unacetylated exon 7)
provides a measure of the
degree of ESX activation. Abnormal levels of ESX (exon 7) acetylation (e.g. as
compared to
that found in a healthy tissue) indicate abnormal regulation of ESX and
indicate a
predilection to the development of cancer.
ESX exon 4 is shown herein to be a potent transactivator comparable to VP 16.
When coupled (e.g. chemically conjugated or expressed as a fusion protein) to
a DNA
binding domain exon 4 is capable of inducing transcription of a target gene or
cDNA. The
target gene or cDNA can be a (e.g., introduced through homologous
recombination or in a
non-integrated vector and/or expression cassette) or an endogenous gene. ).
Transactivation
is most effective when the target gene or cDNA is under the control of a
promoter having an
ETS response element (e.g. an epithelial gene promoter).
The polypeptides of exons 4 and/or 7 can also be used to prepare an affinity
matrix for isolation of nucleic acids and/or polypeptides that interact with
ESX in these
domains. Thus, for example, the exon 4 and/or exon 7 polypeptide can be
attached to a solid
support and then contacted, under appropriate conditions, with target "sample"
(e.g. a cell
lysate). Polypeptides that bind to the exon 4 polypeptide and/or polypeptides
or nucleic
acids that bind to the exon 7 polypeptide will be retained on the support
bound exon
polypeptide while the remainder of the target "sample" is washed ofd The bound
target can
then be released from the affinity matrix.
Labeled exon 4 or exon 7 polypeptides can be used to probe cells, tissues,
etc.
for targets that interact with these polypeptides. In preferred embodiments,
the exon 4
and/or exon 7 polypeptides are labeled with a detectable label. Then a
"sample" can be
probed in vivo, ex vivo, or in situ to identify regions in which the probes
are localized and/or
to identify molecules that interact with these probes.
Cells and/or tissues expressing the ESX gene may be used to monitor
acetylation levels of ESX polypeptides in a wide variety of contexts. For
example, where
the effects of a drug on ESX expression is to be determined the drug will be
administered to
the cell. Acetylation levels, or expression products will be assayed as
described below and
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the results compared results from to organisms, tissues, or cells similarly
treated, but without
the drug being tested.
These uses are intended to be illustrative and not limiting. Other uses, e.g.,
as
suggested herein are within the purview of this invention.
II. The ESX gene and cDNA.
Al The human ESX gene.
Figure 1 provides both nucleic acid (SEQ ID NO:1) and polypeptide (SEQ ID
N0:2) sequence listings for the human ESX cDNA of this invention. In addition,
the human
genomic sequence is provided herein in the sequence listing (SEQ ID NO: 39}.
The
sequence of human ESX consists of an open reading frame of 1113 nucleotides;
an additional
161 and 703 nucleotides of 5'- and 3'-flanking sequence are presented in SEQ
ID N0:3.
The open reading frame of human ESX cDNA encodes for a putative protein of 371
amino
acids and a predicted molecular weight of 41428 Daltons.
B) The murine ESX p, ene.
A 7.8 kb mESX genamic clone was isolated that contains ~2.9 kb of promoter
upstream of ~4.9 kb of DNA incorporating at least 9 exons (see Figure 3 and
SEQ )D
NO:15}. These exons specify a full-length transcript of about 2 kb, with exons
2-9 encoding
the 371 amino acid mESX protein. Comparison of the mouse and human ESX
sequences
revealed the following structural and/or functional domains within a 42 kDa
ESX protein
conserved between mouse and human: an exon 3 encoded POINTED/A-region, found
in a
small subset of all ETS genes; an amphiphathic helix and serine-rich box
encoded by exons
5 and 6; a nucleoplamin-type nuclear targeting sequence encoded by exon 7, and
a helix-
tuna-helix ETS DNA binding domain encoded by exons 8 and 9.
The proximal promoter region of mESX (350 by upstream of the
transcriptional start site, see Figure 6) is 83%, homologous to the hESX
promoter. Conserved
putative response elements within this region include ETS, AP-2, SP1, USF,
Oct, and NF-6B
binding sites which are believed to regulate ESX induction. A conserved CCAAT
box lies
about 80 by upstream of the pyrimidine rich Inr element which specifies ESX
transcript
initiation. Unlike hESX, mESX lacks a TATA box.
Cl Isolation of cDNA and/or probes.
The aucleic acids (e.g., ESX cDNA, or subsequences (probes)) of the present
invention are cloned, or amplified by in vitro methods, such as the polymerase
chain reaction
(PCR), the ligase chain reaction (LCR), the transcription-based amplification
system (TAS),
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the self sustained sequence replication system (SSR). A wide variety of
cloning and in vitro
amplification methodologies are well-known to persons of skill. Examples of
these
techniques and instructions sufficient to direct persons of skill through many
cloning
exercises are found in Bergen and Kimmel, Guide to Molecular Cloning
Techniques,
Methods in Enzymology 152 Academic Press, Inc., San Diego, CA (Bergen);
Sambrook et a1
(1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring
Harbor
Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.); Current Protocols
in
Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint
venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994
Supplement)
(Ausubel); Cashion et al., U.S. patent number 5,017,478; and Carr, European
Patent No.
0,246,864. Examples of techniques sufficient to direct persons of skill
through in vitro
amplification methods are found in Bergen, Sambrook, and Ausubel, as well as
Mullis et al.,
(1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and
Applications
(Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim &
Levinson
(October 1, 1990) C&EN 36-47; The Journal OfIVIHResearch (1991) 3: 81-94;
(Kwoh et
al. (1989} Proc. Natl. Acad Sci. USA 86: 1173; Guatelli et al. (1990) Proc.
Natl. Acad Sci.
USA 87,.1.874; Lomell et al. (1989) .I. Clin. Chem., 35: 1826; Landegren et
al., (1988)
Science, 241: 1077-1080; Van Brunt (1990) Biotechnology, 8: 291-294; Wu and
Wallace,
(1989) Gene, 4: 560; and Barringer et al. (1990) Gene, 89: 117.
In one preferred embodiment, the human ESX cDNA can be isolated by
routine cloning methods. The cDNA sequence provided in SEQ )D NO: 1 can be
used to
provide probes that specifically hybridize to the ESX gene, in a genomic DNA
sample, or to
the ESX mRNA, in a total RNA sample (e.g., in a Southern blot). Once the
target ESX
nucleic acid is identified (e.g., in a Southern blot), it can be isolated
according to standard
methods known to those of skill in the art (see, e.g., Sambrook et al. (1989)
Molecular
Cloning: A Laboratory Manual, 2nd Ed, Vols. 1-3, Cold Spring Harbor
Laboratory; Bergen
and Kimmel (1987) Methods in Enrymology, Vol. 1 S2: Guide to Molecular Cloning
Techniques, San Diego: Academic Press, Inc.; or Ausubel et al. (1987) Current
Protocols in
Molecular Biology, Greene Publishing and Wiley-Interscience, New York).
Methods of
screening human cDNA libraries for the ESX gene are provided in Example 1.
In another prefen ed embodiment, the human ESX cDNA can be isolated by
amplification methods such as polymerase chain reaction (PCR). In a preferred
embodiment, the ESX sequence is amplified from a cDNA sample (e.g., double
stranded
placental cDNA (Clontech)) using the primers fESX-DBD, f-CCGGGACATCCTCA
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TCCACCC-3' (SEQ 1D N0:13)) and 3' ESX-DBD (5'-GTACCTCATGGCCCGGCTCAG-3'
(SEQ ID N0:14)). Preferred amplification conditions include l Ox PCR buffer
(500 mM
KCI, 100 mM Tris, pH 8.3 at room temperature, 15 mM MgCl2, 0.1% gelatin) with
the
amplification run for about 34 cycles at 94°C for 30 sec, 58°C
for 30 sec and 72°C for 60
S sec.
Similarly, using the nucleic acid sequence provided herein (e.g., SEQ 1D
NO:15), one of ordinary skill can routinely isolate the mouse ESX gene, mRNA
or cDNA.
However, in a preferred embodiment, the mouse ESX sequence is amplified from a
nucleic
acid sample (e.g., gDNA or cDNA) using that primers readily derived from the
sequence
listings provided herein. Suitable primers include, but are not limited to
primers (e.g., 20
mers) corresponding to the 5' and 3' termini of the marine ESX cDNA as
described above.
D) Labeling of nucleic acid probes.
Where the ESX cDNA or its subsequences (e.g., exon 4 or 7) are to be used
as nucleic acid probes,~it is often desirable to label the nucleic acids with
detectable labels.
The labels may be incorporated by any of a number of means well known to those
of skill in
the art. However, in a preferred embodiment, the label is simultaneously
incorporated
during the amplification step in the preparation of the sample nucleic acids.
Thus, for
example, polymerase chain reaction (PCR) with labeled primers or labeled
nucleotides will
provide a labeled amplification product. In another preferred embodiment,
transcription
amplification using a labeled nucleotide (e.g. fluorescein-labeled UTP and/or
CTP)
incorporates a label into the transcribed nucleic acids.
Alternatively, a label may be added directly to an original nucleic acid
sample
(e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the
amplification is completed. Means of attaching labels to nucleic acids are
well known to
those of skill in the art and include, for example nick translation or end-
labeling (e.g. with a
labeled RNA) by kinasing of the nucleic acid and subsequent attachment
(ligation) of a
nucleic acid linker joining the sample nucleic acid to a label (e.g., a
fluorophore).
Detectable labels suitable for use in the present invention include any
composition detectable by spectroscopic, photochemical, biochemical,
immunochemical,
electrical, optical or chemical means. Useful labels in the present invention
include biotin
for staining with labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads~),
fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent
protein, and the
like), radiolabels (e.g., 3H, l2sh 3sS, 14C, or 32P), enzymes (e.g., horse
radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA), and colorimetric
labels such
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as colloidal gold or colored glass or plastic (e.g., polystyrene,
polypropylene, latex, etc.)
beads. Patents teaching the use of such labels include U.S. Patent Nos.
3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
It will be recognized that fluorescent labels are not to be limited to single
species organic molecules, but include inorganic molecules, mufti-molecular
mixtures of
organic and/or inorganic molecules, crystals, heteropolymers, and the like.
Thus, for
example, CdSe-CdS core shell nanocrystals enclosed in a silica shell can be
easily
derivatized for coupling to a biological molecule (Bruchez et al. (1998)
Science, 281: 2013-
2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium
selenide)
have been covalently coupled to biomolecules for use in ultrasensitive
biological detection
(Warren and Nie (1998) Science, 281: 2016-2018).
Means of detecting such labels are well known to those of skill in the art.
Thus, for example, radiolabels may be detected using photographic film or
scintillation
counters, fluorescent markers may be detected using a photodetector to detect
emitted light.
Enzymatic labels are typically detected by providing the enzyme with a
substrate and
detecting the reaction product produced by the action of the enzyme on the
substrate, and
colorimetric labels are detected by simply visualizing the colored label.
III. Antibodies to ESX polypeptide(sl.
Antibodies are raised to the ESX polypeptides of the present invention (e.g.
exon 4, exon 7, specific anti-acetylated ESX, specific anti-unacetylated ESX,
etc.), including
individual, allelic, strain, or species variants, and fragments thereof, both
in their naturally
occurring (full-length) forms and in recombinant forms. Additionally,
antibodies are raised
to these polypeptides in either their native configurations or in non-native
configurations.
Anti-idiotypic antibodies can also be generated. Many methods of making
antibodies are
known to persons of skill. The following discussion is presented as a general
overview of
the techniques available; however, one of skill will recognize that many
variations upon the
following methods are known.
Al Antibody Production.
A number of immunogens are used to produce antibodies specifically reactive
with ESX polypeptides. Recombinant or synthetic polypeptides of 10 amino acids
in length,
or greater, selected from amino acid sub-sequences of (ESX) (e.g., SEQ ID
N0:1, exon 4,
exon 7) are the preferred polypeptide immunogen (antigen) for the production
of monoclonal
or polyclonal antibodies. In one class of preferred embodiments, an
immunogenic peptide
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conjugate is also included as an immunogen. Naturally occurring polypeptides
are also used
either in pure or impure form.
Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells (as
described below) and purified using standard techniques. The polypeptide, or a
synthetic
version thereof, is then.injected into an animal capable ofproducing
antibodies. Either
monoclonal or polyclonal antibodies can be generated for subsequent use in
immunoassays
to measure the presence and quantity of the polypeptide.
Methods of producing polyclonal antibodies are known to those of skill in the
art. In brief, an immunogen (antigen), preferably a purified polypeptide, a
polypeptide
coupled to an appropriate carrier (e.g., GST, keyhole limpet hemocyanin,
etc.), or a
polypeptide incorporated into an immunization vector such as a recombinant
vaccinia virus
(see, U.S. Patent No. 4,722,848) is mixed with an adjuvant and animals are
immunized with
the mixture. The animal's immune response to the immunogen preparation is
monitored by
taking test bleeds and determining the titer of reactivity to the polypeptide
of interest. When
appropriately high titers of antibody to the immunogen are obtained, blood is
collected from
the animal and antisera are prepared. Further fractionation of the antisera to
enrich for
antibodies reactive to the polypeptide is performed where desired (see, e.g.,
Coligan (1991)
Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989)
Antibodies: A Laboratory Manual, Cold Spring Harbor Press, N~.
Antibodies, including binding fragments and single chain recombinant
versions thereof, against predetermined fragments of ESX polypeptides are
raised by
immunizing animals, e.g., with conjugates of the fragments with carrier
proteins as described
above. Typically, the immunogen of interest is a peptide of at least about 5
amino acids,
more typically the peptide is 10 amino acids in length, preferably, the
fragment is 15 amino
acids in length and more preferably the fragment is 20 amino acids in length
or greater. The
peptides are typically coupled to a carrier protein (e.g., as a fusion
protein), or are
recombinantly expressed in an immunization vector. Antigenic determinants on
peptides to
which antibodies bind are typically 3 to 10 amino acids in length.
One particularly preferred immunogen is illustrated in the Example 1. In this
example, a peptide fragment consisting of the sixteen carboxy-terminal amino
acids of ESX
was used as an ESX antigen in rabbits. An amino-terminal cysteine was
introduced to allow
coupling of the peptide to a carrier protein (KLH). Anti-ESX antibodies were
obtained by
affinity purification of total IgG from immunized rabbits using an affinity
column to which
the ESX carboxyl terminal peptide fragment was bound.
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Monoclonal antibodies are prepared from cells secreting the desired antibody.
These antibodies are screened for binding to normal or modified polypeptides,
or screened
for agonistic or antagonistic activity, e.g., activity mediated through an ESX
protein.
Specific monoclonal and polyclonal antibodies will usually bind with a KD of
at least about
.1 mM, more usually at least about 50 mM, and most preferably at least about 1
mM or
better.
In some instances, it is desirable to prepare monoclonal antibodies from
various mammalian hosts, such as mice, rodents, primates, humans, etc.
Description of
techniques for preparing such monoclonal antibodies are found in, e.g., Stites
et al. (eds.)
Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,
CA, and
references cited therein; Harlow and Lane, supra; Goding (1986) Monoclonal
Antibodies:
Principles and Practice (2d ed.) Academic Press, New York, NY; and Kohler and
Milstein
(1975) Nature 256: 495-497. Summarized briefly, this method proceeds by
injecting an
animal with an immunogen. The animal is then sacrificed and cells taken from
its spleen,
which are fused with myeloma cells. The result is a hybrid cell or "hybridoma"
that is
capable of reproducing in vitro. The population of hybridomas is then screened
to isolate
individual clones, each of which secrete a single antibody species to the
immunogen. In this
manner, the individual antibody species obtained are the products of
immortalized and
cloned single B cells from the immune animal generated in response to a
specific site
recognized on the immunogenic substance.
Alternative methods of immortalization include transformation with Epstein
Barr Virus, oncogenes, or retroviruses, or other methods known in the art.
Colonies arising
from single immortalized cells are screened for production of antibodies of
the desired
specificity and affinity for the antigen, and yield of the monoclonal
antibodies produced by
such cells is enhanced by various techniques, including injection into the
peritoneal cavity of
a vertebrate (preferably mammalian) host. The polypeptides and antibodies of
the present
invention are used with or without modification, and include chimeric
antibodies such as
humanized marine antibodies.
Other suitable techniques involve selection of libraries of recombinant
antibodies in phage or similar vectors (see, e.g., Huse et al. (1989) Science
246: 1275-1281;
and Ward, et al. (1989) Nature 341: 544-546; and Vaughan et al. (I996) Nature
Biotechnology, 14: 309-314).
Frequently, the polypeptides and antibodies will be labeled by joining, either
covalently or non-covalently, a substance which provides for a detectable
signal. A wide
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variety of labels and conjugation techniques are known and are reported
extensively in both
the scientific aid patent literature. Suitable labels include
radionucleotides, enzymes,
substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent
moieties, magnetic
particles, and the like. Patents teaching the use of such labels include U.S.
Patent Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241. Also,
recombinant immunoglobulins may be produced (see, e.g., Cabilly, U.S. Patent
No.
4,816,567; and Queen et al. {1989) Proc. Nat'1 Acad. Sci. USA 86: 10029-10033.
The antibodies of this invention are also used for affinity chromatography in
isolating ESX polypeptides. Columns are prepared, e.g., with the antibodies
linked to a solid
support, e.g., particles, such as agarose, Sephadex, or the like, where a cell
lysate is passed
through the column, washed, and treated with increasing concentrations of a
mild denaturant,
whereby purified ESX polypeptides are released.
The antibodies can be used to screen expression libraries for particuiar
expression products such as normal or abnormal human ESX protein. Usually the
antibodies
in such a procedure are labeled with a moiety allowing easy detection of
presence of antigen
by antibody binding.
Antibodies raised against ESX polypeptides can also be used to raise anti-
idiotypic antibodies. These are useful for detecting or diagnosing various
pathological
conditions related to the presence of the respective antigens.
BLHuman or humanized (chimericl antibody production.
The anti-ESX antibodies of this invention can also be administered to an
organism (e.g., a human patient) for therapeutic purposes (e.g., to block the
action an ESX
polypeptide or as targeting molecules when conjugated or fused to effector
molecules such
as labels, cytotoxins, enzymes, growth factors, drugs, etc.). Antibodies
administered to an
organism other than the species in which they are raised are often
immunogenic. Thus, for
example, marine antibodies administered to a human often induce an immunologic
response
against the antibody (e.g., the human anti-mouse antibody (HA.MA) response) on
multiple
administrations. The immunogenic properties of the antibody are reduced by
altering
portions, or all, of the antibody into characteristically human sequences
thereby producing
chimeric or human antibodies, respectively.
i) Humanized Lchimeric} antibodies.
Humanized (chimeric} antibodies are immunoglobulin molecules comprising
a human and non-human portion. More specifically, the antigen combining region
(or
variable region) of a humanized chimeric antibody is derived from a non-human
source (e.g.,
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marine) and the constant region of the chimeric antibody (which confers
biological effector
function to the immunoglobulin) is derived from a human source. The humanized
chimeric
antibody should have the antigen binding (e.g., anti-ESX polypeptide)
specificity of the non-
human antibody molecule and the effector function conferred by the human
antibody
S molecule. A large number of methods of generating chimeric antibodies are
well known to
those of skill in the art (see, e.g., U.S. Patent Nos: 5,502,167, 5,500,362,
5,491,088,
5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238,
5,169,939,
5,081,235, 5,075,431, and 4,975,369).
In general, the procedures used to produce these chimeric antibodies consist
, of the following steps (the order of some steps may be interchanged): (a)
identifying and
cloning the correct gene segment encoding the antigen binding portion of the
antibody
molecule; this gene segment (known as the VDJ, variable, diversity and joining
regions for
heavy chains or VJ, variable, joining regions for light chains (or simply as
the V or Variable
region) may be in eitherthe cDNA or genomic form; (b) cloning the gene
segments
encoding the constant region or desired part thereof; (c) Iigating the
variable region with the
constant region so that the complete chimeric antibody is encoded in a
transcribable and
translatable form; (d) ligating this construct into a vector containing a
selectable marker and
gene control regions such as promoters, enhancers and poly(A) addition
signals; (e)
amplifying this construct in a host cell (e.g., bacteria); (f) introducing the
DNA into
eukaryotic cells (transfection) most often mammalian lymphocytes;
Antibodies of several distinct antigen binding specificities have been
manipulated by these protocols to produce chimeric proteins (e.g., anti-TNP:
Boulianne et
al. (1984) Nature, 312: 643; and anti-tumor antigens: Sahagan et al. (1986) J.
Immunol.,
137: 1066). Likewise several different effector functions have been achieved
by linking new
sequences to those encoding the antigen binding region. Some of these include
enzymes
(Neuberger et al. (1984) Nature 3I2: 604), immunoglobulin constant regions
from another
species and constant regions of another immunoglobulin chain (Sharon et al.
(I984) Nature
309: 364; Tan et al., (1985) J. Immunol. 135: 3565-3567).
In one preferred embodiment, recombinant DNA vector is used to transfect a
cell line that produces an anti-ESX antibody. The novel recombinant DNA vector
contains a
"replacement gene" to replace aI1 or a portion of the gene encoding the
immunoglobulin
constant region in the cell line (e.g., a replacement gene may encode all or a
portion of a
constant region of a human immunoglobulin, a specific immunoglobulin class, or
an
enzyme, a toxin, a biologically active peptide, a growth factor, inhibitor, or
a linker peptide
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to facilitate conjugation to a drug, toxin, or other molecule, etc.), and a
"target sequence"
which allows for targeted homologous recombination with immunoglobulin
sequences
within the antibody producing cell.
In another embodiment, a recombinant DNA vector is used to transfect a cell
line that produces an antibody having a desired effector fimction, (e.g., a
constant region of a
human immunoglobulin) in which case, the replacement gene contained in the
recombinant
vector may encode all or a portion of a region of an anti-ESX antibody and the
target
sequence contained in the recombinant vector allows for homologous
recombination and
targeted gene modification within the antibody producing cell. In either
embodiment, when
only a portion of the variable or constant region is replaced, the resulting
chimeric antibody
may define the same antigen and/or have the same effector function yet be
altered or
improved so that the chimeric antibody may demonstrate a greater antigen
specificity,
greater affinity binding constant, increased effector fimction, or increased
secretion and
production by the transfected antibody producing cell line, etc.
Regardless of the embodiment practiced, the processes of selection for
integrated DNA (via a selectable marker), screening for chimeric antibody
production, and
cell cloning, can be used to obtain a clone of cells producing the chimeric
antibody. Thus, a
piece of DNA which encodes a modification for a monoclonal antibody can be
targeted
directly to the site of the expressed immunoglobulin gene within a B-cell or
hybridoma cell
line. DNA constructs for any particular modification may be used to alter the
protein product
of any monoclonal cell line or hybridoma. Such a procedure circumvents the
costly and time
consuming task of cloning both heavy and light chain variable region genes
from each B-cell
clone expressing a useful antigen specificity. In addition to circumventing
the process of
cloning variable region genes, the level of expression of chimeric antibody
should be higher
when the gene is at its natural chromosomal location rather than at a random
position.
Detailed methods for preparation of chimeric (humanized) antibodies can be
found in U.S.
Patent 5,482,856.
iil Human antibodies.
In another embodiment, this invention provides for fully human anti-ESX
antibodies. Human antibodies consist entirely of characteristically human
polypeptide
sequences. The human anti-ESX antibodies of this invention can be produced in
using a
wide variety of methods (see, e.g., Larrick et al., U.S. Pat. No. 5,001,065,
for review).
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In one preferred embodiment, the human anti-ESX antibodies of the present
invention are usually produced initially in trioma cells. Genes encoding the
antibodies are
then cloned and expressed in other cells, particularly, nonhuman mammalian
cells.
The general approach for producing human antibodies by trioma technology
has been described by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg,
U.S. Pat. No.
4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666. The antibody-
producing cell lines
obtained by this method are called triomas because they are descended from
three cells; two
human and one mouse. Triomas have been found to produce antibody more stably
than
ordinary hybridomas made from human cells.
Preparation of trioma cells requires an initial fission of a mouse myeloma
cell
line with unimmunized human peripheral B lymphocytes. This fusion generates a
xenogenic
hybrid cell containing both human and mouse chromosomes (see, Engelman,
supra.).
Xenogenic cells that have lost the capacity to secrete antibodies are
selected. Preferably, a
xenogenic cell is selected that is resistant to 8-azaguanine. Cells possessing
resistance to 8-
azaguanine are unable to propagate on hygoxanthine-aminopterin-thymidine (HAT)
or
azaserine-hypoxanthine (AH) media.
The capacity to secrete antibodies is conferred by a fiuther fusion between
the
xenogenic cell and B-Lymphocytes immunized against an ESX polypeptide or an
epitope
thereof. The B-Lymphocytes are obtained from the spleen, blood or lymph nodes
of human
donor. If antibodies against a specific antigen or epitope are desired, it is
preferable to use
that antigen or epitope thereof as the immunogen rather than ESX polypeptide.
Alternatively, B-lymphocytes are obtained from an unimmunized individual and
stimulated
with an ESX polypeptide, or a epitope thereof, in vitro. In a further
variation, B-lymphocytes
are obtained from an infected, or otherwise immunized individual, and then
hyperimmunized
by exposure to an ESX polypeptide for about seven to fourteen days, in vitro.
The immunized B-lymphocytes prepared by one of the above procedures are
fused with a xenogenic hybrid cell by well known methods. For example, the
cells are
treated with 40-50% polyethylene glycol of MW 1000-4000, at about 37°C
for about S-10
min. Cells are separated from the fusion mixture and propagated in media
selective for the
desired hybrids. When the xenogenic hybrid cell is resistant to 8-azaguanine,
immortalized
trioma cells are conveniently selected by successive passage of cells on HAT
or AH
medium. Other selective procedures are, of course, possible depending on the
nature of the
cells used in fusion. Clones secreting antibodies having the required binding
specificity are
identified by assaying the trioma culture medium for the ability to bind to an
ESX
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polypeptide or an epitope thereof. Triomas producing human antibodies having
the desired
specificity are subcloned by the limiting dilution technique and grown in
vitro in culture
medium, or are injected into selected host animals and grown in vivo.
The trioma cell lines obtained are then tested for the ability to bind an ESX
5 polypeptide or an epitope thereof. Antibodies are separated from the
resulting culture
medium or body fluids by conventional antibody-fractionation procedures, such
as
ammonium sulfate precipitation, DEAF cellulose chromatography and affinity
chromatography.
Although triomas are genetically stable they do not produce antibodies at very
10 high levels. Expression levels can be increased by cloning antibody genes
from the trioma
into one or more expression vectors, and transforming the vector into a cell
line such as the
cell lines typically used for expression of recombinant or humanized
immunoglobulins. As
well as increasing yield of antibody, this strategy offers the additional
advantage that
immunoglobulins are obtained from a cell line that does not have a human
component, and
15 does not therefore need to be subjected to the especially extensive viral
screening required
for human cell lines.
The genes encoding the heavy and light chains of immunoglobulins secreted
by trioma cell lines are cloned according to methods, including the polymerase
chain
reaction, known in the art (see, e.g., Sambrook et al., Molecular Cloning: A
Laboratory
20 Manual, 2nd ed., Cold Spring Harbor, N.Y.,1989; Berger & Kimmel, Methods in
Enrymology, Yol. 152: Guide to Molecular Cloning Techniques, Academic Press,
Inc., San
Diego, Calif., 1987; Co et al. (1992) J. Immunol., 148: 1149). For example,
genes encoding
heavy and light chains are cloned from a trioma's genomic DNA or cDNA produced
by
reverse transcription of the trioma's RNA. Cloning is accomplished by
conventional
25 techniques including the use of PCR primers that hybridize to the sequences
flanking or
overlapping the genes, or segments of genes, to be cloned.
Typically, recombinant constructs comprise DNA segments encoding a
complete human immunoglobulin heavy chain and/or a complete human
immunoglobulin
light chain of an immunoglobulin expressed by a trioma cell line.
Alternatively, DNA
30 segments encoding only a portion of the primary antibody genes are
produced, which
portions possess binding and/or effector activities. Other recombinant
constructs contain
segments of trioma cell line immunoglobulin genes fused to segments of other
immunoglobulin genes, particularly segments of other human constant region
sequences
(heavy and/or light chain). Human constant region sequences can be selected
from various
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reference sources, including but not limited to those listed in Kabat et al.
(1987), Sequences
of Proteins of Immunological Interest, U.S. Department of Health and Human
Services.
In addition to the DNA segments encoding anti-ESX immunoglobulins or
fragments thereof, other substantially homologous modified immunoglobulins can
be readily
designed and manufactured utilizing various recombinant DNA techniques known
to those
skilled in the art such as site-directed mutagenesis (see, e.g., Gillinan &
Smith (1979) Gene,
8: 81-97; Roberts et al. (1987) Nature, 328: 731-734). Such modified segments
will usually
retain antigen binding capacity and/or effector function. Moreover, the
modified segments
are usually not so far changed from the original trioma genomic sequences to
prevent
hybridization to these sequences under stringent conditions. Because, Iike
many genes,
immunoglobulin genes contain separate functional regions, each having one or
more distinct
biological activities, the genes may be fused to fiuictional regions from
other genes to
produce fusion proteins (e.g" immunotoxins) having novel properties or novel
combinations
of properties.
The recombinant polynucleotide constructs will typically include an
expression control sequence operably linked to the coding sequences, including
naturally-
associated or heterologous promoter regions. Preferably, the expression
control sequences
will be eukaryotic promoter systems in vectors capable of transforming or
transfecting
eukaryotic host cells. Once the vector has been incorporated into the
appropriate host, the
host is maintained under conditions suitable for high level expression of the
nucleotide
sequences, and the collection and purification of the human anti-ESX
immunoglobulins.
These expression vectors are typically replicable in the host organisms either
as episomes or as an integral part of the host chromosomal DNA. Commonly,
expression
vectors will contain selection markers, e.g., ampicillin-resistance or
hygromycin-resistance,
to permit detection of those cells transformed with the desired DNA sequences.
In general, prokaryotes can be used for cloning the DNA sequences encoding
a human anti-ESX immunoglobulin chain. E. coli is one prokaryotic host
particularly useful
for cloning the DNA sequences of the present invention. Microbes, such as
yeast are also
useful for expression: Saccharomyces is a preferred yeast host, with suitable
vectors having
expression control sequences, an origin of replication, termination sequences
and the like as
desired. Typical promoters include 3-phosphoglycerate kinase and other
glycolytic
enzymes. Inducible yeast promoters include, among others, promoters from
alcohol
dehydrogenase 2, isocytochrome C, and enzymes responsible for maltose and
galactose
utilization.
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Mammalian cells are a particularly preferred host for expressing nucleotide
segments encoding immunoglobulins or fragments thereof (see, e.g., Winnacker
(1987)
From Genes to Clones, VCH Publishers, N.Y.). A number of suitable host cell
lines capable
of secreting intact heterologous proteins have been developed in the art, and
include CHO
S cell lines, various COS cell lines, HeLa cells, L cells and myeioma cell
lines. Preferably, the
cells are nonhuman. Expression vectors for these cells can include expression
control
sequences, such as an origin of replication, a promoter, an enhancer (Queen et
al. (1986)
Immunol. Rev. 89: 49), and necessary processing information sites, such as
ribosome binding
sites, RNA splice sites, polyadenylation sites, and transcriptionai terminator
sequences.
Preferred expression control sequences are promoters derived from endogenous
genes,
cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like (see,
e.g., Co et al.
(1992),1lmmunol, 148: 1149).
The vectors containing the DNA segments of interest can be transferred into
the host cell by well-known methods, depending on the type of cellular host.
For example,
1 S calcium chloride transfection is commonly utilized for prokaryotic cells,
whereas calcium
phosphate treatment, electroporation, lipofection, bioiistics or viral-based
transfection may
be used for other cellular hosts. Other methods used to transform mammalian
cells include
the use of polybrene, protoplast fusion, liposomes, electroporation, and
microinjection (see,
generally, Sambrook et al., supra).
Once expressed, human anti-ESX immunoglobulins of the invention can be
purified according to standard procedures of the art, including HPLC
purification, fraction
column chromatography, gel electrophoresis and the like (see, generally,
Scopes (1982)
Protein Purification, Springer-Verlag, NY). Detailed protocols for the
production of human
antibodies can be found in U.S. Patent 5,506,132.
Other approaches in vitro immunization of human blood. In this approach,
human blood lymphocytes capable of producing human antibodies are produced.
Human
peripheral blood is collected from the patient and is treated to recover
mononuclear cells.
The suppressor T-cells then are removed and remaining cells are suspended in a
tissue
culture medium to which is added the antigen and autologous serum and,
preferably, a
nonspecific lymphocyte activator. The cells then are incubated for a period of
time so that
they produce the specific antibody desired. The cells then can be fused to
human myeloma
cells to immortalize the cell line, thereby to permit continuous production of
antibody (see
U.S. Patent 4,716,11I).
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In another approach, mouse-human hybridomas which produces human anti-
ESX are prepared (see, e.g., S,S06,132). Other approaches include immunization
of mice
transformed to express human immunoglobulin genes, and phage display screening
(Vaughan et al. supra.).
IV. Production of ESX polvpeptides.
A) De novo chemical synthesis.
The ESX proteins or subsequences thereof (e.g. polypeptides corresponding
to exons 4 or 7) may be synthesized using standard chemical peptide synthesis
techniques.
Where the desired subsequences are relatively short (e.g., when a particular
antigenic
determinant is desired) the molecule may be synthesized as a single contiguous
polypeptide.
Where larger molecules are desired, subsequences can be synthesized separately
(in one or
more units) and then fused by condensation of the amino terminus of one
molecule with the
carboxyl terminus of the other molecule thereby forming a peptide bond.
Solid phase synthesis in which the C-terminal amino acid of the sequence is
I S attached to an insoluble support followed by sequential addition of the
remaining amino
acids in the sequence is the preferred method for the chemical synthesis of
the polypeptides
of this invention. Techniques for solid phase synthesis are described by
Barony and
Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:
Analysis, Synthesis,
Biology. Yol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et
al. (1963) J.
Am. Chem. Soc., 85: 2149-21 S6, and Stewart et al. (1984) Solid Phase Peptide
Synthesis,
2nd ed. Pierce Chem. Co., Rockford, Ill.
B) Recombinant expression.
In a preferred embodiment, the ESX proteins or subsequences thereof (e.g.
polypeptides of exons 4 or 7), are synthesized using recombinant DNA
methodology.
Generally this involves creating a DNA sequence that encodes the fusion
protein, placing the
DNA in an expression cassette under the control of a particular promoter,
expressing the
protein in a host, isolating the expressed protein and, if required,
renaturing the protein.
DNA encoding the ESX proteins or subsequences of this invention can be
prepared by any suitable method as described above, including, for example,
cloning and
restriction of appropriate sequences or direct chemical synthesis by methods
such as the
phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the
phosphodiester method of Brown et al.(1979) Meth. Enzymol. 68: 109-1 S 1; the
diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-
1862; and
the solid support method of U.S. Patent No. 4,458,066.
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Chemical synthesis produces a single stranded oligonucleotide. This may be
converted into double stranded DNA by hybridization with a complementary
sequence, or by
polymerization with a DNA polymerase using the single strand as a template.
One of skill
would recognize that while chemical synthesis of DNA is limited to sequences
of about 100
bases, longer sequences may be obtained by the ligation of shorter sequences.
Alternatively, subsequences may be cloned and the appropriate subsequences
cleaved using appropriate restriction enzymes. The fragments may then be
ligated to
produce the desired DNA sequence.
In one embodiment, ESX proteins of this invention can be cloned using DNA
amplification methods such as polymerase chain reaction (PCR). Thus, for
example, the
nucleic acid sequence or subsequence is PCR amplified, using a sense primer
containing one
restriction site (e.g., Ndel7 and an antisense primer containing another
restriction site (e.g.,
Hind>T>7. This will produce a nucleic acid encoding the desired ESX sequence
or
subsequence and having terminal restriction sites. This nucleic acid can then
be easily
ligated into a vector containing a nucleic acid encoding the second molecule
and having the
appropriate corresponding restriction sites. Suitable PCR primers can be
determined by one
of skill inthe art using the sequence information provided in SEQ II7.NOs:1
and 3.
Appropriate restriction sites can also be added to the nucleic acid encoding
the ESX protein
or protein subsequence by site-directed mutagenesis. The plasmid containing
the ESX
sequence or subsequence is cleaved with the appropriate restriction
endonuclease and then
ligated into the vector encoding the second molecule according to standard
methods.
The nucleic acid sequences encoding ESX proteins or protein subsequences
may be expressed in a variety of host cells, including E. coli, other
bacterial hosts, yeast, and
various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and
myeloma
cell lines. As the ESX proteins are typically found in eukaryotes, a eukaryote
host is
preferred. The recombinant protein gene will be operably linked to appropriate
expression
control sequences for each host. For E. coli this includes a promoter such as
the T7, trp, or
lambda promoters, a ribosome binding site and preferably a transcription
termination signal.
For eukaryotic cells, the control sequences will include a promoter and
preferably an
enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a
polyadenylation sequence, and may include splice donor and acceptor sequences.
The plasmids of the invention can be transferred into the chosen host cell by
well-known methods such as calcium chloride transformation for E. coli and
calcium
phosphate treatment or electroporation for mammalian cells. Cells transformed
by the
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plasmids can be selected by resistance to antibiotics conferred by genes
contained on the
plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, the recombinant ESX proteins can be purified according to
standard procedures of the art, including ammonium sulfate precipitation,
affinity columns,
column chromatography, gel electrophoresis and the like (see, generally, R.
Scopes, (1982)
Protein Purif cation, Springer-Verlag, N.Y.; Deutscher (1990) Methods in
Enrymology Vol.
182: Guide to Protein Purification., Academic Press, Inc. N.Y.). Substantially
pure
compositions of at least about 90 to 95% homogeneity are preferred, and 98 to
99% or more
homogeneity are most preferred. Once purified, partially or to homogeneity as
desired, the
polypeptides may then be used (e.g., as immunogens for antibody production).
One of skill in the art would recognize that after chemical synthesis,
biological expression, or purification, the ESX proteins) may possess a
conformation
substantially different than the native conformations of the constituent
polypeptides. In this
case, it may be necessary to denature and reduce the polypeptide and then to
cause the
polypeptide to re-fold into the preferred conformation. Methods of reducing
and denaturing
proteins and inducing re-folding are well known to those of skill in the art
(See, Debinski et
al. (1993) J. BioL Chem., 268: 14065-14070; Kreitman and Pastan (1993)
Bioconjug. Chem.,
4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).
Debinski et al., for
example, describes the denaturation and reduction of inclusion body proteins
in guanidine-
DTE. The protein is then refolded in a redox buffer containing oxidized
glutathione and L-
arginine.
One of skill would recognize that modifications can be made to the ESX
proteins without diminishing their biological activity. Some modifications may
be made to
facilitate the cloning, expression, or incorporation of the targeting molecule
into a fusion
protein. Such modifications are well known to those of skill in the art and
include, for
example, a methionine added at the amino terminus to provide an initiation
site, or additional
amino acids (e.g., poly His) placed on either terminus to create conveniently
located
restriction~sites or termination codons or purification sequences.
V. Detection of ESX acetylation.
As indicated above, abnormal (e.g., altered or deficient) expression of the
human ESX gene is believed to be a causal factor in the development of various
cancers
(e.g., head, neck, breast, ovary, bladder, colon, etc.). In particular, the
data provided herein
establish the importance of the ESX gene in the etiology of carcinomas,
including epithelial
cancers such as breast cancer. ESX becomes overexpressed at an early stage of
breast
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cancer known as ductal carcinoma in situ, making abnormal expression of ESX a
marker for
early detection of cancers. Of course, early detection can be critical to
treatment efficacy. It
is believed that abnormal expression of the ESX gene influences transcription
of genes that
are regulated by the ESX transcription factor. Thus, it is desirable to screen
for and identify
5 abnormal ESX activity.
ESX, in particular exon 7, is subject to acetylation. Moreover, without being
bound to a particular theory, it is believed that the acetylation of exon 7
(polypeptide)
provides a measure of activation of ESX and abnormal acetylation indicates
abnormal ESX
activation. Thus, in one embodiment, it is desired to assay for ESX
acetylation.
10 A1 Sample collection andprocessing:
Acetylation of the ESX gene product (e.g. exon 7) is preferably detected
and/or quantified in a biological sample. As used herein, a biological sample
is a sample of
biological tissue or fluid that, in a healthy and/or pathological state,
contains an ESX nucleic
acid or polypeptide. Such samples include, but are not limited to, sputum,
amniotic fluid,
15 blood, blood cells (e.g., white cells), tissue or fine needle biopsy
samples, urine, peritoneal
fluid, and pleural fluid, or cells therefrom. Biological samples may also
include sections of
tissues such as frozen sections taken for histological purposes. Often, a
sample will be
obtained from a cancerous or precancerous tissue. Although the sample is
typically taken
from a human patient, the assays can be used to detect ESX genes or gene
products in
20 ~ samples from any mammal, such as dogs, cats, sheep, cattle, and pigs.
The sample may be pretreated as necessary by dilution in an appropriate
buffer solution or concentrated, if desired. Any of a number of standard
aqueous buffer
solutions, employing one of a variety of buffers, such as phosphate, Tris, or
the like, at
physiological pH can be used.
25 B~ Control for physiological state.
As explained herein, expression levels of the ESX gene vary with the
developmental and reproductive state of the organism. Thus, for example, in
mice, ESX
expression is induced early in fetal development (e.g., greater than about 7
days), is
substantially diminished or lost during lactation, and dramatically increases
post-weaning.
30 In light of this variation, it will be appreciated that abnormal levels of
ESX
expression, e.g. as characterized by acetylation state, will be determined
relative to a control
reflecting the sex, developmental state of the animal or human, preferably the
reproductive
state, and/or preferably the particular tissue or cell type as well. Thus, in
a preferred
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embodiment, controls will be matched for one or more of these factors
according to standard
methods known to those of skill in the art.
Cl ESX polvueptide assays.
The expression of the human ESX gene can also be detected and/or quantified
by detecting or quantifying ESX acetylation of the expressed ESX polypeptideor
a subunit
thereof (e.g. exon 7). The acetylated ESX polypeptides (e.g. ratio of
acetylated to non-
acetylated ESX) can be detected and quantified by any of a number of means
well known to
those of skill in the art. These may include analytic biochemical methods such
as
electrophoresis, capillary electrophoresis, high performance liquid
chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography, and the like,
or various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or
double), immunoelectraphoresis, radioimmunoassay (RIA), enzyme-linked
immunosorbent
assays (ELISAs), immunofluorescent assays, western blotting, and the like.
In a particularly preferred embodiment, the acetylated and/or non-acetylated
ESX polypeptides are detected in an electrophoretic protein separation, more
preferably in a
two-dimensional electrophoresis, while in a most preferred embodiment, the
acetylated
and/or non-acetylated ESX polypeptides are detected using an immunoassay.
As used herein, an immunoassay is an assay that utilizes an antibody to
specifically bind to the analyte (acetylated or non-acetylated ESX
polypeptide). The
immunoassay is thus characterized by detection of specific binding of an
acetylated or
nonacetylated ESX polypeptide to an anti-ESX antibody as opposed to the use of
other
physical or chemical properties to isolate, target, and quantify the analyte.
il Electrophoretic assa,~s.
As indicated above, the acetylated and/or non-acetylated ESX polypeptides in
a biological sample can be determined using electrophoretic methods. Means of
detecting
proteins using electrophoretic techniques are well known to those of skill in
the art (see
generally, Scopes (1982) Protein Purification, Springer-Verlag, N.Y.;
Deutscher, (1990)
Methods in Enrymology Vol. 182: Guide to Protein Purification, Academic Press,
Inc.,
N.Y.).
ii1 Immunological binding assays.
In a preferred embodiment, the acetylated and/or non-acetylated ESX
polypeptides are detected and/or quantified using any of a number of well
recognized
immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110;
4,517,288; and
4,837,168). For a review of the general immunoassays, see also Asai (1993)
Methods in Cell
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Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;
Stites and
Terr (1991) Basic and Clinical Immunology 7th Edition. Immunological binding
assays (or
immunoassays) typically utilize a "capture agent" to specifically bind to and
often
immobilize the analyte (in this case ESX polypeptide or subsequence). The
capture agent is
S a moiety that specifically binds to the analyte. In a preferred embodiment,
the capture agent
is an antibody that specifically binds acetylated or non-acetylated ESX
polypeptide(s). The
antibody (anti-ESX) may be produced by any of a number of means well known to
those of
skill in the art as described above.
Immunoassays also often utilize a labeling agent to specifically bind to and
label the binding complex formed by the capture agent and the analyte. The
labeling agent
may itself be one of the moieties comprising the antibodylanalyte complex.
Thus, the
labeling agent may be a labeled ESX polypeptide or a labeled anti-ESX
antibody.
Alternatively, the labeling agent may be a third moiety, such as another
antibody, that
specifically binds to the ~antibody/ESX complex.
1 S In a preferred embodiment, the labeling agent is a second human ESX
antibody bearing a label. Alternatively, the second ESX antibody may lack a
label, but it
may, in turn, be bound by a labeled third antibody specific to antibodies of
the species from
which the second antibody is derived. The second can be modified v~rith a
detectable moiety,
such as biotin, to which a third labeled molecule can specifically bind, such
as enzyme-
labeled streptavidin.
Other proteins capable of specifically binding immunoglobulin constant
regions, such as protein A or protein G may also be used as the label agent.
These proteins
are normal constituents of the cell walls of streptococcal bacteria. They
exhibit a strong
non-irmnunogenic reactivity with immunoglobulin constant regions from a
variety of species
2S (see, generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, and
Akerstrom, et al.
(1985) J. Immunol., 135: 2589-2542).
Throughout the assays, incubation and/or washing steps may be required after
each combination of reagents. Incubation steps can vary from about S seconds
to several
hours, preferably from about S minutes to about 24 hours. However, the
incubation time
will depend upon the assay format, analyte, volume of solution,
concentrations, and the like.
Usually, the assays will be carried out at ambient temperature, although they
can be
conducted over a range of temperatures, such as 10°C to 40°C.
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a) Non-competitive assay formats.
Immunoassays for detecting acetylated and/or non-acetylated ESX
polypeptide may be either competitive or noncompetitive. Noncompetitive
immunoassays
are assays in which the amount of captured analyte (in this case ESX) is
directly measured.
In one preferred "sandwich" assay, for example, the capture agent (anti-ESX
antibodies) can
be bound directly to a solid substrate where they are immobilized. These
immobilized
antibodies then capture ESX present in the test sample. The ESX thus
immobilized is then
bound by a labeling agent, such as a second human ESX antibody bearing a
label.
Alternatively, the second ESX antibody may lack a label, but it may, in turn,
be bound by a
labeled third antibody specific to antibodies of the species from which the
second antibody is
derived. The second can be modified with a detectable moiety, such as biotin,
to which a
third labeled molecule can specifically bind, such as enzyme-labeled
streptavidin.
b Competitive assaY,formats.
In competitive assays, the amount of analyte (acetylated or non-acetylated
ESX) present in the sample is measured indirectly by measuring the amount of
an added .
(exogenous) analyte (acetylated or non-acetylated ESX} displaced (or competed
away) from
a capture agent (anti ESX antibody) by the analyte present in the sample. In
one competitive
assay, a known amount of, in this case, non-acetylated ESX is added to the
sample and the
sample is then contacted with a capture agent, in this case an antibody that
specifically binds
ESX. The amount of non-acetylated ESX bound to the antibody is inversely
proportional to
the concentration of non-acetylated ESX present in the sample.
In a particularly preferred embodiment, the antibody~is immobilized on a
solid substrate. The amount of ESX bound to the antibody may be determined
either by
measuring the amount of acetylated or non-acetylated ESX present in an
ESX/antibody
complex, or alternatively by measuring the amount of remaining uncomplexed
ESX. The
amount of ESX may be detected by providing a labeled ESX molecule.
A hapten inhibition assay is another preferred competitive assay. In this
assay a known analyte, in this case acetylated or non-acetylated ESX is
immobilized on a
solid substrate. A known amount of anti-( acetylated or non-acetylated) ESX
antibody is
added to the sample, and the sample is then contacted with the immobilized
ESX. In this
case, the amount of anti-ESX antibody bound to the immobilized ESX is
inversely
proportional to the amount of ESX present in the sample. Again the amount of
immobilized
antibody may be detected by detecting either the immobilized fraction of
antibody or the
fraction of the antibody that remains in solution. Detection may be direct
where the
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antibody is labeled or indirect by the subsequent addition of a labeled moiety
that
specifically binds to the antibody as described above.
c) Other assay formats.
In a particularly preferred embodiment, Western blot (immunoblot) analysis
S is used to detect and quantify the presence or ratio of acetylated and/or
non-acetylated ESX
in the sample. The technique generally comprises separating sample proteins by
gel
electrophoresis on the basis of molecular weight, transferring the separated
proteins to a
suitable solid support, (such as a nitrocellulose filter, a nylon filter, or
derivatized nylon
filter), and incubating the sample with the antibodies that specifically bind
ESX. The anti-
ESX antibodies specifically bind to ESX on the solid support. These antibodies
may be
directly labeled ar alternatively may be subsequently detected using labeled
antibodies (e.g.,
labeled sheep anti-mouse antibodies) that specifically bind to the anti-ESX.
Other assay formats include liposome immunoassays (LIA), which use
liposomes designed to bind specific molecules (e.g., antibodies) and release
encapsulated
reagents or markers. The released chemicals are then detected according to
standard
techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev. 5:34-41).
dl Scoring of the assay.
The assays of this invention as scored (as positive or negative for acetylated
or non-acetylated ESX polypeptide) according to standard methods well known to
those of
skill in the art. The particular method of scoring will depend on the assay
format and choice
of label. For example, a Western Blot assay can be scored by visualizing the
colored
product produced by the enzymatic label. A clearly visible colored band or
spot at the
correct molecular weight is scored as a positive result, while the absence of
a clearly visible
spot or band is scored as a negative. In a preferred embodiment, a positive
test will show a
signal intensity (e.g., acetylated ESX polypeptide quantity) at least twice
that of the
background and/or control and more preferably at /east 3 times or even at
least 5 times
greater than the background and/or negative control.
e) Reduction of non-specific binding.
One of skill in the art will appreciate that it is often desirable to reduce
non-
specific binding in immunoassays. Particularly, where the assay involves an
antigen or
antibody immobilized on a solid substrate it is desirable to minimize the
amount of non-
specific binding to the substrate. Means of reducing such non-specific binding
are well
known to those of skill in the art. Typically, this involves coating the
substrate with a
proteinaceous composition. In particular, protein compositions such as bovine
serum
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albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered
milk
being most preferred.
1~ Labels.
The particular label or detectable group used in the assay is not a critical
aspect of the invention, so long as it does not significantly interfere with
the specific binding
of the antibody used in the assay. The detectable group can be any material
having a
detectable physical or chemical property. Such detectable labels have been
well-developed
in the field of immunoassays and, in general, most any label useful in such
methods can be
applied to the present invention. Thus, a label is any composition detectable
by
spectroscopic, photochemical, biochemical, immunochemical, electrical, optical
or chemical
means. Useful labels in the present invention include magnetic beads (e.g.
Dynabeads~),
fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, and
the like),
radiolabels (e.g., 3H, lzsh 3sS, laC, or 32P), enzymes (e.g., horse radish
peroxidase, alkaline
phosphatase and others commonly used in an ELISA}, and colorimetric labels
such as
colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene,
latex, etc.) beads.
The label may be coupled directly or indirectly to the desired component of
the assay according to methods well known in the art. As indicated above, a
wide variety of
labels may be used, with the choice of label depending on sensitivity
required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and
disposal provisions.
Non-radioactive labels are often attached by indirect means. Generally, a
ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand
then binds to
an anti-ligand (e.g., streptavidin) molecule which is either inherently
detectable or covalently
bound to a signal system, such as a detectable enzyme, a fluorescent compound,
or a
chemiluminescent compound. A number of ligands and anti-ligands can be used.
Where a
ligand has a natural anti-ligand, for example, biotin, thyroxine, and
cortisol, it can be used in
conjunction with the labeled, naturally occurring anti-ligands. Alternatively,
any haptenic or
antigenic compound can be used in combination with an antibody.
The molecules can also be conjugated directly to signal generating
compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of
interest as
labels will primarily be hydrolases, particularly phosphatases, esterases and
glycosidases, or
oxidoreductases, particularly peroxidases. Fluorescent compounds include
lluorescein and
its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
Chemiluminescent
compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
For a review
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of various labeling or signal producing systems which may be used, see, U.S.
Patent No.
4,391,904.
Means of detecting labels are well known to those of skill in the art. Thus,
for example, where the label is a radioactive label, means for detection
include a scintillation
counter or photographic film as in autoradiography. Where the label is a
fluorescent label,
it may be detected by exciting the fluorochrome with the appropriate
wavelength of light and
detecting the resulting fluorescence. The fluorescence may be detected
visually, by means
of photographic filin, by the use of electronic detectors such as charge
coupled devices
(CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be
detected by
providing the appropriate substrates for the enzyme and detecting the
resulting reaction
pmduct. Finally simple colorimetric labels may be detected simply by observing
the color
associated with the label. Thus, in various dipstick assays, conjugated gold
often appears
pink, while various conjugated beads appear the color of the bead.
Some a~say formats do not require the use of labeled components. For
instance, agglutination assays can be used to detect the presence of the
target antibodies. In
this case, antigen-coated particles are agglutinated by samples comprising the
target
antibodies. In this format, none of the components need be labeled and the
presence of the
target antibody is detected by simple visual inspection.
Substrates.
As mentioned above, depending upon the assay, various components,
including the antigen, target antibody, or anti-human antibody, may be bound
to a solid
surface. Many methods for immobilizing biomolecules to a variety of solid
surfaces are
known in the art. For instance, the solid surface may be a membrane (e.g.,
nitrocellulose), a
microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass
or plastic), a
dipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and the like), a
microcentrifuge
tube, or a glass or plastic bead. The desired component may be covalently
bound or
noncovalently attached through nonspecific bonding.
A wide variety of organic and inorganic polymers, both natural and synthetic
may be employed as the material for the solid surface. Illustrative polymers
include
polyethylene, polypropylene, poly(4-methylbutene), polystyrene,
polymethacrylate,
polyethylene terephthalate), rayon, nylon, polyvinyl butyrate), polyvinylidene
difluoride
(PVDF), silicones, golyformaldehyde, cellulose, cellulose acetate,
nitrocellulose, and the
like. Other materials which may be employed, include paper, glasses, ceramics,
metals,
metalloids, semiconductive materials, cements or the like. In addition, are
included
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substances that form gels, such as proteins (e.g., gelatins),
lipopolysaccharides, silicates,
agarose and polyacrylamides can be used. Polymers which form several aqueous
phases,
such as dextrans, polyalkylene glycols or surfactants, such as phospholipids,
long chain (12-
24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where
the solid
surface is porous, various pore sizes may be employed depending upon the
nature of the
system.
In preparing the surface, a plurality of different materials may be employed,
particularly as laminates, to obtain various properties. For example, protein
coatings, such
as gelatin can be used to avoid non-specific binding, simplify covalent
conjugation, enhance
signal detection or the like.
If covalent bonding between a compound and the surface is desired, the
surface will usually be polyfimctional or be capable of being
polyfunctionalized. Functional
groups which may be present on the surface and used for linking can include
carboxylic
acids, aldehydes, amino~groups, cyano groups, ethylenic groups, hydroxyl
groups, mercapto
groups and the like. The manner of Iinlting a wide variety of compounds to
various surfaces
is well known and is amply illustrated in the literature (see, e.g., Chibata
(1978) Immobilized
Enzymes, Halsted Press, New York, and Cuatarecasas (1970) J. Biol. Chem. 245:
3059).
In addition to covalent bonding, various methods for noncovalently binding
an assay component can be used. Noncovalent binding is typically nonspecific
absorption of
a compound to the surface. Typically, the surface is blocked with a second
compound to
prevent nonspecific binding of labeled assay components. Alternatively, the
surface is
designed such that it nonspecifically binds one component but does not
significantly bind
another. For example, a surface bearing a lectin such as Concanavalin A will
bind a
carbohydrate containing compound but not a labeled protein that lacks
glycosylation.
Various solid surfaces for use in noncovalent attachment of assay components
are reviewed
in U.S. Patent Nos. 4,447,576 and 4,254,082.
F) Evaluation of ESX acetylation levels and/or abnormal expression.
One of skill will appreciate that abnormal expression levels or abnormal
expression products (e.g., mutated transcripts, truncated or non-sense
polypeptides) are
identified by comparison to normal expression levels and normal expression
products.
Normal levels of expression or normal expression products can be determined
for any
particular population, subpopulation, or group of organisms according to
standard methods
well known to those of skill in the art. Typically this involves identifying
healthy organisms
and/or tissues (i. e. organisms and/or tissues without ESX expression
dysregulation or
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neoplastic growth) and measuring expression levels of the ESX gene (as
described herein) or
sequencing the~gene, mRNA, or reverse transcribed cDNA, to obtain typical
(normal)
sequence variations. Application of standard statistical methods used in
molecular genetics
permits determination of baseline levels of expression, and normal gene
products as well as
S significant deviations from such baseline levels.
Preferably, normal levels of expression are determined using a control
organism or tissue that is in a physiological milieu that is similar to that
of the test sample.
For example, ESX expression can be influenced by age of the organism,
pregnancy,
menopause, and day of menstrual cycle, among other factors. Therefore, it is
preferred to
choose as a control tissue one that is at a similar stage as the tissue being
tested for abnormal
ESX expression. For example, a tissue known to be healthy can be obtained from
the same
organism from which the test tissue is obtained.
VI. Detection kits.
The present invention also provides for kits for the diagnosis of organisms
(e.g., patients) with a predisposition (at risk) for carcinomas, including
epithelial cancers.
The kits preferably include one or more reagents for determining the presence
or absence or
degree of acetylation of ESX, for quantifying expression of the ESX, gene, or
for detecting
an abnormal ESX gene (amplified or rearranged), or expression products of an
abnormal
ESX gene. Preferred reagents include nucleic acid probes that specifically
bind to the
normal ESX gene, cDNA, or subsequence thereof, probes that specifically bind
to abnormal
ESX gene (e.g., ESX genes containing premature truncations, insertions, or
deletions),
antibodies that specifically bind to normal ESX polypeptides (e.g. acetylated
or non-
acetylated) or subsequences thereof, or antibodies that specifically bind to
abnormal ESX
polypeptides or subsequences thereof. The antibody or hybridization probe may
be free or
immobilized on a solid support such as a test tube, a microtiter plate, a
dipstick and the like.
The kit may also contain instructional materials teaching the use of the
antibody or
hybridization probe in an assay for the detection of a predisposition for ESX.
The kits may include alternatively, or in combination with any of the other
components described herein, an anti-ESX antibody. The antibody can be
monoclonal or
polyclonal. The antibody can be conjugated to another moiety such as a label
and/or it can
be immobilized on a solid support (substrate).
The kits) may also contain a second antibody for detection of ESX
polypeptide/antibody complexes or for detection of hybridized nucleic acid
probes. The kit
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may contain appropriate reagents for detection of labels, positive and
negative controls,
washing solutions, dilution buffers and the like.
VII. Gene and/or cDNA activation or squelching using ESX
A) Gene and/or cDNA upregulation ltransactivation).
In another embodiment, this invention provides methods of activation
(upregulating) or inactivating (downregulating gene expression). It is
demonstrated herein
(e.g. Example 7) that ESX, in particular exon 4, is a potent transactivator.
When attached to
a nucleic acid (e.g. DNA) binding domain. Thus, constructs comprising ESX, ESX
exon 4,
or variants thereof, attached (e.g. chemically conjugated or recombinantly
expressed) can be
used to target and upregulate selected genes. The target genes (or cDNAs) can
be
endogenous or heterologous genes. They can be integrated into the host genome
or can be
non-integrated (e.g. extra-chromosomal).
In preferred embodiments, the target genes/cDNAs are under the control of a
promoter comprising an The method of claim 8, wherein said gene or cDNA is
under the
control of promoter having an ETS response element. The promoter can be an a
naturally
occuring promoter having such a response element (e.g. an epithelial gene
promoter) or it
can be a promoter engineered to contain such a response element.
Epithelial gene promoters are known to those of skill in the art. Indeed, ESX
contains one such promoter and it is unique among transcription factors
generally, and Ets
factors specifically, for its restricted expression in terminally
differentiated epidermal cells
(Andreoli et al. (1997) Nucleic Acids Res., 25, 4287-4295; Choi et al. (1998)
J. Biol.
Chem., 273: 110-I 17). In stratified epithelium, ESX is thought to
transactivate such genes
as the transforming growth factor-(3 type II receptor (TGF-(3RI1), Endo-
A/keratin-8, and
several markers of epidermal cell differentiation including transglutaminase
3, SPRR2A, and
profilaggrin (Andreoli et al. (1997) Nucleic Acids Res., 25, 4287-4295; Choi
et al. (1998) J.
Biol. Chem., 273: 110-117; Oettgen et al. (1997) Mol. Cell. Biol., 17: 4419-
4433; Tytnms et
al. (I997) Oncogene, 15, 2449-2462).
Nucleic acid binding proteins (domains) include, but are not limited to DNA
binding proteins such as Fis, LacI, lambda cI, lambda cro, LexA, TrpR, ArgR,
AraC, CRP,
FNR, OxyR, IHF, GaIR, MaIT, LRP, SoxR, SoxS, sigma factors, chi, T4 MotA, P1
RepA,
p53, NF-kappa-B, GAL4, and the like. A large number of nucleic acid binding
proteins are
described in the TransFac database (see also (1997) Nucleic Acids Res. 25(1)
265-268).
Methods of coupling the nucleic acid binding domain to the ESX polypeptide
are well known to those of skill in the art. Chemical conjugation is
preferably by way of a
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linker. A "linker" as used herein, is a molecule that is used to join the
targeting molecule to
the effector molecule. The linker is capable of forming covalent bonds to both
the DNA
binding domain and to the ESX polypeptide. Suitable linkers are well known to
those of
skill in the art and include, but are not limited to, straight or branched-
chain carbon linkers,
heterocyclic carbon linkers, or peptide connectors. The linkers may be joined
to the
constituent amino acids through their side groups (e.g., through a disulfide
linkage to
cysteine). However, in a preferred embodiment, the connectors will be joined
to the alpha
carbon amino and carboxyl groups of the terminal amino acids.
Many procedures and linkers molecules for attachment of various
polypeptides are known (see, e.g., European Patent Application No. 188,256;
U.S. Patent
Nos. 4,545,985 and 4,894,443, 4,671,958, 4,659,839, 4,414,148, 4,699,784;
4,680,338;
4,569,789; and 4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47: 4071-
4075;
Waldmann (1991) Science, 252: 1657). .
In a preferred embodiment, the nucleic binding domain and the ESX
polypeptide are expressed as a fusion protein. Methods of preparing fusion
proteins are well
known to those of skill in the art and are illustrated in Example 7.
B) Gene and/or cDNA downregulation fs4uelchingl.
In another embodiment, the ESX polyeptide (e.g. exon 4 polypeptide) can be
used to down regulate gene expression. It is demonstrated herein that exon 4
polypeptides
are capable of squelching (downregulating) activity of a target gene or cDNA.
Squelching
occurs when a potent transactivator reduces the expression of a gene or cDNA
(e.g. a co-
transfected reporter plasmid in a transient transfection assay), with the
resulting decline in
activity believed to be due to sequestration of GTFs and reduction in their
effective
concentration (Natesan et al. (1997) Nature, 390: 349-350).
Without being bound to a particular theory, it is believed that squelching is
mediaged by high-affinity binding of ESX exon 4 polypeptide to a limiting
component of the
basic transcriptional machinery, TATA-binding protein (TBP). When TBP is
recruited by is
sequestered by excess ESX (e.g. exon 4) unbound to DNA, squelching of TBP-
dependent
gene expression can occur. Thus provision of a cell with excess ESX exon 4
(e.g.
transfecting the cell with an ESX exon 4-expressing vector) can down-regulate
a gene or
cDNA. As with transactivation, preferred target genes/cDNAs have a native or
engineered
Ets response element.
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VIII. Probes.
In another embodiment, the ESX polypeptides of ESX exon 4 and/or ESX
exon 7 can be used as probes to identify in vivo, in vitro, or in situ
naturally occuring
molecules or test agents that interact with these ESX domains. Thus, far
example, the
polypeptide expressed by ESX exon 4 interacts with other intracellular
proteins (e.g., TATA-
binding protein (TBP)). Similarly, ESX exon 7 can interact with proteins (e.g.
histone acetyl
transferase.(HAT) domain from the nuclear co-regulatory factor, pCAF,
(pCAF/HAT)) and
nucleic acids (it is a sequence-nonspecific DNA-binding motif that recognizes
and stabilizes
architecturally irregular DNA structures like the H-DNA form thought to be
present within
the GGA mirror-repeat and nuclear matrix binding region of the erbB2 proximal
promoter).
Thus, ESX exon 4 and/or ESX exon 7 can be used to probe organisms, tissues
and cells for binding proteins and/or nucleic acids. In a preferred
embodiment, this is
accomplished simply by labeling the ESX exon 4 or exon 7 polypeptide with a
detectable
label, treating the sample animal, tissue, or cell and detecting or isolating
the ESX probe to
I S localize and/or isolate molecules interacting with the probe.
IX. Affinity matrix for isolating exon 4 and/or exon 7 binding molecules.
Alternatively, the ESX exon 4 and/or exon 7 polypeptides can be used e.g. in
an affinity matrix (e.g. affinity column) to isolate targets (e.g. proteins or
nucleic acids that
interact with these ESX domains). Briefly, in one embodiment, affinity
chromatography
involves immobilizing (e.g. on a solid support) polypeptides comprising the
ESX exon 4
and/or exon 7 polypeptides. Cells, cellular lysate, or cellular homogenate, or
other samples
are then contacted with the immobilized polypeptide which then binds to its
components of
the sample that interact with that polypeptide. The remaining material is then
washed away
and the bound molecules) can then be released from the ESX polypeptide(s) for
further use.
Methods of performing affinity chromatography are well known to those of skill
in the art
(see, e.g., U.S. Patent Nos: 5,710,254, 5,491,096, 5,278,061, 5,110,907,
4,985,144,
4,385,991, 3,983,001, etc.).
Suitable matrix materials include, but are not limited to paper, glasses,
ceramics, gels, aerogels, metals, metalloids, polacryloylmorpholide, various
plastics and
plastic copolymers such as NylonTM, TeflonTM, polyethylene, polypropylene,
poly(4-
methylbutene), polystyrene, polystyrene, polystyrene/latex, polymethacrylate,
polyethylene
terephthalate), rayon, nylon, polyvinyl butyrate), polyvinylidene difluoride
(PVDF),
silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and
thelike, and
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other materials generally known to be suitable for use in affinity columns
(e.g. HPLC
columns).
X. ESX modulation/therapeutics.
The ESX polypeptide appears to be an extremely strong gene transactivator,
as revealed by GAL4 fusion studies showing that the ESX amino acid sequences
encoded by
ESX exon 4 are as powerful as the transactivating sequences of VP16, one of
the strongest
transactivators known and most often used as a positive control in GAL4 fusion
studies.
These studies indicate that ESX is most likely "turning on" rather than
"turning off' all the
genes under its control (e.g., growth factor receptors such as erbB2, and
extracellular matrix
proteases such as MMPs, and UPA). Up-regulation of ESX will therefore turn on
(e.g.,
transactivate) genes under ESX control, while down-regulation of ESX will turn
off genes
under ESX control.
Al Screening for ESX modulation.
As indicated earlier, ESX controls a number of functions including, but not
limited to in remodeling ductal epithelium and in regulating gene programs
involved with
this process (e.g. extracellular matrix degradation, apoptosis, etc.). In
particular extracellular
matrix degradation control or apoptosis appear to be essential for enhanced
tumor cell
invasion and metastasis. Modulation of such functions is useful in both a
research and a
therapeutic context. Thus, in one embodiment, this invention provides methods
of screening
for agents that modulate (e.g., up=regulate (turn on or increase} or down-
regulate (tum off or
decrease) ESX expression or ESX polypeptide activity.
In a preferred embodiment, such such methods involve contacting a cell or an
isolated system (e.g. a solution) containing an endogenous or heterologous ESX
exon 4 or
exon 7 DNA or polypeptide with the agent that is to be screened for ESX
modulatory
activity and detecting binding of that agent to ESX exon 4 and/or exon 7
nucleic acid or
polypeptide. Alternatively a change in ESX acetylation (e.g. exon 7
acetylation) can be
detected.
Methods of assaying for binding interactions are well known to those of skill
in the art. Such methods include, for example, DNA bending assays (see, e.g.,
Wechsler and
Dang (1992) Proc. Natl. Acad Sci. USA, 89: 7635-7639 with modifications to
prevent
anomalous results described by McCormick et al. (1996) Proc. Natl. Acad Sci.
USA, 93:
14434-14439), and more traditional binding assays such as transcription factor
binding
assays (see, e.g., U.S. Patents 5,350,835 and 5,563,036). It was a discovery
of this invention
that the minimal ESX domain necessary for ESX-mediated transactivation is
encoded by
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exon 4 (aa 129-159), an acidic domain containing a central lysine residue (K-
145).
Subsequent mutations of this domain have established that the central K-145 is
essential and
provides nearly 1000-fold transactivation potency (relative to a neutral
residue placed there).
A database search revealed that the exon 4-encoded domain is homologous to the
essential
S core domain of all known Topoisomerase I molecules (cf. Stewart et al,
(1996) J. Biol.
Chem. 271: 7602-760$; Pommier (1996) Sem. Oncology 23: 3-10). Since human Topo-
I is a
critical intracellular target for the newest and most exciting family of
camptothecin-like
anticancer agents (like Topotecan, CPT-11, 9AC, etc.; see reviews).
This information not only provides important data regarding the molecular
transactivation mechanism of ESX, but it suggests that this particular ESX
domain may be
used to search for or screen (from libraries, e.g., combinatorial libraries of
synthctic
chemicals and/or natural products) for even newer and more effective and
selective
anticancer agents. Existing .Topo-I agents target a very different, C-terminal
cpnserved
domain in the Topo-I enzyme. Prior to this invention there was no specific
function
attributed to the highly conserved Topo-I Core domain which is homologous to
the ESX
transactivation domain.
These data also shed light on the fimctioning of Topo-I (and new ways to
inhibit it) as they do on the fimctioning of ESX. In this regard, this
invention provides, in
one embodiment, methods of screening for a therapeutic lead compound. The
methods
involve providing a nucleic acid encoding a polypeptide of ESX exon 4 or a
polypeptide
sequence of ESX exon 4; (ii) contacting the compound to the nucleic acid or
polypeptide
sequence; and (iii) detecting binding of the compound to the nucleic acid or
polypeptide
sequence. Compounds that specifically bind to the exon 4 nucleic acid and/or
polypeptide
are expected to provide lead compounds for therapeutic evaluation and/or
development.
Suitable binding assays are described herein and are also well known to those
of skill in the
art.
B) ESX Modulators for screening.
Virtually any compound can be screened for ESX modulatory activity.
However, it will be appreciated that some compounds are expected to show ESX
modulatory
activity and these compounds may be preferentially screened. Such compounds
include, but
are not limited to compounds that specifically target and bind to ESX exon 4
and/or exon 7
nucleic acids or polypeptides (e.g., ESX muteins, or ESX antisense molecules).
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il ESX muteins.
' It was a discovery of this invention that full-length ESX bends DNA by as
much as 80 degrees upon DNA-binding. In contrast, when only the DNA-binding
portion of
ESX (see, Fig. 5), or any other ETS protein is assessed, only 6-20 degrees of
DNA bending
is observed (as reported by NMR and X-ray crystallography studies on other
truncated ETS
proteins). This indicates that a mutated version of a full DNA bending ESX
construct can
act as a "dominant-negative" transcription factor or fused to a known
repression module to
produce an agent that will silence ESX regulated genes and turn off potential
gene programs
necessary for tumor cell invasion and metastasis. Using the sequence
information provided
herein (e.g., Fig. 5) ESX polypeptide variants can be routinely produced.
For example, it is demonstrated herein that the central Klas of exon 4 (aa 129-
159) of is essential for ESX transactivation activity and provides nearly 1000-
fold
transactivation potency (relative to a neutral residue placed there. The
mutation of Ktas to a
neutral residue will provide an inactivating (competitive) mutein.
Methods of making other such polypeptide variants (muteins) are well known
to those of skill (see, e.g:, U.S. Patents 5,486,463, 5,422,260, 5,116,943,
4,752,585,
4,518,504). Screening of such polypeptides (e.g., in DNA binding assays or for
competitive
inhibition of full-length normal ESX polypeptides) can be accomplished with
only routine
experimentation. Using high-throughput methods, as described herein, literally
thousands of
agents can be screened in only a day or two.
iiZ Antisense molecules.
ESX gene regulation can be downregulated or entirely inhibited by the use of
antisense molecules. An "antisense sequence or antisense nucleic acid" is a
nucleic acid is
complementary to the coding ESX mRNA nucleic acid sequence or a subsequence
thereof.
Binding of the antisense molecule to the ESX mRNA interferes with normal
translation of
the ESX poiypeptide.
Thus, in accordance with preferred embodiments of this invention, preferred
antisense molecules include oligonucleotides and oligonucleotide analogs that
are
hybridizable with ESX messenger RNA (preferably with exon 4 or exon 7). This
relationship
is commonly denominated as "antisense." The oligonucleotides and
oligonucleotide analogs
are able to inhibit the function of the RNA, either its translation into
protein, its translocation
into the cytoplasm, or any other activity necessary to its overall biological
function. The
failure of the messenger RNA to perform all or part of its function results in
a reduction or
complete inhibition of expression of ESX polypeptides.
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In the context of this invention, the term "oligonucleotide" refers to a
polynucleotide formed from naturally-occurring bases and/or cyclofuranosyl
groups joined
by native phosphodiester bonds. This term effectively refers to naturally-
occurring species
or synthetic species formed from naturally-occurring subunits or their close
homologs. The
term "oligonucleotide" may also refer to moieties which function similarly to
oligonucleotides, but which have non naturally-occurring portions. Thus,
oligonucleotides
may have altered sugar moieties or inter-sugar linkages. Exemplary among these
are the
phosphorothioate and other sulfur containing species which are known for use
in the art. In
accordance with some preferred embodiments, at least one of the phosphodiester
bonds of
the oligonucleotide has been substituted with a structure which functions to
enhance the
ability of the compositions to penetrate into the region of cells where the
RNA whose
activity is to be modulated is located. It is preferred that such
substitutions comprise
phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or
cycloalkyl
structures. In accordance with other preferred embodiments, the phosphodiester
bonds are
substituted with structures which are, at once, substantially non-ionic and
non-chiral, or with
structures which are chiral and enantiomerically specific. Persons of ordinary
skill in the art
will be able to select other linkages for use in the practice of the
invention.
Oligonucleotides may also include species which include at least some
modified base forms. Thus, purines and pyrimidines other than those normally
found in
nature may be so employed. Sinularly, modifications on the furanosyl portions
of the
nucleotide subunits may also be effected, as long as the essential tenets of
this invention are
adhered to. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-
substituted
nucleotides. Some specific examples of modifications at the 2' position of
sugar moieties
which are useful in the present invention are OH, SH, SCH3, F, OCH3, OCN,
O(CHz)[n]NH2
or O(CHZ)[n]CH3, where n is from 1 to about 10, and other substituents having
similar
properties.
Such oligonucleotides are best described as being functionally
interchangeable with natural oligonucleotides or synthesized oligonucleotides
along natural
lines, but which have one or more differences from natural structure. All such
analogs are
comprehended by this invention so long as they function effectively to
hybridize with
messenger RNA of ESX to inhibit the function of that RNA.
The oligonucleotides in accordance with this invention preferably comprise
from about 3 to about 50 subunits. It is more preferred that such
oligonucleotides and
analogs comprise from about 8 to about 25 subunits and still more preferred to
have from
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about 12 to about 20 subunits. As will be appreciated, a subunit is a base and
sugar
combination suitably bound to adjacent subunits through phosphodiester or
other bonds. The
oligonucleotides used in accordance with this invention may be conveniently
and routinely
made through the well-known technique of solid phase synthesis. Equipment for
such
synthesis is sold by several vendors, including Applied Biosystems. Any other
means for
such synthesis may also be employed, however, the actual synthesis of the
oligonucleotides
is well within the talents of the routineer. It is also will known to prepare
other
oligonucleotide such as phosphorothioates and alkylated derivatives.
iii) Combinatorial libraries le:~ , small organic molecules).
Conventionally, new chemical entities with useful properties are generated by
identifying a chemical compound (called a "lead compound") with some desirable
property
or activity, creating variants of the lead compound, and evaluating the
property and activity
of those variant compounds: However, the current trend is to shorten the time
kale for all
aspects of drug discovery. Because of the ability to test large numbers
quickly and
efficiently, high throughput screening (HTS) methods are replacing
conventional lead
compound identification methods.
In one preferred embodiment, high throughput screening methods involve
providing a library containing a large number of potential therapeutic
compounds (candidate
compounds). Such "combinatorial chemical libraries" are then screened in one
or more
assays, as described below to identify those library members (particular
chemical species or
subclasses) that display a desired characteristic activity. The compounds thus
identified can
serve as conventional "lead compounds" or can themselves be used as potential
or actual
therapeutics.
A combinatorial chemical library is a collection of diverse. chemical
compounds generated by either chemical synthesis or biological synthesis by
combining a
number of chemical "building blocks" such as reagents. For example, a linear
combinatorial
chemical library such as a polypeptide (e.g., mutein) library is formed by
combining a set of
chemical building blocks called amino acids in every possible way for a given
compound
length (i.e., the number of amino acids in a polypeptide compound). Millions
of chemical
compounds can be synthesized thmugh such combinatorial mixing of chemical
building
blocks. For example, one commentator has observed that the systematic,
combinatorial
mixing of 100 interchangeable chemical building blocks results in the
theoretical synthesis
of 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop
et al.
(1994) 37(9): 1233-1250).
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Preparation and screening of combinatorial chemical libraries is well known
to those of skill in the art. Such combinatorial chemical libraries include,
but are not limited
to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J.
Pept. Prot. Res.,
37: 487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesis is
by no means
the only approach envisioned and intended for use with the present invention.
Other
chemistries for generating chemical diversity libraries can also be used. Such
chemistries
include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26
Dec. 1991),
encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), random bio-
oligomers
(PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No.
5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al.,
(1993) Proc.
Nat. Acad Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara et al.
(1992) J.
Amer. Chem. Soc. I 14: 6568), nonpeptidal peptidomimetics with a Beta-D-
Glucose
scaffolding (Hirschmann et al., (1992) .I. Amer. Chem. Soc. 114: 9217-9218),
analogous
organic syntheses of small compound libraries (Chen et al. (1994) J. Amer.
Chem. Soc. 116:
2661), oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidyl
phosphonates
(Campbell et al., (1994) J. Org. Chem. 59: 658). See, generally, Gordon et
al., (1994) ,L
Med Chem. 37:1385, nucleic acid libraries (see, e.g., Strategene, Corp.),
peptide nucleic
acid libraries (see, e.g., U.S. Patent 5,539,083) antibody libraries (see;
e.g., Vaughn et al.
(1996) Nature Biotechnology, 14(3): 309-314), and PCT/LTS96/10287),
carbohydrate
libraries (see, e.g., Liang et al. (I996) Science, 274: 1520-1522, and U.S.
Patent 5,593,853),
and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993)
C&EN, Jan
18, page 33, isoprenoids U.S. Patent 5,569,588, thiazolidinones
and.metathiazanones U.S.
Patent 5,549,974, pyrrolidines U.S. Patents 5,525,735 and 5,519,134,
morpholino
compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially
available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY,
Symphony,
Raisin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus,
Millipore,
Bedford, MA).
A number of well known robotic systems have also been developed for
solution phase chemistries. These systems include automated workstations like
the
automated synthesis apparatus developed by Takeda Chemical Industries, LTD.
(Osaka,
Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark
Corporation,
Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the
manual
synthetic operations performed by a chemist. Any of the above devices are
suitable for use
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with the present invention. The nature and implementation of modifications to
these devices
(if any) so that they can operate as discussed herein will be apparent to
persons skilled in the
relevant art. In addition, numerous combinatorial libraries are themselves
commercially
available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos,
Inc., St.
S Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek
Biosciences, Columbia, MD, etc.).
Cl High-throughput screening.
Any of the assays for compounds modulating ESX gene expression and/or
ESX protein activity (e.g., binding activity) described herein are amenable to
high
throughput screening. Preferred assays thus detect enhancement or inhibition
of ESX gene
transcription, inhibition or enhancement of ESX polypeptide expression,
inhibition or
enhancement of DNA binding by ESX polypeptide, or inhibition or enhancement of
expression of native genes (or reporter genes) under control of the ESX
polypeptide.
High throughput assays for the presence, absence, or quantification of
1 S particular nucleic acids or protein products are well known to those of
skill in the art.
Similarly, binding assays and reporter gene assays are similarly well known.
Thus, for
example, U.S. Patent 5,559,410 discloses high throughput screening.methods for
proteins,
U.S. Patent 5,585,639 discloses high throughput screening methods for nucleic
acid binding
(i.e., in arrays), while U.S. Patents 5,576,220 and 5,541,061 disclose high
throughput
methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available
(see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries,. Mentor,
OH; Becl~an
Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.).
These systems
typically automate entire procedures including all sample and reagent
pipetting, liquid
2S dispensing, timed incubations, and final readings of the micmplate in
detectors) appropriate
for the assay. These configurable systems provide high throughput and rapid
start up as well
as a high degree of flexibility and customization. The manufacturers of such
systems
provide detailed protocols the various high throughput. Thus, for example,
Zymark Corp.
provides technical bulletins describing screening systems for detecting the
modulation of
gene transcription, ligand binding, and the like.
XI. In vivo administration of ESX modulators.
The ESX polypeptides, ESX polypeptide subsequences (e.g. exon 4 and/or
exon 7), anti-ESX antibodies, anti-ESX antibody-effector (e.g., enzyme, toxin,
hormone,
growth factor, drug, etc.) conjugates or fusion proteins, or other ESX
modulators of this
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invention are useful for parenteral, topical, oral, or local administration,
such as by aerosol
or transdermally, for prophylactic and/or therapeutic treatment. The
pharmaceutical
compositions can be administered in a variety of unit dosage forms depending
upon the
method of administration. For example, unit dosage forms suitable for oral
administration
include powder, tablets, pills, capsules and lozenges. It is recognized that
the ESX
polypeptides and related compounds described of, when administered orally,
must be
protected from digestion. This is typically accomplished either by complexing
the protein
with a composition to render it resistant to acidic and enzymatic hydrolysis
or by packaging
the protein in an. appropriately resistant carrier such as a liposome. Means
of protecting
proteins from digestion are well known in the art.
The pharmaceutical compositions of this invention are particularly useful for
topical administration to cancers, in particular epithelial cancers, and their
precursors (such
as ductal carcinoma in situ, DCIS). In another embodiment, the compositions
are useful for
parenteral administration, such as intravenous administration or
administration into a body
cavity or lumen of an organ. The compositions for administration will commonly
comprise
a solution of the ESX polypeptide, antibody or antibody chimera/fusion
dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety
of aqueous
carriers can be used, e.g., buffered saline and the like. These solutioiLS are
sterile and
generally free of undesirable matter. These compositions may be sterilized by
conventional,
well known sterilization techniques. The compositions may contain
pharmaceutically
acceptable auxiliary substances as required to approximate physiological
conditions such as
pH adjusting and buffering agents, toxicity adjusting agents and the.like, for
example,
sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium
lactate and
the like. The concentration of chimeric molecule in these formulations can
vary widely, and
will be selected primarily based on fluid volumes, viscosities, body weight
and the like in
accordance with the particular mode of administration selected and the
patient's needs.
Thus, a typical pharmaceutical composition for intravenous administration
would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about
100 mg per
patient per day may be used, particularly when the drug is administered to a
secluded site
and not into the blood stream, such as into a body cavity or into a lumen of
an organ.
Substantially higher dosages are possible in topical administration. Actual
methods for
preparing parenterally administrable compositions will be known or apparent to
those skilled
in the art and are described in more detail in such publications as
Remington's
Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton,
Pennsylvania (1980).
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The compositions containing the present ESX polypeptides, antibodies or
antibody chimera/fusions, or a cocktail thereof (i.e., with other proteins),
can be
administered for therapeutic treatments. To treat an epithelial cancer
characterized by
overexpression of ESX, one can administer an anti-ESX antibody or an abnormal
ESX
protein that is not biologically active. Such inactive ESX polypeptides can,
for example,
interfere with binding of native ESX polypeptide to its DNA binding site, or
to RNA
polymerase or other protein through which the ESX transcription factor
activity is mediated.
In therapeutic applications, compositions are administered to a patient
suffering from a disease (e.g., an epithelial cancer) in an amount sufficient
to cure or at least
partially arrest the disease and its complications. An amount adequate to
accomplish this is
defined as a "therapeutically effective dose." Amounts effective for this use
will depend
upon the severity of the disease and the general state of the patient's
health. Single or
multiple administrations of the compositions may be administered depending on
the dosage
and frequency as required and tolerated by the patient. In any event, the
composition should
provide a sufficient quantity of the proteins of this invention to effectively
treat the patient.
Among various uses of the ESX polypeptides, polypeptide subsequences,
anti-ESX.antibodies and anti-ESX-effector chimeras/fusions of the present
invention are
treatment a variety of disease conditions, including cancers such as cancers
of the breast,
head, neck, ovary, bladder, colon, and the like.
XII. Cellular transformation and gene therapy.
The present invention provides packageable human ESX nucleic acids (e.g.
nucleic acids encoding exon 4, and/or exon 4 attached to a nucleic acid
binding domain)) for
the transformation of cells in vitro and in vivo. These packageable nucleic
acids can be
inserted into any of a number of well known vectors for the transfection and
transformation
of target cells and organisms as described below. The nucleic acids are
transfected into
cells, ex vivo or in vivo, through the interaction of the vector and the
target cell. The nucleic
acid, under the control of a promoter, then expresses the ESX protein
construct thereby
upregulating or downregulating the target gene. For treatment of conditions
characterized by
excessive ESX expression, the ESX construct can be one that downregulates the
target gene
(e.g. a polypeptide comprising exon 4 without a nucleic acid binding domain).
Conversely,
in conditions characterized by under expression of a target gene, the cells)
can be
transfected with a construct comprising a DNA binding domain specific to a
domain
adjacent to or in proximity of the target gene attached to an ESX exon 4
transactivator. This
will be most useful where the target gene promoter contains an Ets response
element.
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Such gene therapy procedures have been used to correct acquired and
inherited genetic defects, cancer, and viral infection in a number of
contexts. The ability to
express artificial genes in humans facilitates the prevention and/or cure of
many important
human diseases, including many diseases which are not amenable to treatment by
other
therapies. As an example, in vivo expression of cholesterol-regulating genes,
genes which
selectively block the replication of HIV, and tumor-suppressing genes in human
patients
dramatically improves the treatment of heart disease, A1DS, and cancer,
respectively. For a
review of gene therapy procedures, see Anderson (1992) Science 256: 808-813;
Nabel and
Felgaer (1993) TIBTECH 11: 2I 1-217; Mitani and Caskey (1993) TIBTECH 11: 162-
166;
Mulligan (1993) Science 926-932; Dillon (1993) TIBTECH 11: 167-175; Miller
(/992)
Nature 357: 455-460; Van Brunt (1988) Biotechnology 6(10): 1149-1154; Vigne
(1995)
Restorative Neurology and Neuroscience 8: 35-36; Kremer and Perricaudet (1995)
British
Medical Bulletin 51(1) 31-44.; Haddada et al. (1995) in Current Topics in
Microbiolo~r and
Immunology, Doerfler and Btihm (eds) Springer-Verlag, Heidelberg Germany; and
Yu et al.,
(/994} Gene Therapy I:13-26.
Delivery of the gene or genetic material into the cell is the first critical
step in
gene therapy treatment of disease. A large number of delivery methods are well
known to
those of skill in the art. Such methods include, for example liposome-based
gene delivery
(Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988)
BioTechniques
6(7): 682-691; Rose U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and
Felgner et
al. (1987) Proc. Natl. Acad Sci. USA 84: 7413-7414}, and replication-defective
retroviral
vectors harboring a therapeutic polynucleotide sequence as part of the
retroviral genome
(see, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg
(1992) J. IVIHRes.
4:43, and Cornetta et aI. ( 1991 } Hum. Gene Ther. 2: 215). Widely used
retroviral vectors
include those based upon marine leukemia virus (MuLV), gibbon ape leukemia
virus
(GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus
(HIV), and
combinations thereof (see, e.g., Buchscher et al. (1992} J. Virol. 66(5) 2731-
2739; Johann et
al. (1992) J: Virol. 66 (5}:1635-1640; Sommerfelt et al., (1990) Virol. 176:
58-59; Wilson et
al. (1989) J. Virol. 63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-
2224; Wong-Staal et
al., PCT/US94/05700, Rosenburg and Fauci (1993) in Fundamental Immunology,
Third
Edition Paul (ed) Raven Press, Ltd., New York and the references therein, and
Yu et al.,
(1994) Gene Therapy supra).
AAV-based vectors are also used to transduce cells with target nucleic acids,
e.g., in the in vitro production of nucleic acids and peptides, and in in'
vivo and ex vivo gene
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therapy procedures (see, West et al. {I987) Virology 160: 38-47; Carter et al.
(1989) U.S.
Patent No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human
Gene
Therapy 5: 793-801; Muzyczka (1994) J. Clin. Invst. 94: 1351 and Samulski
(supra) for an
overview of AAV vectors. Construction of recombinant AAV vectors are described
in a
number of publications, including Lebkowski, U.S. Pat. No. 5,173,414;
Tratschin et al.
(I985) Mol. Cell. Biol. 5(11): 3251-3260; Tratschin, et al. (1984) Mol. Cell.
Biol., 4:2072-
2081; Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470;
McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol., 63: 3822-3828.
Cell lines that
can be transformed by rAAV include those described in Lebkowski et al. (1988)
Mol. Cell.
Biol., 8:3988-3996.
A1 Ex vivo transformation of cells.
Ex vivo cell transformation for diagnostics, research, or for gene therapy
(e.g.,
via re-infusion of the transformed cells into the host organism) is well known
to those of
skill in the art. In a preferred embodiment, cells are isolated from the
subject organism,
transfected with the constructs) of this invention, and re-infused back into
the subject
organism (e.g., patient). Various cell types suitable for ex vivo
transformation are well
known to~those of skill in the art. Particular preferred cells are progenitor
or stem cells (see,
e.g., Freshney et al., (1994) Culture ofAnimal Cells, a Manual ofBasic
Technique, third
edition Wiley-Liss, New York) and the references cited therein for a
discussion of how to
isolate and culture cells from patients).
For some embodiments, stem cells are used in ex-vivo procedures for cell
transformation and gene therapy. One advantage for some applications to using
stem cells is
that they can be differentiated into other cell types in vitro, or can be
introduced into a
mammal (such as the donor of the cells) where they will engraft in the bone
marrow.
Methods for differentiating CD34+ cells in vitro into clinically important
immune cell types
using cytokines such a GM-CSF, IFN-g and TNF-a are known (see, Inaba et al.
(1992) .J.
Exp. Med 176, 1693-1702).
Stem cells are isolated for transduction and differentiation using known
methods. For example, in mice, bone marrow cells are isolated by sacrificing
the mouse and
cutting the leg bones with a pair of scissors. Stem cells are isolated from
bone marrow cells
by panning the bone marrow cells with antibodies which bind unwanted cells,
such as CD4+
and CD8+ (T cells), CD45+ (pang cells), GR-1 (granulocytes), and Iad
(differentiated
antigen presenting cells). For an example of this protocol see, Inaba et al.
{1992) J. Exp.
Med. 176, 1693-1702.
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. In humans, bone marrow aspirations from iliac crests are performed e.g.,
under general anesthesia in the operating room. The bone marrow aspirations is
approximately 1,000 ml in quantity and is collected from the posterior iliac
bones and crests.
if the total number of cells collected is less than about 2 x 108/kg, a second
aspiration using
the sternum and anterior iliac crests in addition to posterior crests is
performed. During the
operation, two units of irradiated packed red cells are administered to
replace the volume of
marrow taken by the aspiration. Human hematopoietic progenitor and stem cells
are
characterized by the presence of a CD34 surface membrane antigen. This antigen
is used for
purification, e.g., on affinity columns which bind CD34. After the bone marrow
is
harvested, the mononuclear cells are separated from the other components by
means of ficol
gradient centrifugation. This is performed by a semi-automated method using a
cell
separator (e.g., a Baxter Fenwal CS3000+ or Terumo machine). The light density
cells,
composed mostly of mononuclear cells are collected and the cells are incubated
in plastic
flasks at 37°C for 1.5 hours. The adherent cells (monocytes,
macrophages and B-Cells) are
discarded. The non-adherent cells are then collected and incubated with a
monoclonal anti-
CD34 antibody (e.g., the marine antibody 9C5) at 4°C for 30 minutes
with gentle rotation.
The final concentration for the anti-CD34 antibody is 10 pg/ml. After two
washes,
paramagnetic microspheres (Dyna Beads, supplied by Baxter Immuriotherapy
Group, Santa
Ana, California) coated with sheep antimouse IgG (Fc) antibody are added to
the cell
suspension at a ratio of 2 cells/bead. After a further incubation period of 30
minutes at 4°C,
the rosetted cells with magnetic beads are collected with a magnet.
Chymopapain (supplied
by Baxter Immunotherapy Group, Santa Ana, California) at a final concentration
of 200
U/m1 is added to release the beads from the CD34+ cells. Alternatively, and
preferably, an
affinity column isolation procedure can be used which binds to CD34, or to
antibodies bound
to CD34 (see, the examples below). See, Ho et al. (1995) Stem Cells 13 (suppl.
3): 100-105.
See also, Brenner (1993) Journal ofHematotherapy 2: 7-17. In another
embodiment,
hematopoetic stem cells are isolated from fetal cord blood. Yu et al. (1995)
Proc. Natl.
Acad Sci. USA, 92: 699-703 describe a preferred method of transducing CD34+
cells from
human fetal cord blood using retroviral vectors.
For some purposes, non-stem cells are preferred for ex vivo treatments using
ESX nucleic acids. For example, where it is desirable to have the ESX product
expressed
transiently, mortal cells that do not differentiate are preferred carriers of
ESX nucleic acids.
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B) In vivo transformation.
Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing
therapeutic nucleic acids can be administered directly to the organism for
transduction of
cells in vivo. Administration is by any of the routes normally used for
introducing a
molecule into ultimate contact with blood or tissue cells. The packaged
nucleic acids are
administered in any suitable manner, preferably with pharmaceutically
acceptable carriers.
Suitable methods of administering such packaged nucleic acids are available
and well known
to those of skill in the art, and, although more than one route can be used to
administer a
particular composition, a particular route can often provide a more immediate
and more
effective reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method used to
administer the
composition. Accordingly, there is a wide variety of suitable formulations of
pharmaceutical
compositions of the present invention.
Formulations suitable for oral administration can consist of {a) liquid
solutions, such as an effective amount of the packaged nucleic acid suspended
in diluents,
such as water, saline or PEG 400; (b) capsules, sachets or tablets, each
containing a
predetermined amount of the active ingredient, as liquids, solids, granules or
gelatin; (c)
suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms
can include
one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato
starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and other
excipients,
colorants, fillers, binders, diluents, buffering agents, moistening agents,
preservatives,
flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible
carriers.
Lozenge forms can comprise the active ingredient in a flavor, usually sucrose
and acacia or
tragacanth, as well as pastilles comprising the active ingredient in an inert
base, such as
gelatin and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in
addition to the active ingredient, carriers known in the art.
The packaged nucleic acids, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can be
"nebulized'~ to be
administered via inhalation. Aerosol formulations can be placed into
pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Suitable formulations for rectal administration include, for example,
suppositories, which consist of the packaged nucleic acid with a suppository
base. Suitable
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suppository bases include natural or synthetic triglycerides or paraffin
hydrocarbons. In
addition, it is also possible to use gelatin rectal capsules which consist of
a combination of
the packaged nucleic acid with a base, including, for example, liquid
triglycerides,
polyethylene glycols, and paraffin hydrocarbons.
Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection solutions,
which can contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation
isotonic with the blood of the intended recipient, and aqueous and non-aqueous
sterile
suspensions that can include suspending agents, solubilizers, thickening
agents, stabilizers,
and preservatives. In the practice of this invention, compositions can be
administered, for
example, by intravenous infusion, orally, topically, intraperitoneally,
intravesically or
intrathecally. Parenteral administration and intravenous administration are
the.preferred
methods of administration. The formulations of packaged nucleic acid can be
presented in
unit-dose or mufti-dose sealed containers, such as ampoules and vials.
Injection solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described. Cells transduced by
the packaged
nucleic acid as described above in the context of ex vivo therapy can also be
administered
intravenously or parenterally as described above.
The dose administered to a patient, in the context of the present invention
should be sufficient to effect a beneficial therapeutic response in the
patient over time. The
dose will be determined by the efficacy of the particular vector employed
and.the condition
of the patient, as well as the body weight or surface area of the patient to
be treated. The
size of the dose also will be determined by the existence, nature, and extent
of any adverse
side-effects that accompany the administration of a particular vector, or
transduced cell type
in a particular patient.
In determining the effective amount of the vector to be administered in the
treatment or prophylaxis, the physician evaluates circulating plasma levels of
the vector,
vector toxicities, progression of the disease, and the production of anti-
vector antibodies. In
general, the dose equivalent of a naked nucleic acid from a vector is from
about 1 mg to 100
mg for a typical 70 kilogram patient, and doses of vectors which include a
retroviral particle
are calculated to yield an equivalent amount of therapeutic nucleic acid.
For administration, inhibitors and transduced cells of the present invention
can be administered at a rate determined by the LD-50 of the inhibitor,
vector, or transduced
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cell type, and the side-effects of the inhibitor, vector or cell type at
various concentrations, as
applied to the mass and overall health of the patient. Administration can be
accomplished
via single or divided doses.
In a preferred embodiment, prior to infusion, blood samples are obtained and
saved for analysis. Between 1 x 108 and 1 x 1012 transduced cells are infused
intravenously
over 60-200 minutes. Vital signs and oxygen saturation by pulse oximetry are
closely
monitored. Blood samples are obtained 5 minutes and 1 hour following infusion
and saved
for subsequent analysis. Leukopheresis, transduction and reinfusion can be
repeated are
repeated every 2 to 3 months. After the first treatment, infusions can be
performed on a
outpatient basis at the discretion of the clinician. If the reinfusion is
given as an outpatient,
the participant is monitored for at least 4, and preferably 8 hours following
the therapy.
Transduced cells are prepared for reinfusion according to established
methods. See, Abrahamsen.et al. (1991) J. Clin. Apheresis, 6: 48-53; Carter et
al. (1988) J.
Clin. Arpheresis, 4:113117; Aebersold et al. (1988) J. Immunol. Meth., 112: 1-
7; Muul et
al. (1987) J. Immunol. Methods,101: 171-181 and Carter et al. (1987)
Transfusion 27: 362-
365. After a period of about 2-4 weeks in culture, the cells should number
between 1 X 108
and 1 x 1012. In this regard, the growth characteristics of cells vary from
patient to patient
and from cell type to cell type. About 72 hours prior to reinfusion of the
transduced cells, an
aliquot is taken for analysis of phenotype, and percentage of cells expressing
the therapeutic
agent.
EXAMPLES
The following examples are offered to illustrate, but not to limit the present
invention.
EaampIe 1: Cloning and Expression of a Human ESX Gene.
This example describes the isolation of a complete human ESX cDNA
sequence that encodes a putative protein of 371 amino acids. Briefly, a highly
conserved
eight amino acid motif within the carboxy (C)-terminal region of the ETS
domain was
identified and this motif was used to search a database of human epithelium
expressed
sequence tags (SSTs). The database (dbEST) contained >250,000 largely
anonymous ESTs
(Lennon et al. (1996) Genomics 33: 151-152. This search identified a partial
cDNA
sequence from fetal liver-spleen (GenBank locus T78501). Within this same
database, were
found two other unidentified but nearly identical partial sequences from
normal mammary
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epithelium (GenBank locus 873021) and adult pancreas (GenBank locus T27397).
Human
placental polyA+ mRNA was used to generate a full-length cDNA sequence.
Experimental procedures.
Cloning of EST cDNA.
The Basic Local Alignment Search Tool (BLAST) was used to search a
database of expressed sequence tags (EST) using nucleotides derived from human
Ets-2 that
encode a highly conserved eight amino acid motif within the carboxy terminal
region of the
ETS domain (MIV~.'EKLSR). The BLAST algorithm is described in Altschul et al.
(1990) J.
Mol. Biol. 215: 403. This search identified a partial cDNA sequence from fetal
liver-spleen
(GenBank locus T78501) as a putative new member of the Ets family that was
named ESX.
Made available by LM.A.G.E. Consortium and commercially obtained (Research
Genetics,
Inc.), this 1.1 kb partial cDNA sequence derived from fetal liver-spleen
contains a polyA
tail, approximately 0.7 kb of 3' untranslated sequence and a 5' region
encodingthe C-
terminal 126 amino acids of ESX. Re-sequencing of T78501 revealed several
errors in its
original GenBank sequence that would have disrupted the reading frame. A 5'
RACE
procedure (Frohman (1990) .RACE: Rapid amplification of cDNA ends, p 28 in PCR
Protocols: A guide to methods and applications, Innis, et al., Eds. Academic
Press, San
Diego, CA) was performed using the Marathon cDNA amplification kit (Clontech
Laboratories, Inc.) using placental polyA mRNA to clone the remaining 5'
portion of ESX
cDNA, which was estimated to be approximately 0.8 kb. Automated DNA sequencing
of
three independent clones of the expected length yielded identical results and
5' cDNA
termination sites within 30 bases of one another. Melding these sequences with
the amended
T78501 sequence produced the open reading frame as shown in SEQ ID NO:1. To
identify
ESX domain homologies, performed BLAST searches of the SWISS-PROT and PIR
protein
databases were performed.
ESX polweptide production. DNA bindinE assay. and DNA footprintinE
assay.
Using primers incorporating the initiating methionine or the termination
codon of ESX and designed with NheI and HindIII sites, respectively, PCR
amplification
was performed on double stranded placental cDNA (Clontech) to produce a full-
length ESX
cDNA product which was subsequently cloned into the NheI and HindIII sites of
a pRSETA
His-tag expression plasmid (Invitrogen). Following sequence verification, an
ESX
expression clone in BL21 (DE3)pLysS cells was used to produce ESX protein
following 8M
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urea bacterial extraction, purification on ProBond resin (Invitrogen), and
dialysis against
PBS containing 10% glycerol. SDS polyacrylamide gel analysis indicated a 42
kDa pmtein
with >90% purity.
Electrophoretic mobility shift assay (EMSA) was performed as previously
described (Scott et al. (1994) J. Biol. Chem. 269: 19848-19858), using
approximately one ng
of ESX protein per condition and 0.3 pmol of end-labeled TAS probe (+cold
competitor).
TAS is a duplexed 31-mer oligonucleotide from the HER2/neu promoter, extending
from -50
by to -20 by relative to the major transcriptional start site, that includes
an Ets response
element.
DNase I footprinting was performed on a 125 by BssHl1//SmaI fragment from
the HER2/neu promoter, labeled on the antisense strand at the SmaI site.
Reactions
contained ~10 ng of ESX protein with 1 unit of DNase-I acting for 1 min at
room
temperature. Reaction products containing ESX were electrophoresed on a 6%
denaturing
gel alongside a control~reaction lane (minus ESX, lane C).
Trans-activation of Ets-responsive gene expression by ESX.
Cultured COS cells were transiently cotransfected by calcium phosphate
precipitation as previously described (Scott et al. (1994) J. Biol. Chem. 2b9:
19848-19858)
using pcDNAl/Amp (Invitrogen) to express full-length ESX protein and either
the
thymidine kinase minimal piomoter-CAT vector (pBLCATS, from American Type
Culture
Collection) enhanced with 3 tandem (head-to-tail) upstream copies of TAS
(p3TA5-
BLCATS) or a 700 by AflII/NcoI fragment from the HER2/neu promoter (containing
two
other putative Ets response elements upstream of the TAS sequence) inserted
into pCAT-
Basic (Promega) to give pHER2-CAT. Mutant reporter plasmids, p3TA5P-BLCATS and
pHER2m-CAT, were similarly constructed with the former possessing a GGAA to
GAGA
mutation within each of the tandem repeats and the latter retaining the two
upstream
promoter response elements intact but possessing a GGAA to TTAA Ets response
element
mutation within the TAS sequence. Transfections, using 0.5 mg of reporter and
5 mg of
expression plasmid, were repeated at least three times with the mean values
(+SD) of CAT
reporter activity (arbitrary units) as shown.
Chromosomal localization.
Metaphase chromosomal localization and interphase copy number of ESX
were determined by FISH analysis with a genomic ESX P1 clone, using a
previously
described technique (Stolcke et al. (1995) Genomics 26: 134-137).
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Northern hybridization.
Total cellular RNA was prepared by guanidinium isothiocyanate extraction
(pH 5.5) as described previously (Scott et al., supra.) and blotted onto nylon
membranes
following electrophoresis through I % formaldehyde agarose gels (~ 20 mg per
lane). All
blots were probed with a randomly primed 400 by cDNA fragment from the C-
terminal ESX
coding region, and given final washes at 65°C in 0.2x SSC. Short
exposure of the
autoradiograph was used to demonstrate HRG induction of ESX in the
overexpressing SK-
BR-3 cells.
Detection of ESX expression by in situ hybridization
ESX sense and antisense riboprobes for in situ hybridization were generated
by 35S-labeling and run-off transcription using T7 or T3 RNA polymerase,
respectively,
from pT7T3 (Pharmacia) containing a 700 by fragment of 3' untranslated ESX
cDNA.
Using previously described techniques (Wilkinson (1992) In situ hybridization:
a practical
approach, IRI, Press, Oxford), tissue hybridization and autoradiography were
performed on
thin sections of paraffin-embedded samples of normal mammary epithelium (n=3)
and DCIS
breast tumors (n=10). Samples were chosen according to their previously
determined
HER2/neu overexpression and amplification status (Liu et al. (1992) Oncogene
7: 1027-
1032) and for their RNA integrity and comparable levels of glyceraldehyde-3-
phosphate
dehydrogenase (GAPDH) expressian, as determined by preliminary in situ
hybridization
with an antisense probe for GAPDH. The hybnidizations show only the antisense
riboprobe
signals resulting from ESX transcripts in the underlying hematoxylin-
counterstained
epithelial cells. ESX sense riboprobe was used to control for non-specific
hybridization and
autoradiography background signal using adjacent sections from each sample.
The density
ofi~s background signal (from sense riboprobe) was nearly identical for the
representative
samples shown in this figure, representing less than one-tenth the antisense
riboprobe signal
density over the epithelial cells and comparable to that over the acellular
stromal component
of each sample.
Preparation of anti-ESX antiserum.
A peptide fragment consisting of the sixteen carboxy-terminal amino acids of
ESX was synthesized for use as an ESX antigen in rabbits. An amino-terminal
cysteine was
introduced to allow coupling of the peptide to a carrier protein (KLH). To
obtain anti-ESX
antibodies, total IgG from immunized rabbits was affinity purified on a column
to which the
ESX carboxy-terminal peptide fragment was bound.
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Results and Discussion.
CloainQ of a human ESX cDNA.
The nucleotide and deduced amino acid sequences of a human ESX cDNA
are shown in Figure 1. The cDNA includes an open reading frame that encodes a
371 amino
acid ESX protein as shown in Figure 2A. The C-terminal ETS DNA binding domain
of ESX
(aa 274-354) contains 27 of the 38 most highly conserved (consensus) residues
found in the
DNA-binding domain of all Ets family members (Figure 2D). This domain in ESX
has its
greatest homology with the Drosophila E74/human Elf 1 subfamily (nearly 50%
identity,
70% similarity), although ESX has no homology with E?4/Elf 1 outside the Ets
DNA
binding domain. The most obvious structural differences distinguishing ESX
from other Ets
family members are the five non-conservative changes in its DNA-binding domain
consensus residues, including three within the first helix (al) that enhance
basicity in a
region likely to make critical contact with the minor groove phosphate
backbone of bound
DNA (Werner et al. (1995) Cell 83: 761-771; Kodandapani et al. (1996) Nature
380: 456-
460). Therefore, ESX may be assigned to the E74/Elf 1 subfamily on the basis
of its
sequence homology within the ETS domain (Lautenberger et al. (1992) Oncogene
7: 1713-
1719; Laudet et al.(1993) Biochem. Biophys. Res. Commun. 190: 8-14; Degnan et
al. {1993)
Nucl. Acids Res. 21: 3479-3484; Wasylyk et al. (1993) Eur. .T. Biochem. 211: 7-
18;
Janknecht and Nordheim (I993) Biochem. Biophys. Acta. 1155: 346-356). In
contrast to its
two other subfamily members, however, ESX possesses an amino (I~-terminal A-
region or
Pointed domain, a helix-loop-helix structure that has been conserved from
Drosophila to
humans and retained within subfamilies remote to E74/Elf 1 (Lautenberger et
al., supra.;
Wasylyk et al., supra.; KTambt (1993) Development 11.7: 163-176). The A-region
in ESX
(aa 64-103) is most similar to that found in Ets-1 {aa 69-106) with 65%
similarity and 40%
identity, including 7 of 9 consensus A-region residues (Figure 2B).
Additional features within ESX highlight the known plasticity of Ets proteins
in regions outside of their ETS domain, reflecting >500 million years of
evolutionary
recombination and exon shuffling (Lautenberger et al., supra.; Laudet et al.,
supra.; Degnan
et al., supra.; Wasylyk et al., supra.). ESX has one of the shortest C-
terminal tails (16 aa) of
all Ets family members. While this terminal sequence has no significant
homology to any
known eukaryotic gene product, it is over 50% identical and 85% similar to a
highly
conserved element within the Ross River (aa 194-207) and Semliki Forest (aa
197-210)
vines-encoded nsPl protein, which is required for membrane-bound initiation of
RNA
synthesis, replication and the subsequent pathogenicity of these New World RNA
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alphaviruses (Strauss and Strauss (1994) Microbiological Rev. 58: 491-562).
Contained
within the N-terminal flanking region of the ESX DNA-binding domain is a
serine-rich track
of 51 residues (aa 188-238) that is 35% identical to the conserved polyserine
transactivating
domain of the lymphocyte-restricted HMG-box protein, SOX4 (aa 370-420)
(VandeWetering et al. (1993) EMBO J. 12: 3847-3854). Polyserine domains are
known to
act as strong transactivators, presumably, as in the case of p65NF-kB (aa 530-
560), by
foaming amphipathic helical structures in which the serines are clustered
opposite a
hydrophobic face (Seipel et al. (1992) EMBO J. 11: 4961-4968; Schmitz and
Baeuerle
(1991) EMBO J. 10: 3805-3817), as shown in a helical wheel model of the serine
box in
ESX (Figure 2C).
ESX binding to and transactivation of I3ER2/neu Ets response element.
Earlier studies have demonstrated that the HElZ2/neu oncogene, which is
activated by overexpressiow in >40% of DCIS early breast tumors (Liu et al.,
supra.),
contains a highly conserved Ets responsive element in its proximal promoter
(Scott et al.,
supra.). Therefore, an oligonucleotide (TA5) containing the Ets response
element from
HER2/neu was used to assess DNA-binding and transactivation by ESX.
Bacterially
expressed~full-length ESX demonstrates high-affinity, sequence-specific
binding to TA5 by
electrophoretic mobility shift assay (EMSA), as shown in Figure 8A. Unlike
EMSA results
for other Ets proteins known to contain flanking regions that restrict DNA-
binding (Jonsen et
al. (i996) Mol. Cell. Biol. 16: 2065-2073), full-length ESX binds DNA with
comparable
affinity to that of truncated ESX (aa 271-371), consisting primarily of the
ESX DNA-
binding domain. As seen with other Ets factors, DNA probes with mutations in
the GGAA
Ets core of TA5 fail to compete against TA5 for ESX binding, while those with
mutations
flanking the GGAA core are relatively effective at competing for ESX binding.
To confirm that ESX binds DNA in an Ets-like manner, ESX footprinting was
performed on a larger HER.2/neu promoter fragment overlapping the TA5 sequence
and its
GGAA core response element. Characteristic of DNA-bound Ets proteins, ESX
produces a
DNase-I hypersensitive site embedded within a footprint on the antisense
strand of the core
response element (Figure 8B).
The transactivating potential of ESX was then determined by cotransfecting
COS cells with an ESX expression plasmid and either of two different Ets-
responsive
reporter genes: a minimal promoter construct enhanced by 3 tandem head-to-tail
copies of
TA5 from the HER2/neu promoter, or -~0.7 kb of the wild-type HER2/neu promoter
driving
the chloramphenicol acetyl transferase (CAT) reporter gene. Exogenously
introduced ESX
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significantly increases CAT expression from both constructs, but only when the
core Ets
response element is intact and not mutated, confirming the Ets-specific
transactivating
potential of ESX (Figure 8C).
Chromosomal localization.
To obtain further insight into the evolutionary mechanisms of Ets dispersion
during the metazoan radiation of this multigene family, we mapped the
chromosomal
location of the human ESX gene and found that the gene is located next an
unrelated
subfamily member. About 10 of the known human Ets genes have been
chromosomally
mapped and half of these occur as a tandem linkage of dissimilar subfamily
members at two
general loci (21 q22 for Ets2, Erg, and GABPa; 11 q23 for Ets 1 and Fli 1 ),
supporting a
proposed model in which duplication of an ancestral Ets was followed by
duplication and
transposition of the Ets pair to another chromosome (Lautenberger et al.,
supra.; Laudet et
al., supra.; Degnan et al., supra.; Wasylyk et al., supra.).
An ESX clone isolated from an arrayed P1 library was used to map ESX to
chromosome 1q32 by fluorescence in situ hybridization (FISH) (Figure 8D).
Since SAP1
(also known as ELK4, a member of the SAP/Elk/Net subfamily) was recently
mapped to
1q32 (Shipley et al. (1994) Genomics 23: 710-711; Giovane et al. (1995)
Genomics 29: 769-
772), ESX and SAP1 now represent the third known set of tandemly linked human
Ets
genes. While the chromosomal location of Elf I (subfamily homolog of ESX) is
not
presently known, it is tempting to speculate that it will be linked to another
SAP/EIk/Net
subfamily member, in accordance with the evolutionary model for the generation
of the Ets-
1/Fli-1 and Ets-2/Erg loci.
Southern blotting suggested the presence of excess ESX gene copies in
several breast cancer cell Iines known for their amplification of HER2/neu
(e.g. SK-BR-3,
BT-474). Therefore, FISH analysis was also performed on these cells. As shown
in
Figure8D, ESX amplification in these cell lines results predominantly from an
increase in
chromosome 1 q copy number (aneusomy). While gene amplification is not thought
to be a
common mechanism by which Ets proto-oncogenes become activated (Wasylyk et
al.,
supra.; Janknecht and Norheim, supra.), multiple copies of DNA sequences
mapping across
the 1q32 locus can be observed in about 50% of early breast tumors {Isola et
al. (1995) Am.
J. Pathol. 147: 905-911). Apart from two other more centromeric proto-
oncogenes on this
chromosome arm, SKI at Iq22-24 and TRK at 1q23-24 (Chaganti et al. (1986)
Cytogenet.
Cell Genet. 43: 181-186; Moms et al. (1991) Oncogene 6: 1093-1095, ESX and
SAP1
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represent likely oncogene candidates accounting for this lq amplification in
human breast
tumors.
Expression of ESX.
Many human Ets exhibit a tissue-restricted pattern of gene expression, with
some family members showing greater tissue specificity than others (Wasylyk et
al., supra.;
Janknecht and Norheim, supra.). Northern blots of normal human tissue
demonstrate that
ESX mRNA expression is restricted to tissues of epithelial origin, with little
if any
expression detectable in testes, ovary, brain, skeletal muscle, or lympho-
hematopoietic
tissues (spleen, thymus, white blood cells). PEAS, by comparison, the only
other
epithelium-restricted Ets, is expressed in a subset (5 of 9) of the ESX-
positive tissues (data
not shown); expression of both in normal heart leaves open to question the
endo-, myo-, or
peri-cordial component of this tissue that is the source of ESX and PEAS
transcripts.
When a panel of human breast cancer cell lines was compared for ESX
expression with normal human mammary epithelial cells (HMEC), ESX mRNA was
IS increased in the HER2/neu-positive tumor lines and not increased in the
HER2/neu-negative
lines. Two immortalized but non-transformed mammary cell lines (HBL 100, MCF i
OA)
expressed-ESX mRNA at levels similar to or below that of HMEC. To explore the
possible
relationship between ESX overexpression and HER2/neu activation, ESX mRNA was
measured in cultured SK-BR-3 cells after treatment with the ligand heregulin-b
11-244
(HRG), known to inittiate mitogenic signaling in these cells by activation of
HER2/neu
receptor tyrosine kinase in association with ErbB3 (Holmes et al. (1992)
Science 256: 1205
1210; Li et al. (i996) Oncogene 12: 2473-2477). ESX mRNA increased within 15
min of
HRG treatment, achieving peak levels between 60 and I20 min. These results
indicate that
ESX induction is an immediate early gene response to HER2/neu activation,
supporting a
signaling link between ESX and HER2/neu gene function.
Since HER2/neu activation occurs early during human breast tumorigenesis
and with development of DCIS, evidence of early ESX overexpression was
screened for by
in situ hybridization in DCIS tumor samples previously characterized as
HERZ/neu-positive
with regard to amplification and overexpression relative to that of normal
breast epithelium
(Liu et al., supra.). ESX expression was restricted to normal and malignant
mammary
ductal epithelium with no ESX expression detectable in breast stroma,
including its reticulo-
endothelial cell and inflammatory/lymphocytic cell components. Consistent with
ESX
overexpression observed in HER2/neu amplified breast cancer lines, ESX
transcript levels in
HER2/neu-positive DCIS were markedly increased relative to that of normal
breast
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epithelium. These tissue hybridization studies indicate that overexpression of
ESX, as with
HER2/neu, may occur early during development of human breast tumors.
Since ESX can transactivate the HER2/neu promoter, one potential
mechanistic link may be explored by interfering with transcriptional
regulation at the Ets
response element on this promoter (Noonberg et al. (1994) Gene 149: 123-126).
Also,
preliminary studies suggest that activated HER2/neu increases Ets-mediated
gene expression
via Ras signaling and that this can lead to feedback upregulation of Ets
transcription (Galang
et al. (1996) J. Biol. Chem. 271: 7992-7998; O'Hagan et al. (1995) Amer.
Assoc. Cancer
Res. 37: 3575. Thus, there is compelling rationale to establish the prevalence
and
mechanistic role of ESX overexpression in breast tumors as well as other human
malignancies of epithelial origin.
Anti-ESX antibodies.
In a Westerwblot analysis, anti-ESX polyclonal antibodies prepared as
described above specifically recognized purified recombinant ESX protein (~42
kD), as well
as a similar sized protein in whole cell extracts. The intensity of the ESX
band in samples
prepared from whole cell extracts was correlated with cellular ESX mRNA
levels.
The anti-ESX antibodies also function to immunoprecipitate a single ~42 kD
ESX protein band from 35S metabolically labeled cells.
Example 2: Cloning and analysis of marine ESX.
A 8FIX2 genomic library from strain 129 mouse DNA was screened using a
5' cDNA probe from hESX to isolate a clone from which a 7,751 by fragment was
subcloned into Bluescript and sequenced. A fully encoding mESX cDNA clone was
derived
from total RNA of 129 mouse ES cells by reverse transcription PCR (RT-PCR)
using
specific primers extending 5' and 3' from the putative ATG-start and TAA-stop
codons,
respectively of the genomic sequence. A Bluescript subclone containing this
1,116 by
mESX cDNA was similarly sequenced. All sequencing was performed on an ABI
Prism
Automated DNA sequences (model 377) using 3'-dye labeled ddNTP terminators.
The full
length mouse ESX genomic sequence is provided in SEQ >D NO: 11.
Alignment of genomic and cDNA mESX sequences as well as comparison of
mESX vs hESX homologous sequences were used to determine exon and intron
boundaries
(see, Fig. 7). Conserved marine and human promoter elements as well as
putative amino
acid domain homologies were identified from PIR-protein, SWISS-PROT, and
PROSITE
databases by GCG computer search (Genetics Computer Group, Wisconsin Package
3.0,
Madison, WI).
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A 7.8 kb mESX genomic clone was isolated that contains ~2.9 kb of promoter
upstream of ~4.9 kb of DNA incorporating at least 9 exons. These specify a
full-length
transcript of ~2 kb, with exons 2-9 encoding the 371 amino acid mESX protein
(see Fig. 3).
The following putative structural and/or functional domains within the 42
kDA ESX protein were conserved between mouse and human (Fig. 4):
An Exon 3 encoded POINTED/A-region found in a small subset of all Ets;
An amphilic helix and serine rich box encoded by exons 5 and 6;
A nucleoplasmin-type nuclear targeting sequence encoded by exon 7; and
A helix-turn-helix Ets DNA-binding domain encoded by exons 8 and 9.
A comparison of the human ESX and mouse ESX genomic DNA structure is shown in
Figure 6.
The proximal promoter region of mESX (350 by upstream of transcriptional
start site) was 83% homologous to the hESX promoter (Fig. 5). Conserved
putative
response elements within this region include Ets, AP-2, SP1, USF, Oct, and NF-
6B binding
sites. A conserved CCAAT box lies ~80 by upstream of the pyrimidine-rich Inr
element
which specifies ESX transcript initiation. Unlike hESX, mESX lacks a TATA box.
The comparison of mESX and hESX genomic and cDNA sequences supports
a modular model of ESX primary structure in which putative protein domains,
first
suggested by homology with other proteins, are now shown to be highly
conserved and
derived from individual exons or exon pairs.
Example 3: Embryo and mammary epithelia cell expression of ESX.
Whole mount analysis of mammary gland morphology was performed as
described by Smith (1996) Breast Cancer Res. Treat., 39: 21-31. Endogenous
mESX
transcripts were detected by Northern blotting using a 5' specific mESX cDNA
probe.
Mouse embryos exhibited progressive induction of mESX transcription after
7 days of age, with 17 day levels approximately 10-fold higher than those of
11 day old
embryos. ESX mRNA, undetectable in virgin mouse mammary glands, was induced
during
pregnancy in association with progressing ductal morpohogenesis, branching and
lobuloalveolar differentiation. ESX then declined to undetectable levels
during lactation, but
increased dramatically with 3 days of weaning when milk secretion stops,
alveolar
epithelium involutes by apoptosis, and glandular remodeling occurs leaving a
more mature
ductal epithelium system ready for subsequent pregnancy. These data suggest
that ESX has
a primary role in directing ductal epithelial proliferation and migration in
preparation for
lobuloalveolar differentiation.
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Example 4: Transgenic hESX model
MMTV-hESX transgenic mice were produced by implanting foster mothers
with fertilized eggs microinjected with a full-length hESX expression
construct, driven by
the MMTV LTR and containing the polyA signaling and splicing sequence from
SV40).
(The MMTV promoter is well described (Huang et al. ( 1981 ) Cell, 27: 245-
255). In
addition, the use of MMTV-LTR for targeted expression of transgenes to the
mammary
gland of mice and other animals is described in detail in Webster and Muffler,
(1994) Sem.
Cancer Biol., 5: 69-76). hESX transgene expression was detected using a probe
specific for
the SV40 polyA sequence and confirmed by nested RT-PCR analysis using 5'
primers
specific for hESX and 3' primers specific for the SV40 polyA sequence.
Founder (Fo) lines created as described in Example 3, were tested for
transgene presence. Fourteen of fortyone animals carried the transgene. The
Founder
animals were then mated and 155 day pregnant Ft females were then tested for
mammary
gland expression of hESX mRNA. Total RNA was extracted from the mammary glands
of
15 day pregnant MMTV-hESX transgenic Fl mice. A northern blot of 10 pg of the
RNA
was probed for sequences specific to the SV40 polyA-containing hESX
transcript.
Mammary gland morphology in an MMTV-hESX expressing transgenic
mouse appeared abnormal, showing retardation of lobuloalveolar development
during
pregnancy (15 day, first pregnancy). This morphologic abnormality suggests
that failure to
turn of ESX in progenitor epithelial cells and alveolar buds leads to
continued ductal growth
with interrupted mammary gland maturation.
Examule 5: ESX is a transcriptional activator
To prove that ESX upregulates genes (vs. transeriptionally repressing them),
many different hESX-Gal4 fusion constructs were produced in which the DNA-
binding
domain (DBD) of the yeast Gal4 was chimerically expressed with various
portions of human
ESX (see, Fig. 9) (for a general description of the method see, e.g., White
and Parker (1993)
Analysis of cloned Factors, In .Transcription Factors: a practical approach;
D.S. Latchman,
ed.; IRL Press at Oxford Univ. Press, Oxford). These fission constructs were
then co-
transfected into human breast cancer cells along with a Gal4 binding
luciferase reporting
expression construct in order to find either an ESX transactivating or
repressing domain. A
similar Gal4-VP16 construct was used as positive control since the VP16
transactivating
domain from Herpes Simplex virus is acknowledged to be one of the strongest of
all known
transactivators.
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ESX transactivated as strongly as VP16 (+++++) (see, Fig. 9) and the
minimal ESX domain necessary for this activity is encoded by exon 4 (aa 129-
159), an
acidic domain containing a central lysine residue (K-145). Subsequent
mutations of this
domain established that the central K-145 is essential and provides nearly
1000-fold
transactivation potency (relative to a neutral residue placed there).
A database revealed that the exon 4-encoded domain is homologous to an
essential core domain of all known Topoisomerase I molecules (Stewart et al.
(1996) ,I. Biol.
Chem. 271:7602-7608; Pommier (1996) Sem. Oncology 23: 3-10). Since human Topo-
I is a
critical intracellular target for the newest and most exciting family of
camptothecin-like
anticancer agents (like Topotecan, CPT-11, 9AC, etc.; see reviews), this
information not
only provides important clues as to the molecular transactivation mechanism of
ESX, but it
indicates that this particular ESX domain may be used to search for or screen
(from Libraries
of chemicals or natural products) for even newer and more effective and
selective anticancer
agents.
Existing Topo-I agents target a very different, C-terminal conserved domain
in the Topo-I enzyme; as yet, there is no specific function assigned to the
highly conserved
Topo-I Core domain which is homologous to the ESX transactivation domain.
Example 6: The epithelium-specific Ets transcription factor ESX is associated
with
mammary Qland development and involution
In this example, in order to study mammary gland expression of the
epithelium-restricted Ets factor, ESX, mouse cDNA and genomic sequences were
cloned and
a 350 by proximal promoter region with >80% mouse-human homology was
identified
that mediates ESX induction by serum, heregulin (HItG) or epidermal growth
factor (EGF).
ESX mRNA expression progressively increases during embryonic mouse development
from
day 7, is detectable in virgin mammary glands and shows little if any change
during
pregnancy, then declines to barely detectable levels following 3 days of
lactation. Similarly,
cultured HC11 cells from midpregnant mouse mammary epithelium show an increase
in
ESX expression upon reaching lactogenic competency (in the presence of EGF or
HRG),
with a decline to barely detectable levels upon exposure to Iactogenic
hormones which
induce milk protein (~i-casein) expression. In contrast, involuting mouse and
rat mammary
glands show maximal ESX expression. High ESX Levels are also seen in the
invoiuting
ventral prostate gland of rats. These findings, including the persistance of
upregulated ESX
in fully regressed mammary glands, suggest that ESX expression can be induced
by soluble
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growth factors and is maximally upregulated in those partially committed
epithelial cells that
are destined to' survive both the apoptotic and remodelling phases of
glandular involution.
Introduction.
Ets transcription factors regulate stage- and tissue-specific gene programs in
fetal development and are overexpressed or rearranged in a variety of
vertebrate and human
malignancies (reviewed in Wasylyk and Nordheim (1997) Ets transcription
factors:
partners in the integration of signal responses. In Transcription Factors in
Eukaryotes
(Papavassiliou AG, ed.) pp. 253-286, Springer-Verlag GmbH & Co. KG.,
Heidelberg,
Germany; Hromas Klemsz (1994) Int. J. Hematol. 59: 257-265; and Macleod et al.
(1992)
Trends Biochem. Sci. 17: 251-256.). We recently cloned and characterized a
novel 42 kDa
Ets factor, ESX Epithelial-restricted with Serine boX~, which is
transcriptionally
upregulated in a subset of early breast tumors and breast cancer cell lines
and is thought to
transactivate the Ets responsive mammary gland oncogene, erbB2 (Scott et al.
.(1994) J.
Biol. Chem. 269: 19848=19858; Chang et al. (1997) Oncogene 14: 1617-1622).
Subsequent
to this report, four groups have published on the potential biological and
developmental
importance of this epithelium-specific Ets factor (variably named ESE-1, Elf
3, Jen, or ERT;
now identified ESX in HUGO/GDB:6837498) in non-mammary epithelial systems,
where
ESX is thought to transactivate such genes as the transforming growth factor-
(3 type II
receptor (TGF-(3RII), Endo-A/keratin-8, and several markers of epidermal cell
differentiation including transglutaminase 3, SPRR2A, and profilaggrin
(Oettgen et al.
(1997) Mol. Cell. Biol. 17: 4419-4433; Tymms et al. (1997) Oncogene 15: 2449-
2462;
Andreoli et al. (1997) Nucleic Acid Res. 25: 4287-4295; Choi S-G et al. (1998)
J. Biol.
Chem. 273: 110-117.).
While its expression profile suggests that ESX is associated with
development of both simple and stratified epithelium (Davies and Garrod (1997)
BioEssays
19: 699-704),. detailed studies have been performed only in the latter and
these have shown
that ESX is unique among transcription factors generally, and Ets factors
specifically, for its
restricted expression in the most terminally differentiated of epidermal cells
(Oettgen et al.
(1997) Mol. Cell. Biol. 17: 4419-4433; Andreoli et al. (1997) Nucleic Acid
Res. 25: 4287-
4295). A limited in situ analysis of normal human mammary tissue demonstrated
low but
heterogeneous levels of ESX transcript expression restricted to the polarized
simple
epithelium of ductules and terminal ductal-lobular units (5). To evaluate ESX
expression
during all differentiation stages of mammary epithelium, mouse ESX cDNA and
genomic
sequences were first cloned and compared to their human counterparts, and then
used to
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study postnatal rodent models of mammary gland development. The inductive
influences
controlling ESA expression were explored by transient transfection of an ESX
promoter-
reporter construct into a breast cancer cell line (SKBr3) responsive to
heregulin-~3 (HRG)
and epidermal growth factor (EGF). RNA samples probed for ESX expression were
derived
S from different stages of cultured HC11 mouse mammary epithelial cells, first
made
competent for lactogenesis and then hormonally induced to synthesize (3-casein
(Ball et al.
(1988) EMBO,I. 7: 2089-2095; Marte et a1 (1995) Mol. Endocrinol. 9: 14-23).
These
results were compared to ESX Northern blots of virgin, pregnant, lactating,
and involuting
mouse mammary glands. Lastly, mouse and rat mammary glands collected during
involution were also compared to the involuting ventral prostate gland of rats
to demonstrate
that maximal induction of ESX occurs during this stage of glandular
regression, suggesting
an association with epithelial apoptosis.
Methods.
Comparison of marine and human ESX genomic and cDNA sequences
1S A ~.FDGI 129SV mouse genomic library (Stratagene) was screened using a S',
cDNA probe from hESX (S; Genbank accession number U66894) to isolate a clone
from
which a~7.7S kb BamHI fragment was subcloned into pBluescript SK (Stratagene).
Upon
full sequencing this genomic clone was found to contain 3.6 kb of sequence
upstream from
the ATG-start codon (beginning exon Z), about 2.9 kb upstream of the
transcriptional start
site. The deduced organization of 9 exons (8 coding) and 8 introns spanning
4.9 kb of
genomic sequence was subsequently found to be similar to that reported by
Tymms et aL
(1997) Oncogene 1S: 2449-2462. This mESX genomic sequence was compared to a
previously isolated and fully sequenced 1.8 kb BgIII-BgIII human genomic clone
containing
1.S kb of hESX promoter sequence upstream of exon 1 and the S' half of intron
1. A 1.1 kb
2S Bluescript subclone encoding the entire mESX cDNA was derived from 129SV
mouse ES
cell total RNA by RT-PCR using specific primers extending S' and 3' from the
respective
ATG-start and TAA-stop codons in the genomic sequence, and the entire cDNA
subclone
was sequenced. All sequencing was performed on an ABI Prism Automated DNA
Sequences (model 377) using 3'-dye labelled ddNTP terminators. Computer
alignments of
genomic and cDNA mESX and hESX sequences were performed, and comparison of
genomic and cDNA mESX sequences were used to determine exon and intron
boundaries.
Conserved marine and human promoter elements as well as putative amino acid
domain
homologies were identified from PIR-protein, SWISS-PROT and PROSITE databases
by
GCG computer search (Genetics Computer Group, Wisconsin Package 3.0, Madison,
Wn.
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Growth factors and tissue culture conditions.
Recombinant human EGF was commercially obtained {Sigma). Recombinant
human HRG isoforms were kindly provided (Amgen; (31 isoform 177-228) or
commercially
obtained (NeoMarkers; full-length ~i 1 isoform), with no significant
difference in activity
S detected between the truncated and full-length (31 isoforms. SICBr3, MCF-7
and MDA-435
breast cancer cell lines (Chang et al. (1997) Oncogene 14: 1617-1622, Daly et
al. (1997)
Cancer ReS. 57: 3804-3811), and NIH3T3 mouse fibroblasts, were all maintained
in DMEM
medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS).
HC11
cells, derived from midpregnant BALB/c mouse mammary gland tissue, were
maintained in
culture using a growth medium consisting of RPMI-1640, 10% heat-inactivated
fetal calf
serum, 5 pg/ml bovine insulin, and either 2 nM HRG or 2 nM EGF (Ball et al.
(1988)
EMBO J. 7: 2089-2095.12. Marte et al. (1995) Mol. Endocrinol. 9: 14-23). HC11
cells
were induced into lactogenie competency by culturing them in growth media and
then
maintaining them at ccirifluency for 3 d (Marte et al. (1995) Mol. Endocrinol.
9: 14-23).
These competent cultures were then induced to terminally differentiate and
produce ~i-casein
by incubation for 1-6 d in DIP induction medium (RPMI-1640, 5 pg/ml ovine
prolactin, 5
~g/ml insulin, and 1 ~t.M dexamethasone).
Northern blot analysis of cell and tissue RNA samples
RNA samples included commercial blots of polyA-RNA from 7d to 17d
mouse embryos (Clontech) and total RNA extracted from HC 11 cell cultures,
excised mouse
(BALB/c) and rat (Sprague Dawley) inguinal mammary glands (virgin, pregnant,
lactating,
and involuting), and excised rat ventral prostate glands (pre- or post-
castration). Extractions
of total RNA were performed on snap frozen (liquid nitrogen) cell pellets or
excised glands
(Biellce et al. (1997) Cell Death and Differentiation 4: 114-124), using
either the
guanidinium isothiocyanate or Trizol (Gibco BRL) methods. When indicated,
polyA-
enriched RNA from mammary or prostate tissue was prepared using oligo dT-
cellulose
(Boehringer Mannheim). Either 10 IZg of total RNA or S p.g poly(A)-enriched
RNA/sample
were electrophoresed into 1 % agarose gels and transferred onto either nylon
(Zeta-probe,
BioRad) or nitrocellulose filters which were then LJV crosslinked using a
Strataiinker 1800
(Stratagene). After ethidium bromide or acridine orange staining to quantitate
transfer of
185 and 28S RNA, filters were hybridized with a randomly primed and 32aP-dATP
labelled
300 by cDNA fragment from the N-terminal mESX coding region, and given final
washes at
65° C in 0.2x SSC prior to autoradiography.
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ESX promoter activation in transient transfection assay
Luciferase (luc) reporter constructs (in pGL2-Basic Vector; Promega)
containing either 0.4 kb (-349 by to +61 bp) of mESX proximal promoter having
>80%
homology to hESX (mESX-luc) or 1.1 kb (-349 by to +704 bp) of proximal
promoter with
additional 5' untranslated sequence up to the ATG initiation codon (mESXL-
luc), were
constructed by PCR amplification from the marine genomic clone. Transient
transfection of
mESX-luc reporter (1 p.g DNA) in 6 pl of Lipofectamine (Gibco BRL) in serum-
free
medium (SFM) was performed into replicate tissue culture wells containing 60%
confluent
(1x145) cells. After 5 h, the Lipofectamine containing media was replaced with
SFM and
12 h later cell cultures were induced with 10% serum-containing media + grog
factor
(HRG or EGF) at the indicated concentrations. The transiently transfected cell
cultures were
then harvested at 0- 24 h following serum + growth factor induction, extracts
prepared and
luciferase activity measured as recommended by the vendor (Promega).
Results.
We isolated a 7.8 kb mESX genomic clone containing 4.9 kb of sequence
specifying 9 exons (exons 2-9, coding) and 8 introns, consistent with the
recently described
genomic structure of mESX (Tymms et al. (1997) Oncogene 15: 2449-2462).
Alignment
of this genomic sequence with that determined from the 1.1 kb cDNA clone
allowed us to
compare the primary structures of marine and human ESX as shown in Figure 10.
This
comparison of the primary structures reveals 87% amino acid identity, and also
maps the 7
exon boundaries within the encoded 371 amino acids of ESX.
Comparing the 2.9 kb of mESX promoter-containing sequence with that of a
formerly cloned 1.5 kb hESX promoter-containing genomic fragment (Chang et al.
{1997)
Oncogene 14: 1617-1622), and aligning both with reference to exon 1 and a
previously
determined hESX 5'UTR sequence (Oettgen et al. (1997) Mol. Cell Biol. 17: 4419-
4433),
showed <SO% by homology between the most upstream genomic sequences (-I500 by
to -
350 bp). In contrast, the proximal ESX promoter regions {-350 by to +50 bp)
showed 83%
homology at the nucleotide level between mouse and human genes, demonstrated
in Figure
11. The notable features in this proximal promoter region include conservation
of 6 different
consensus response elements (Ets, AP-2, SP 1/GC box, USF, Oct, NF-xB), a CCAAT
box at
-75 bp, and a putative pyrinnidine-rich initiator element (Inr) capable of
specifying transcript
initiation from the TATA-less marine promoter.
To verify that this homologous region of the proximal promoter can confer
growth factor induced transcriptional upregulation of mESX, as is known to
occur in hESX
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overexpressing breast cancer cells (Chang et al. (1997) Oncogene 14: 1617-
1622), the
activities of two different mESX promoter-reporter constructs (0.4 kb mESX-luc
and 1.1 kb
mESXL-luc) were assessed by transient transfection into cultured cells
expressing negligible
(IVIH3T3), low (MCF-7, MDA-435) or high (SKBr3) levels of endogenous ESX
(Id.).
Since no significant differences in promoter activity were observed between
the 1.1 kb
mESXL-luc and the 0.4 kb mESX-luc constructs, the smaller mESX-luc construct
was used
for all consequent experiments. The negligible and low ESX expressing cell
lines
consistently showed minimal reporter activity unresponsive to culture
stimulation by serum
+ growth factors (N»i3T3, MDA-4S3) or estradiol (MCF-7). In contrast, mouse
rlIFi3T3
cells engineered to overexpress human ErbB receptor pairs (ErbB2 + ErbB 1=
NE2/1 cells;
ErbB2 + ErbB3 = NE2/3 cells; ErbB2 + ErbB4 = NE3/4 cells) and with
intracellular
signaling upon appropriate ErbB ligand stimulation, showed ligand inducible
increases in
mESX promoter activity in the presence of serum. NE2/1 cells produced mESX-luc
reporter
upregulation in response to EGF while NE2/3 and NE2/4 cells responded
similarly to HRG
(data not shown), demonstrating the functionality of this ectopic mESX
promoter within
mouse cells activated by human ErbB receptors.
SKBr3 cells, which overexpress ErbB2 and also moderately express ErbBl
and ErbB3 receptors, were used to study mESX promoter induction since they
were known
to produce an immediate increase in endogenous ESX transcripts following
culture exposure
to HRG (5).. Treatment of SKBr3 for various intervals (0-24 h) produced serum
and growth
factor (HIZG, EGF) inducible increases in mESX-luc reporter activity. Serum
supplementation alone produced a 3- to 4-fold maximal induction of promoter
activity which
peaked within 8 h of treatment and then declined to near serum-free basal
promoter activity
by 24 h. When these SKBr3 cells were treated with 1 nM HRG in addition to
serum-
supplementation, a 7-fold peak induction over basal promoter activity was
observed at 8 h,
and promoter activity was still elevated nearly 4-fold over basal levels 24 h
after treatment.
HRG concentrations from 0.1 nM to 2 nM produced comparable enhancements in
mESX
promoter activity after 8 h, ranging from 2- to 3-fold over the peak activity
produced by
serum alone. At this same time point, concentrations of EGF up to 4 nM (in
serum-
supplemented media) also enhanced mESX promoter activity to a similar
(although slightly
lessor) degree as HRG (Figure 3B). In the absence of serum, neither HRG nor
EGF
produced any significant mESX promoter induction. Insulin (5 ~g/ml + serum-
supplementation) had no significant impact on mESX promoter activity in SKBr3
cells.
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Epithelial-specific ESX mRNA expression has been shown for various mouse
tissues after fetal day 17, but not during earlier embryonic development or
during adult
mouse mammary gland differentiation (Tymms et al. (1997) Oncogene 15: 2449-
2462). As
shown on the Figure 4 Northern blot, mouse embryos exhibit progressive
induction of a 2.2
kb ESX transcript after fetal day 7 with 17 day transcript levels about 10-
fold higher than
those of 11 day old embryos, which is consistent with the earliest onset of
epithelial
differentiation and progressive fetal growth of epithelial organs and tissues.
Of interest,
prior.to day 17 embryos show no detectable evidence of the alternatively
spliced larger ESX
transcript (3.8- 4.1 kb) noted in later stage fetal and adult organs and
malignant tissues
(Chang et al. (1997) Oncogene 14: 1617-1622; Oettgen et al. (1997) Mol. Cell.
Biol. 17:
4419-4433; Tymms et al. (1997) Oncogene 15: 2449-2462; Andreoli et al. (1997)
Nucleic
Acid Res. 25: 4287-4295).
Postembryondc mammary gland expression of ESX was evaluated in 3
separate experiments where RNA was isolated from mouse glands taken at various
stages of
differentiation including that of virgin, pregnant, lactating, and involuting
mammary glands.
In general, a basal level of ESX expression was seen in virgin and first-
pregnancy glands,
which declined to undetectable levels after 2-3 days of lactation, and. then
increased to
maximal levels following weaning and involution. In a representative stage-
specific
Northern blot profile of ESX expression RNA samples from 8-12 day involuting
glands
revealed persistently high ESX expression. These later time points are beyond
the active
phases of mammary gland involution and after most of the molecular and
histologic
correlates of apoptosis and tissue remodelling have already peaked (Bielke et
al. (1997) Cell
Death and D~erentiation 4: 114-124; Strange et al. (1992) Development 115: 49-
58; Wilde
et al. (1997) Mammary apoptosis: physiological regulation and molecular
determinants. In
Biological Signalling and the Mammary Gland, Wilde CJ, Peaker M and Taylor E,
eds.), pp
103-114, Hannah Research Institute, Ayr, Scotland). A fully regressed mouse
mammary
gland resected 8 weeks after weaning also showed maximal ESX expression
comparable to
peak transcript levels observed within the first 12 days of involution.
A panel of rat mammary gland RNA samples was also probed and confirmed
this stage-specific profile of ESX expression {Figure 6). Rat mammary ESX
expression was
basal during pregnancy, undetectable during lactation, and showed re-induction
to maximal
levels within 3 days of weaning and involution. Given the fact that normal
prostate
expresses ESX {5) and that regressing prostatic tissue shows morphological and
biochemical features similar to involuting mammary tissue (14), we looked for
changes in
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ESX expression during castration-induced involution of the adult rat ventral
prostate. As
with the rodent mammary tissue, rat prostate expression of ESX appears highest
during
glandular involution.
Detailed studies in stratified epithelium have shown that ESX expression is
restricted to the most terminally differentiated epidermal keratinocytes
(Oettgen et al.
(1997) Mol. Cell. Biol. 17: 4419-4433; Andreoli et al. (1997) Nucleic Acid
Res. 25: 4287-
4295). Since ESX transcripts decline to undetectable levels during lactation,
when the
mammary gland is composed of fully differentiated secretory epithelium, we
tried to
simulate this in vivo observation using cultured HC 11 cells which can be
induced into
lactogenic competency on exposure to growth factors, and then hormonally
stimulated to
differentiate and produce the milk protein, ~i-casein (Ball et al. (1988) EMBO
J. 7: 2089-
2095; Marte et al. (1995) Mol. Endocrinol. 9: 14-23). Proliferating HC11 cells
express
basal levels of ESX until they reach confluence and a state of lactogenic
competence (2-3 d
after culture confluence), at which point ESX expression increases
dramatically. Upon
growth factor (HRG or EGF) withdrawal and administration of lactogenic
hormones (DIP
induction medium), these competent and terminally differentiating cells
express increasing
amounts of (3-casein while ESX transcript levels fall concurrently to basal
levels.
Discussion.
The >30 known metazoan members of the Ets family of transcription factors
are recognized for their roles in embryonic development and tissue maturation
where they
direct stage-specific and tissue-restricted programs of gene expression,
targeted by a highly
conserved ~85 amino acid Ets DNA binding domain {Wasylyk and Nordheim (1997)
Ets
transcription factors: partners in the integration of signal responses. In
Transcription
Factors in Eukaryotes (Papavassiliou AG, ed.) pp. 253-286, Springer-Verlag
GmbH & Co.
KG., Heidelberg, Germany; Hromas Klemsz (1994) Int. J. Hematol. 59: 257-265;
Macleod
et al. (1992) Trends Biochem. Sci. 17: 251-256). As a new member of this
family, the 371
amino acid ESX transactivator possesses a typical Ets DNA binding domain
located in its C-
terminal region (Chang et al. (1997) Oncogene 14: 1617-1622; Oettgen et al.
(I997) Mol.
Cell. Biol. 17: 4419-4433; Tymms et al. (1997) Oncogene 15: 2449-2462;
Andreoli et al.
(1997) Nucleic Acid Res. 25: 4287-4295). Outside of this conserved DNA binding
domain,
ESX contains several other structural motifs not found in other Ets proteins,
a situation
thought to result from >500 million years of evolutionary recombination and
exon shuffling
(Id). The present findings support a domain-based modular structure for ESX as
shown by
the overall high degree of amino acid sequence homology (87% identity) between
mouse
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and human ESX, and the fact that ail putative domains within ESX are encoded
by only one
or two exons (Figure 1). In addition to the exon 8- and 9-encoded Ets DNA
binding domain,
these other structural modules include the exon 3-encoded Pointed (B-region)
domain, the
exon 5- and 6-encoded amphipathic helix and serine-rich box, and the exon 7-
encoded
bipartite nuclear targeting sequence {Chang et al. (1997) Oncogene 14: 1617-
1622).
Similar patterns of epithelial-specific ESX mRNA expression have been
noted in human and mouse tissues (Tymms et a1 (1997) Oncogene 15: 2449-2462),
suggesting common mechanisms of transcriptional control and promoter
regulation in both
the marine and human genes. Results from this study indicate that mouse and
human ESX
promoters share a high degree of homology (83% nucleotide identity) over a
relatively short
region extending ~0.4 kb upstream from the putative transcriptional start site
(+1) and just
beyond a conserved pair of Ets binding sites adjacent to an AP-2 consensus
response
element (Figure 2). The mESX promoter lacks the TATA box sequence present in
hESX (-
41 bp). However, both promoters have a typical CCAAT box located ~75 by
upstream of a
conserved pyrimidine-rich type Inr, making it likely that both mESX and hESX
function as
TATA-less promoters. No significant differences in promoter activity were
observed
between the 1.1 kb mESXL-luc and the 0.4 kb mESX-luc constructs, suggesting
that the 0.7
kb of 5' untranslated region (LTTR) between the Inr and ATG initiation colon
(beginning
exon 2) does not contain strong promoter regulatory elements.
In addition to conserved Ets and AP-2 response elements, both the marine
and human ESX proximal promoters share consensus elements for SP lIGC, USF,
Oct, and
NF-~B. Any combination of these response elements could account for the
development-
and tissue-specific profile of ESX expression common to both mouse and human
tissues
{Tymms et al. (I997) Oncogene 15: 2449-2462). These same response elements
likely
contribute to the differential upregulation of ESX promoter activity observed
between high
(SKBr3) and low (MCF-7,11~A-435) ESX expressing cell lines, and in SKBr3 and
ErbB
receptor overexpressing N1H3T3 cells (NE211, NE213, NE214) upon exposure to
serum and
growth factors (HRG, EGF). HRG, in particular, appears to synergistically
enhance ESX
proximal promoter activity 2- to 3-fold over the primary 3-fold stimulatory
effect of serum-
supplementation alone, consistent with our previous report of HRG induced
upregulation of
ESX mRNA in cultured SKBr3 cells (Chang et al. (1997) Oncogene 14: 1617-1622).
Additional studies are underway to determine the mechanisms and response
elements
mediating serum, HRG, and EGF induction of ESX promoter activity in these
cells. The
dramatic changes in ESX mRNA levels observed during normal mammary epithelial
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differentiation in vitro and in vivo may also be mediated by these same growth
factor
responsive promoter elements.
Mammary epithelium not only requires membrane activated ErbB receptor
family members for nozmal ductal development (Xie et al. (1997) Mol.
Endocrinology i 1:
1766-1781) but also the ErbB receptor ligands, EGF and HRG, that are potent in
vivo
stimulators of mammary epithelial proliferation and differentiation
(Vonderhaar (1987) J.
Cell. Physiol. 132: 581-584; Coleman et al. (1988) Dev. Biol. 127: 304-315;
Jones et al.
(1996) Cell Growth Di, fJ' 7: 1031-1038). The in vivo situation can be
simulated in vitro
using HC11 cell cultures, in which both HRG and EGF are mitogenic and either
can be used
to promote HC 11 lactogenic competency, a state of commitment essential for
subsequent
hormonal induction of terminal differentiation and milk expression (Ball et
al. (1988)
EMBO J. 7: 2089-2095; Marte et al. (1995) Mol. Endocrinol. 9: 14-23). The
mechanisms
associated with lactogenic competency are incompletely understood but are
partially
mediated by responses~to increased cell-cell interactions and to a reorganized
extracellular
matrix (Chammas et al. (1994) J. Cell Science 107: 1031-1040, Lochter and
Bissell (1997)
Mammary gland biology and the wisdom of extracellular matrix. In Biological
Signalling
and the Mammary Gland, Wilde CJ, Peaker M and Taylor E, eds.), pp 77-92,
Hannah
Research Institute, Ayr, Scotland.). Our present study demonsirates that in HC
11 cells,
growth factor promoted lactogenic competency is associated with a dramatic
upregulation in
ESX expression.
The changes in ESX expression associated with in vitro induction of HC11
terminal differentiation mimicked some but not all the features of ESX
transcript profiles
observed during in vivo mammary gland development. Pregnancy represents a
developmental stage in which epithelial cell proliferation and increasing
commitment to
terminal differentiation occur. Unlike the ESX upregulation observed when
proliferating
HC11 cells become lactogenically competent, glands from sexually mature virgin
and lst-
pregnancy mice showed no significant variation in their level of ESX
expression. However,
with in vivo terminal differentiation of mouse and rat mammary epithelium into
milk
producing lobilloalveolar units, there was a marked decline in ESX expression
(Figures 5
and 6) consistent with the fall in ESX transcript levels observed with
hormonal induction of
(3-casein expression in competent HC11 cells. This dramatic decline in ESX
expression
upon terminal differentiation of mammary epithelial cells in vitro and in vivo
is in unique
contrast to stratified epithelial systems where ESX expression is upregulated
and restricted
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to the most terminally differentiated forms of epidermal keratinocytes
(Oettgen et al. ( 1997)
Mol. Cell. Biol: 17: 4419-4433; Andreoli et al. (1997) Nucleic Acid Res. 25:
4287-4295).
ESX may now be added to a small but growing list of epithelial genes known
to be repressed during lactogenesis and then dramatically upregulated with
weaning and
initiation of mammary gland involution (Biellce et al. {1997) Cell Death and
Differentiation
4: 114-124; Strange et al. (1992) Development 115: 49-58; Wilde et al. (1997)
Mammary
apoptosis: physiological regulation and molecular determinants. In Biological
Signalling
and the Mammary Gland, Wilde CJ, Peaker M and Taylor E, eds.), pp 103-114,
Hannah
Research Institute, Ayr, Scotland, Marti et al. (1995) Cell Death and
Differentiation 2: 277-
283; Lund et al. (1996) Development 122: 181-193; Lochter and Bissell (1997)
Mammary
gland biology and the wisdom of extracellular matrix. In Biological Signalling
and the .
Mammary Gland, Wilde CJ, Peaker M and Taylor E, eds.), pp 77-92, Hannah
Research
Institute, Ayr, Scotland). Increasing ESX transcript levels are evident in the
involuting
mammary glands of both mouse and rat beginning as early as 1-2 days after
weaning. In the
rat gland this induction reaches peak levels within 4 days, while in the mouse
gland
expression is maximal by 8 days and remains high for at least 8 weeks, a point
when
apoptosis.and remodelling are completed and the gland is fully regressed. The
persistence
of high ESX transcript levels in fully regressed mammary glands suggests that
the
involutional induction of ESX is occurring in newly committed epithelial cells
that are
destined to survive both apoptotic and remodelling phases of involution.
Future in situ
analysis will address the possibility that ESX upregulation occurs in a
subpopulation of
partially committed and pluripotential ductal epithelium poised to regenerate
a fully
differentiated milk producing gland with the next cycle of pregnancy and
lactation.
Molecular markers that potentially distinguish virgin mammary epithelium from
partially or
terminally differentiated ductal-lobular elements are of both biological and
medical interest,
as they might ultimately serve to identify women whose breast tissue is more
or less
vulnerable to malignant transformation (Chepko and Smith (1997) Tissue & Cell
29: 239-
253; Russo and Russo (1997) Endocrine-Related Cancer 4: 7-21).
Like the mammary gland, prostatic tissue is subject to involutional changes
and its epithelium regresses in a reversible manner following surgical
castration or
pharmacologically induced androgen ablation. While the regressing ventral
prostate shows
morphological and biochemical features of epithelial apoptosis analogous to
those of
involuting mammary gland, unlike the latter it shows little evidence of tissue
remodelling
with slight induction of ECM proteinases of the matrix metallopmteinase
(M1V1P) and serine
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protease families (Bielke et al. (1997) Cell Death and DiJJ''erentiation 4:
114-124). In
contrast, the transition from lactating to involuting mammary gland is well
characterized by
two distinct phases of apoptosis, an early proteinase-independent phase and a
prominant
proteinase-dependent later stage (Land et al. (1996) Development 122: 181-
I93). In the
initial phase (days 1-3 after weaning), the gland's alveoli and supporting
mesenchyme
remain largely intact, but chromatin cleavage and DNA laddering become
detectable along
with induction of the same apoptosis-associated genes upregulated during
prostatic
involution (Bielke et al. (1997) Cell Death and Di, fj''erentiation 4: 114-
124.; Strange et al.
(1992) Development 115: 49-58; Wilde et al. (1997) Mammary apoptosis:
physiological
regulation and molecular determinants. In Biological Signalling and the
Mammary Gland,
Wilde CJ, Peaker M and Taylor E, eds.), pp 103-114, Hannah Research institute,
Ayr,
Scotland, Marti et al. (1995) Cell Death and Differentiation 2: 277-283; Lund
et al. (1996)
Development 122: 181-193;. Lochter and Bissell (1997) Mammary gland biology
and the
wisdom of extracellular matrix. In Biological Signalling and the Mammary
Gland, Wilde
CJ, Peaker M and Taylor E, eds.), pp 77-92, Hannah Research Institute, Ayr,
Scotland, Nishi
et al. (1996) Prostate 28: 139-152). During the second stage (days 3-10 after
weaning),.
massive.apoptotic cell loss (~50% of the gland's cellularity and >95% of all
alveolar
epithelium) results in collapse and dissolution of all milk producing glands,
necessitating a
much more extensive protease-mediated ECM remodelling process than that
required by the
involuting prostate. Despite these differences between involuting breast and
prostate glands,
ESX transcript levels increased in both in a similar manner. Maximal
upregulation of
prostatic ESX occurred within 2-4 days of hormonal ablation, concurrent with
increases in
other apoptosis-associated prostatic transcripts (e.g. sulphated giycoprotein-
2, tissue
transglutaminase, p53, DDC-4, TGF-ail, TGF-(3RI>] previously demonstrated in
these same
RNA samples (Bielke et al. (1997) Cell Death and D fferentiation 4: 114-124)
or by other
groups (Nishi et al. (1996) Prostate 28: 139-152).
While a number of ECM proteases are known to be transcriptionally
regulated by Ets factors (Higashino et al. (1995) Oncogene 10: 1461-1463;
D'Orazio et al.
(1997) Gene 201: 179-187), the early and comparable extent of ESX upregulation
observed
during involution of prostate and mammary glands and the persistance of
upregulated ESX
in fully regressed mammary glands suggest that this Ets family transactivator
may be
regulating other genes in addition to proteases in cells destined to survive
both the apoptotic
and remodelling phases of glandular involution. In this regard,
transglutaminase 3 and TGF-
/3RII are two of the few genes identified to date as being transcriptionally
upregulated by
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ESX (Oettgen et ah (1997) Mol. Cell. Biol. 17: 4419-4433; Andreoli et al.
(1997) Nucleic
Acid Res. 25: 4287-4295; Choi S-G et al. (1998) J. Biol. Chem. 273: 110-I 17).
The former
is closely related to tissue transglutaminase which, along with TGF-~3RII, are
upregulated in
concert with ESX during involution. Thus, our findings should not only
stimulate the
search for ESX regulated genes associated with involution and apoptosis, but
also provide
greater incentive to identify ECM and growth factor sensitive response
elements within the
ESX promoter that account for its transcriptional upregulation during prostate
and mammary
gland involution.
Example 7: Exon 4-encoded acidic domain in the epithelium-restricted Ets
Factor.
ESX, confers potent transactivatinQ capacity and binds to TATA-binding
Protein (TBP~
The Ets.gene family has a complex evolutionary history with many family
members known to regulate genetic programs essential for differentiation and
development
and some known for their involvement in human tumorigenesis. To understand the
biological properties associated with a recently described epithelium-
restricted Ets factor
ESX, an ~11 kb fragment from the 1 q32.2 genomically localized human gene was
cloned and
analyzed. Upstream of the ESX promoter region in this genomic fragment lies
the terminal
exon of a newly identified gene that encodes a ubiquitin-conjugating enzyme
variant, UEV-
1. Tissues expressing ESX produce a primary 2.2 kb transcript along with a 4.1
kb
secondary transcript arising by alternate poly(A) site selection and uniquely
recognized by a
genomic probe from the 3' terminal region of the 1 I kb clone. Endogenous
expression of
ESX results in a 42 kDa nuclear protein having 5 fold greater affinity for the
chromatin-
nuclear matrix compartment as compared to other endogenous transcription
factors like AP-
2 and the homologous Ets factor, ELF-1. Exon mapping of the modular structure
inferred
from ESX cDNA and construction of GAL4(DBD)-ESX expression constructs were
used to
identify a transactivating domain encoded by exon 4 having comparable potency
to the
acidic transactivation domain of the viral transcription factor, VP16. This
exon 4-encoded
31 amino acid domain in ESX was shown by mutation and deletion analysis to
possess a 13
residue acidic transactivation core which, based on modeling anal circular
dichroism
analysis, is predicted to form an amphipathic 0-helical secondary structure.
Using
recombinant GST-ESX (exon 4) fusion proteins in an in vitro pull-down assay,
this ESX
transactivation domain was shown to bind specifically to one component of the
general
transcription machinery, TATA-binding protein (TBP). Transient transfection
experiments
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confirmed the ability of this TBP-binding transacdvation domain in ESX to
squelch
heterologous promoters independent of any promoter binding as efficiently as
the
transactivation domain from VP 16.
Materials and Methods.
Cells lines, recombinant ESX protein, and anti-ESX polyclonal antibody
Human breast cancer cell lines SKBr3, MDA-468, MDA-231, ZR-75-1 and
the African green monkey kidney line, COS-7, were obtained from the American
Type
Culture Collection (Manassas, VA) and maintained as recommended. Recombinant
ESX
protein was prepared and purified as previously described (Chang et al. (1997)
Oncogene,
14: 1617-1622). To produce an affinity purified anti-ESX polyclonal, a
synthetic I7 amino
acid ESX peptide (N-terminal cysteine plus 16 C-terminal amino acids of ESX)
coupled to
KLH was injected into a New Zealand White SPF female rabbit (Animal Pharm
Services,
Inc). Total IgG was obtained from 8 week post-immunization rabbit serum using
a
commercial Protein A purification kit (Pierce). Antibody purification from the
total IgG was
performed using the antigenic 17 amino acid ESX peptide coupled to an Affi-Gel
10 matrix .
according to the manufacturer's recommendations (BioRad).
Northern and Western analyses.
For Northern blotting, total cell RNA (10 pg/sample lane) prepared from
freshly harvested cell the by guanidinium isothiocyanate method was
electrophoresed into
1 % agarose gels and transferred onto membranes that were then hybridized with
3zP-probes
labeled, washed and autoradiographed as previously described (Chang et al.
(1997)
Oncogene, 14: 1617-1622, Example 6). The 5' HindIII probe was prepared from a
431 by
Hind>ZI-Pstl fragment, the 3' Hind)ZI probe prepared from a 349 by Scal-
HindllI fragment
and the ESX probe prepared from ESX cDNA. For Western blotting, whole-cell or
nuclear
extracts were boiled in sample loading buffer (1% SDS, 20% glycerol, 100 mM
DTT, 50
mM Tris pH 6.8) and then eiectrophoresed into 9% sodium dodecyl sulphate
polyacrylamide
gels (SDS-PAGE) and transferred onto membranes (Immobilon-P, Millipore) by
electroblotting at zoom temperature (Hoeffer, 250 mA, 1.5 h). Protein-bound
membranes
were blocked in PBS containing 5% dried milk and 0.1% Tween 20, incubated in
the same
buffer with anti-ESX polyclonal (above) or commercially obtained antibodies
that recognize
ELF-1, p65NF-OB, SP1, or AP-2 (Santa Cruz Biotechnology), and then incubated
with a
secondary IgG antibody conjugated to horseradish peroxidase (Sigma). Protein
bands were
visualized using the SuperSign.al chemiluminescent substrate (Pierce).
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Isolation and sequencing of the 11 kb ESX~enomic fragment.
The ESX containing 11 kb HindIII fragment identified in Southern blots was
isolated from HindIII digestion fragments of the P 1 clone originally used to
chromosomally
rnap ESX (Chang et al. (1997) Oncogene, 14: 1617-1622) and was subcloned into
the
HindIll site of pcDNAI/Amp (Invitrogen). All DNA manipulations were carried
out by
using standard methods (Ausubel et al. (1989) Current Protocols in Molecular
Biology. John
Wiley & Sons: New York; Sambrook, et al. (1989) Molecular Cloning -A
Laboratory
Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,
New
York). The 11 kb HindIII clone was fiuther analysed by subcloning genomic
BamHI, BgIII,
and StuI fi~agments into pBluescript (Stratagene), and the resulting plasmid
clones were
sequenced with primers derived from the human ESX cDNA (Chang et al. (1997)
supra.).
The 5'-upstream region was sequenced by primer walking. All genomic DNA
fragments
were sequenced from both directions by automated DNA sequencing with an ABI
sequences.
GAL4 cbnstructs encoding deleted and mutated ESX fusion proteins
All GAL4(DBD)-ESX chimeras were constructed by cloning a set of deletion
ESX PCR products in frame with the DNA-binding domain (aa 1-147) of GAL4 into
a pM .
vector (Clontech) for the expression of ESX fusion proteins in transiently
transfected SKBr3,
MCF-7 and COS-7 cells. Since the GAL4(DBD)-ESX chimeras produced relatively
similar
reporter gene results in each of the 3 cell lines, only the SKBr3 data is
shown for the
mapping of ESX's transactivation. The plasmid pM contains the DNA-binding
domain
(DBD) of the yeast GAL4 protein driven by the SV40 early promoter. The
GAL4(DBD)-
ESX full-length fusion constructs, and all the N- and C-terminal deletion
constructs of ESX
were constructed by PCR amplification of the appropriate fragment of hESX
derived from
the plasmid, pcDNAI/Amp-ESX (Chang et al. (1997) supra.), using Pfu polymerise
(Stratagene). PCR primers containing an EcoRl site (5' primer) and HindllI
site (3' primer)
were used to create in frame PCR fragments for cloning between the EcoRI and
Hind)II sites
of pM to produce plasmids encoding GAl:,4(DBD)-ESX(1-30), GAIr4(DBD)-ESX(1-
63),
GAL4(DBD)-ESX(1-103), GAL4(DBD)-ESX(1-128), and GAL,4(DBD)-ESX(1-156),
GAL4(DBD)-ESX(1-268), GAIr4(DBD)-ESX(55-103), GALA(DBD)-ESX(55-128),
GAL4(DBD)-ESX(55-156), GAlr4(DBD)-ESX(55-268), GAL4(DBD)-ESX(104-15~,
GA1:.4(DBD)-ESX(104-159), GAL,4(DBD)-ESX(104-199), GAL4(DBD)-ESX(104-229),
GALA(DBD)-ESX(129-156), GAi~t(DBD)-ESX(129-159), GALA(DBD)-ESX(129-199),
GAI,4(DBD)-ESX(129-229) and GAL4(DBD)-ESX(157-268). All PCR product inserts
were verfied by restriction mapping and sequencing. The luciferase reporter
gene construct
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pG5EIbLUC (Chang and Gralla (1993) Mol. Cell. Biol., 13: 7469-7475) was used
to
determine the transactivation activities of GAL4(DBD)-ESX fusion constructs.
pG5EIbLUC
is a reporter plasmid with five GAL4 response elements, an inverted CCAAT box,
and a
TATA element controlling the luciferase (LUC) reporter gene. The internal
control pkasmid
pCH110 (Pharmacia) was used to monitor transfection efficiency.
All GAL4(DBD)-ESX(129-159) fusion protein point mutations were
introduced by a two-step PCR protocol using two complementary mutagenic
primers, two
flanking primers and Pfu polymerase as previously described (Ausubel et al.
(1989) supra.).
Briefly, a first PCR was performed with a set of one mutagenic primer plus one
flanking
primer. The two products from this first PCR reaction were gel-purified and
used as
template in a second PCR reaction employing only the flanking primers.
Sequences for
these mutagenic primers are available upon request. The integrity of akk
fusion constructs
was confirmed by DNA sequencing.
Transient transfection assays.
All plasmids used for transfection assays were prepared using plasmid Maxi
Kits from Qiagen. Transient transfection experiments were carried out with
Lipofectamine
reagent (Gibco BRL) according to the manufacturer's recommendations. Briefly,
subconfluent cells in 60 mm diameter plates were transfected with a total of 4
pg DNA
consisting of 0.5 wg of reporter plasmid, 2.5 pg of expression plasmid and 1
~g of the
internal control plasmid. The composition of transfected piasmids are
described in each
figure legend. Following transfection (48 h), cells were washed twice with PBS
and
harvested. Cell extracts were prepared and luciferase assays were carried out
using a
luciferase assay kit (Promega) and quantitated using a Turner Designs
luminometer (model
TD-20e). Transfection efficiency was monitored by measuring the f3-
galactosidase activity
from a co-transfected pCH110 pkasmid (Pharmacia). For each experiment, at
keast three
independent transfections were performed and results are dispkayed as the mean
plus SEM
luciferase acitivity in relative light units. Additionally, the expression
level for all
GAL4(DBD)-ESX fusion constructs was verified by Western blotting of lysates
from
transfected cells probed with a monoclonal antibody against the GAL4 DNA
binding domain
(Santa Cruz Biotechnology):
GST pull-down assays.
Plasmid pGEX-6P-1-ESX(129-159), which encodes GST-exon 4 (amino
acids 129-159) and plasmid pGEX-6P-1-ESX(129-159)L143P which encodes GST-exon
4
with the leucine to proline substitution at position 143 were constructed by
cloning PCR-
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amplified EcoRI XhoI fi~agment containing residues 129 to 159 from pM-hESX(129-
159)
and pM-hESX(129-159)L143P respectively into pGEX-6P-1 vector (Pharmacia)
cleaved at
the EcoRI and XhoI sites. Glutathione S-transfease (GST), GST-exon 4 and GST-
exon
4(L143P) proteins were expressed in E. coli BL21 (Novagen) and purified by
using the Bulk
GST Purification Module in accordance with the manufacturer's protocol
(Pharmacia). The
quality and quantity of the proteins were verified by SDS-PAGE followed by
Commassie
staining. Following rebinding of purified GST, GST-exon 4 and GST-exon
4(L143P) to
glutathione-Sepharose beads, binding reactions with commerically available TBP
(Promega)
and TFIIB (Promega) were performed in binding buffer (130 mM NaCI, 50 mM Tris
pH 7.5,
1 mM DTT, 0.1% NP-40) at 4°C. The glutathione GST, GST-exon 4 and GST-
exon
4(L143P) beads were washed extensively in binding buffer followed by elution
in binding
buffer adjusted tol M NaCI for TF1IB or successive elutions at 0.5 M, 0.8 M,
1.3 M and 2.0
M NaCI for TBP. Aliquotes.of the NaCI eluates were subjected to SDS-PAGE,
electrophoretically transferred to nitrocellulose membranes, probed with an
antibody to
either TBP (Santa Cruze Biotechnology) or TFIIB (Promega) and analyzed for
immunoreactivity with an enhanced-chemiluminescence detection system (Pierce).
Circular dichroism spectroscopy.
A 25 amino acid peptide from exon 4 of ESX, 131-
SSSDELSWIIELLEKDGMAFQEALD-155 (SEQ ID NO: ~, was synthesized on an
automated synthesizer and purified by high-performance liquid chromatography.
Secondary
structural predictions were carried out using the Chou-Fasman algorithm (Chou
and Fasman,
1978). Solutions of the peptide were prepared for circular dichroism (CD)
measurements in
10 mM Tris HCI, pH 7.4 mixed with 0-SO% methanol in increments of 10%; the
final
peptide concentration was 0.1 mg/ml. Samples were studied at room temperature
in a
circular quartz cell of 1 mm pathlength. Data were recorded over the range 192-
260 nm in a
Jasco model 720 spectropolarimeter (Jasco Inc., Euston, MD), continuously
flushed with dry
nitrogen. CD readings in millidegrees were converted to mean residue
ellipticity [Q]r in
degrees cm2 dmol'~, using a mean residue mass of 113 Da. The a-helix content
was
estimated by comparison with a reference set of 33 proteins using the variable
selection
method (VARSLC) (Johnson (1990) Proteins, 7, 205-214).
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Results.
Organization of the ESX gene locus: exon-intros structure and putative
UEV gene upstream of the ESX promoter.
Cloning of the ESX gene locus was undertaken to further our goal of
understanding the functional domains of the ESX protein. Southern blot
examination of
normal as well as several human breast cancer cell lines with 5' and 3' ESX
cDNA probes
identified a single 11 kb HindIII band. This 11 kb fragment was isolated,
subcloned and
sequenced from the HindIII digestion fragments of the PI clone initially used
to localize the
ESX gene to 1q32 (Chang et al. (1997) supra.). A computer search of this
genomic
sequence (submitted to GenBank) for known ESX cDNA sequences localized the
primary
2.2 kb ESX transcript (Andreoli et a1 (1997) Nucleic Acids Res., 25, 4287-
4295, Chang et
al. (1997) supra., Choi et al. (1998) J. Biol. Chem., 273: 110-117; Oettgen et
al. (1997)
Mot. Cell. Biol., 17: 4419-4433) to 9 exons spanning ~5 kb of the genomic DNA,
with exons
8 and 9 coding for the C-terminally located Ets DNA binding domain (Figure
12A). The
exonic sequences are identical to the published human cDNA sequence (GenBank
Accession
No. U66894). The exon-intros splice sites are in good agreement with splice
site consensus
sequences (Table 1), and agree precisely in their positioning to those
obtained for the mouse
homolog of ESX (Example 6, Tyrtuns et al. (1997) Oncogene, 15: 2449-2462).
Figure 12B
compares the predicted ESX amino acid sequence and location of exon-intros
junctions
within the Ets DNA-binding domain to that of other Ets factors whose genornic
organization
are published. Both amino acid identities and exon-intros junction alignments
confirm the
well established evolutionary conservation of ETS-1 subfamily members (ETS-1,
ETS-2,
PNT) across species ranging from Drosophita to human (Klambt C. (1993)
Development,
117: 163-176; Watson et al. (1988) Virology, 164: 99-105). In contrast to this
situation in
the ETS-1 subfamily, ESX and EltF demonstrate only 43% amino acid identity
within the
Ets domain but share a single and identically placed exon-intros boundary that
terminates
the a3 helix of this domain (Change et al. (1997) supra.; Figure 12B).
As described in Example 6, the proximal 350 by of the ESX promoter is
>80% identical to that of the corresponding mouse promoter region, while the
mouse and
human promoter sequence between -1500 by and -350 by share <50% identity. Two
longer
Alu elements are located upstream of the region of diverging human-mouse
promoter
homology while two shorter Alu elements are located within introns 2 and 8.
Incidentally
noted were two small CpG islands of 100 by size near exons 1 and 7 (Figure
12A).
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Table 1. Sizes of exons and introns and sequences at exon/intron junctions of
the human
ESX gene.
Exon Sequence at exon/intron Intron
junctions
no.size 5' splice donor 3' splice acceptorno. sizeas interrupted
~P) ~P)
1 112 ACTCCG/gtagga.... ccacag/GTAGCC 1 422
2 171 GTACAG/gtgggt . ttgcag/AGAAGG 2 657 Glu-55
. . .
3 222 ACCTCA/gtgagt . tgtcag/CTTCCA 3 165 Thr-129
. . .
4 93 CCTTTG/gtgaga.... tcccag/ACCAGG 4 203 Asp-160
120 CCGCAG/gtgaga . ccccag/GGACTG 5 187 Gly-200
. . .
6 90 CCAGCG/gtgagt . ccacag/ATGGTT 6 145 Asp-230
. . .
7 117 AGCACG/gtgagc.... ttgcag/CGCCCA 7 530 Ala-269
8 196 CATGAG/gtgagc.... accccag/GTACTA 8 869 Arg-334
9 797 9
Surprisingly, a BLAST search of ESTs compiled by TIGR identified a 1.3 kb-
S tentative human contig (THC213038) with complete identity in its 3' terminal
sequence to a
589 by region ~4 kb upstream of the ESX transcription initiation site (Figure
12A),
suggesting the presence of a terminal exon from another gene in this upstream
region. As
well, a 5'-TG-3' dinucieotide in the genomic sequence immediately upstream of
the
homology break with THC213038 suggested the presence of a splice acceptor site
at this
location. A BLAST search of GenBank revealed that the THC213038 sequence was
identical to the terminal 3' untranslated region of a recently described
ubiquitin-conjugating
E2 enzyme variant, UEV-1 (Sancho et al. (1998) Mol. Cell. Biol., 18: 576-589).
Northern
analysis using a genomic probe from this 5' HindllI region overlapping with
THC213038
demonstrated a widespread pattern of expression for this putative UEV gene in
contrast to
the known epithelium-restricted pattern of ESX expression. While this Northern
blot result
is consistent with the published pattern of UEV-1 expression (Sancho et al.
(1998) supra.),
the reported chromosome location of UEV-1 on 20q13.2 is not consistent with
the well
documented mapping of ESX to 1q32.2 (Chang et al. (1997) supra.; Oettgen et
al. (1997)
Genomics, 45: 456-457; Tymms et al. (1997) Oncogene, 15, 2449-2462), although
one
reported LTEV-1 cDNA clone (MAC4) did identify a lq locus when mapped by
fluorescence
in situ hybridization (Sancho et al. (1998) supra.).
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Polv(Al site selection produces either 2.2 kb or 41 kb ESX transcripts
A BLAST search for ESTs in the 900 by region between the termination of
the primary 2.2 kb ESX transcript and the downstream 3' HindIII site
identified two contigs,
THC203540 and THC209689 (Figure 12A). These overlap by 20 by which is
insufficient to
meet TIGR's 40 by overlap criteria for contiguousness but are otherwise
identical to the
genomic sequence from this region. Northern blots probed with a 349 by
fragment from the
3' HindllI region identified a 4.1 kb transcript identical in position and
expression pattern as
that obtained with cDNA probes from the 2.2 kb ESX transcript. THC203540
extends 300
by beyond the 3' HindIII site and terminates with the variant and weak
polyadenylation
signals ATTAAA and GATAAA, respectively. Together, therefore, THC203540 and
THC209689 contain 1.2 kb of sequence that undoubtedly comprise the 3'
untranslated
portion of the larger 4.1 kb ESX transcript previously reported (Andreoli et
al. (1997)
Nucleic Acids Res., 25, 4287-4.295; Chang et al. (1997) supra.; Example 6;
Oettgen et al.
(1997) Mol. Cell. Biol.; 17: 4419-4433; Tymms et al. (1997) Oncogene, 15, 2449-
2462) and
now thought to arise by alternate poly(A) site selection. Additionally, all
the clones
contributing to these two THCs were derived from epithelial sources,
consistent with the
reported, epithelial expression pattern of ESX. It is interesting to note that
a run of 12
consecutive A's in the 3' untranslated region of the 4.1 kb ESX transcript is
apparently
responsible for the oligo dT priming that generated 3 of the 4 clones
contained in
THC209687 and possibly explains the 2.7 kb ESX cDNA described in an earlier
report
(Choi et al. (1998) J. Biol. Chem., 273: 110-1 I7).
Chromatin-nuclear matrix association by endogenous ESX erotein
Using an affinity purified rabbit polyclonal antibody directed against the 17
C-terminal amino acids of human ESX, Western analyses on whole cell extracts
from human
breast cancer cell lines expressing high (SICBr3, MDA-468), medium (ZR-75-1)
or low
(MDA-231) levels of the 2.2 kb ESX transcript showed that these cells express
proportional
amounts of a ~42 kDa ESX protein, although this polyclonal also recognizes
several other
proteins whose epitopes were competed by the C-terminal ESX peptide. The
possibility was
explored that ESX might reside within a nuclear compartment since some
transcription
factors (e.g. Spl, p65NF-xB, steroid hormone receptors) are known to be
preferentially
retained in a high-salt resistant chromatin-pellet fraction of the nucleus
containing the
nuclear matrix (Raziuddin et al. (1997) J. Biol. Chem., 272: 15715-15720; Van
Wijnen et
aL (1993) Biochemistry, 32: 8397-8402). SKBr3 nuclei were used to produce NE
(nuclear
extract) and NP (nuclear pellet) fractions which were compared by Western
analyses for
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their relative abundance of ESX, Sp 1, p6SNF-xB, AP-2 and the ESX-homologous
Ets factor,
ELF-1. Cell nuclei were extracted in a standard Dignam buffer {.42 M NaCI)
with the
extract partitioned from the chromatin-nuclear matrix residue by high-speed
centrifugation
(NE fraction). Following removal of the Dignam extract, the chromatin-nuclear
matrix
S pellet was then completely solubilized by heating (80°C) in RIPA
buffer (NP fraction).
Multiple Western blots were prepared by loading gels with identical NE and NP
sample
volumes, and then blotted with, specific antibodies. As known components of
the nuclear
matrix, p6SNF-xB and Spl served as positive controls. Upon scanning
densitometry
measurement of their Western bands, p65NF-xB and Spl were found to have NP/NE
ratios
of 1:1.7 and 1:3, respectively, consistent with previous studies confirming
their general
propensity for retention by the chromatin-nuclear matrix (Raziuddin et al.
(1997) supra.;
Van Wijnen et al.(1993) supra.). In contrast, AP-2 and ELF-1 were found to
have NP/NE
ratios of i :12 and 1:1 S, demonstrating their lack of chromatin-nuclear
matrix association.
As shown Western Blot's and indicated by an NP/NE ratio of 1:2.5, ESX appeared
to be
1 S preferentially retained in the chromatin-nuclear matrix fraction. Given
the considerable
homology (~SO% identity and 70% similarity) in amino acid sequences between
ESX and
ELF-1 in their Ets DNA-binding domains (Chang et al. (1997) supra:), the S-
fold greater
chromatin-nuclear matrix affinity of ESX relative to ELF-1 probably reflects
the strong
localizing function of another domain positioned outside of the ESX DNA-
binding domain.
ESX transactivating domain encoded by exon 4.
To look for ESX transactivating domains, a series of C-terminally deleted
ESX sequences were fused 3' to the DNA binding domain (DBD} of GAL4 and these
GAL4{DBD)-ESX fusion constructs were tested for their capacity to
transactivative a
GAL4(DBD) responsive reporter in transiently transfected SKBr3 cells. As shown
in Figure
2S 13, C-terminal deletions of ESX which preserved exon 4 (aa 129-1 S9)
demonstrated a
transactivating capacity comparable to the GAL4(DBD)-VP16 positive control,
whereas
deletions lacking exon 4 produced near background reporter activity. We had
initially
suggested that the serine-rich box of ESX (aa 189-229), encoded by exons S and
6 and
homologous to the poiyserine transactivating domain of the SOX4 gene, might
serve as the
ESX transactivating domain (Chang et a1 (1997) supra.). However, the results
shown in
Figure 13 indicate that the serine-rich box of ESX has no detectable
transactivating activity,
at least in the context of these GAL4(DBD) assays. The suppression from
optimal reporter
activity observed with those GAL4(DBD)-ESX fusion constructs containing the
highly basic
exon 7 (aa 230-268) domain might be explained by its putative HMG/-like A/T
hook
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(Andreoli et al. (1997) Nucleic Acids Res., 25, 4287-4295; Oettgen et al.
(1997) Mol. Cell.
Biol., 17: 4419-4433). This exon 7-encoded DNA-binding capacity could result
in
sequestration of the GAL4(DBD)-ESX fusion proteins into a chromatin-nuclear
matrix
compartment that would diminish its exposure to the co-transfected reporter
gene. To define
more precisely the ESX transactivating domain, GAI,4(DBD) fusion constructs
were
examined that contained only exon 3 (aa 55-128), exon 4 (aa 129-159), exons 3-
4 (aa 55-
159), exons 4-5 (aa 129-199), exons 4-6 (aa 129-229) and exons 4-7 (aa 129-
268). The
results shown in Figure 13 establish that ESX exon 4 autonomously encodes a
potent
transactivating domain of comparable strength to VP16. Exon 3, encoding the A-
region or
Pointed domain, exhibited no transactivating potential in agreement with
previous studies
among ETS-1 subfamily members possessing this conserved domain (Schneikert et
al.,
1992). Addition of exon 3, exon 5, or exons 5-6 to the exon 4 transactivation
domain
produced nearly equivalent activity as exon 4 alone, although addition of exon
7 attenuated
this activity. Finally, to'demonstrate the essential requirement of exon 4 for
transactivation
two fusion constructs lacking exon 4, GAL4(DBB)-exons 5-9 and GAIr4(DBD)-exons
5-7,
were tested and found to possess no significant transactivating capacity
{Figure 13).
Acidic core and candidate transactivati~ motifs in~ axon 4.
Activation domains are typically characterized by the nature of their amino
acid composition; thus, with 7 D or E residues out of 31 (22%), the axon 4
domain may be
classified as an acidic activation domain. The relevance of these acidic
residues to
transactivation was determined by alanine substitutions of these residues into
the
GAlr4{DBD)-axon 4 construct as either single or double mutations. ~ Figure 14A
demonstrates that while mutation of the two C-terminally located acidic
residues
(E152A/D155A) had no impact upon transactivation, mutations in the other
acidic pairs
D134A/E135A and E144A/D146A Beverly diminished transactivating capacity (to
<$%
control activity) while the single mutation E141A reduced reporter activity by
~60%.
Interestingly, the single D 134A mutation eliminated axon 4 transactivating
capacity while
mutation of the adjacent acidic residue (E135A) left the transactivating
capacity of axon 4
undiminished. Mutation of K145, the only basic amino acid within axon 4, to Q
had no
significant effect on axon 4 transactivation while the double mutation
KI45Q/D146A
reduced transactivating capacity to 20% of control {Figure 14A).
Interestingly, an N-terminal 6 amino acid element of axon 4 {129-TSSSED-
134) harbors two overlapping casein kinase II consensus sequences (S/TXXD/E).
Since
transactivation by Myb and PU.1 are known to be modulated by casein kinase II
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phosphorylation (Oelgeschl~ger et al. (1995) Mol. Cell. Biol., 15: 5966-5974;
Pongubala et
al. (1993) Science, 259: 1622-1625), the mutations S131R and S132A were
introduced into
the GAL4(DBD)-ESX (1-156) fusion construct to address the possibility that ESX
activation
is dependent upon serine phosphorylation within exon 4. As shown in Figure 13,
replacement of these exon 4 serines (S131R/S132A) had no significant impact on
transactivation relative to the unmutated GAL4(DBD)-ESX (1-156) fusion
control,
suggesting that serine phosphorylation is not involved in transactivation by
exon 4. Also of
interest, the motif 150-FXX~~ø-154 (X = any amino acid, ~ = any hydrophobic
amino acid)
present in the C-terminal region of exon 4 is a known recognition element for
TAFII31 that
is also critical for VP16 transactivation, suggesting that it generally
fimctions as a
recognition motif within acidic transactivators (Uesugi et al. (1997) Science,
277, 1310-
1313). Therefore, mutational analysis of this motif was performed to establish
its relevance
to exon 4 transactivating capacity. When incorporated into the GAL4-(DBD)-exon
4 (129-
159) fusion construct, neither the single FISOA mutation or the double
FISOA/L154A
I S mutations resulted in significant ( >20%) reduction in ESX transactivating
capacity (Figure
SB). As a positive control, inactivation of this motif by introduction of
F479A/L483A
double mutations into the GAL4(DBD)-VP16 fusion construct resulted in complete
loss of
VP16 transactivating capacity, as previously described (Id). Thus, despite the
presence of a
similar FXX~~ motif in the acidic activation domains of VP16 and ESX exon 4,
only the
VP16 domain appears to depend on this recognition element.
Since mutations in the two C-terminal acidic residues of ESX exon 4 as well
as within the C-terminally located FXXc~~ motif produced no significant loss
of
transactivation capacity, progressive C-terminal deletions in exon 4 were
undertaken to
delineate a minimal ESX transactivation domain. Stop codons were introduced at
amino
acid positions 158, 148 and 145 to generate GAL4 (DBD)-exon 4 fusion
constructs with
exon 4 peptide lengths of 27, 19 and 16 amino acids, respectively. The two
longer N-
terminal exon 4 constructs had reduced though still quite potent
transactivating capacities
(88% and 52% of control activity), while the 16 amino acid exon 4 fusion
construct (129-
144) with deleted D146 residue exhibited only 13% of control activity (Figure
14A). Lastly,.
a I3 amino acid GAL4(DBD)-exon 4 fusion construct (aa 134-147) with deleted N-
terminal
serines and possessing only the most essential of the exon 4 acidic residues
showed 45% of
control exon 4 activity, suggesting that much of the exon 4 transactivating
capacity results
from this acidic central core of 13 amino acids.
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a-helical structure of acidic core in ESX transactivatinE domain
While the ability of acidic domains to support transactivation has been well
established, elucidation of the structural basis for this transactivating
function remains
largely unclear (LJesugi et al. (1997) supra.). Previous structural studies of
the acidic
activation domains of GCN4 and p65NF-xB by circular dichroism (CD) and nuclear
magnetic resonance (NMR) spectroscopy revealed that these domains, while
unstructured in
aqueous solution at neutral pH, assumed respectively (3 sheet under acidic
conditions and a-
helical conformation in less polar solvents (Schmitz et al. (1994) J. Biol.
Chem., 269:
25613-2562011 Van et al. (1993) Cell, 72: 587-594). Based on protein modeling
algorithms,
the 13 amino acid acidic core of the ESX exon 4 transactivation domain, 134-
DELSWIIELLEKD-146 (SEQ ID NO: ~, is predicted to form an a-helical structure
(Chow
and Fasman. (1978) Ann. Rev. Biochem., 47: 251-276). A helical wheel
projection of these
13 residues suggests its amphipathic character, with hydrophilic residues
D134, 5137, E141,
E144 and K145 distributed on one face and hydrophobic residues L136, I139,
I140 and
L143, on the other (Figure 15A). To assess the impact of a helix destabilizing
mutation in
the middle of exon 4 on its transactivating capacity, L143 was mutated to the
helix-breaking
amino acid proline. As shown in Figure 145A, the L143P mutation was incapable
of
transactivation, suggesting the importance of this exon 4 helical structure on
its
transactivating function.
CD spectroscopy, which allows sensitive detection of secondary structure
elements in proteins and peptides, was used to probe the structure of the 25
amino acid exon
4 transactivation domain (aa 131 to 155). The spectral profile obtained for
the 25-residue
peptide in the presence of 50% methanol is shown in Figure 15B. In the absence
of
methanol the spectral minimum close to 200 nm shows the main secondary
structural feature
to be a random coil, although the signal approaching 222 nm indicates a small
degree of a-
helix. With increasing methanol concentration, however, there was a
strengthening of the a-
helical signal at 222 nm and a shift of the lower wavelength minimum towards
208 nm,
clearly indicating greater a-helical structure in the more hydrophobic
environment. This
observation that the a-helical content of the exon 4 domain depends on
hydrophobic
conditions suggests that hydrophobic interactions in the full-length ESX
protein serve to
stabilize the exon 4 helical structure and its transactivating function.
Binding of the ESX transactivating domain to TBP.
Transactivators like VP16 are known to enhance transcription by recruiting
one or more proteins into a preinitiation complex with RNA pol II, this
complex containing
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general transcription factors (GTFs) including the TATA-binding protein (TBP)
and assorted
TBP-associated factors (TAFs) {Chang and Gralla (1993) Mol. Cell. Biol., I3:
7469-7475;
Ptashne and Gann (1997) Nature, 386: S69-577; Pugh BF. (1997) Transcription
Factors in
Eukaryotes. Papavassiliou AG (ed), Springer-Verlag: Heidelberg, Germany, pp 37-
S0;
S Uesugi et al. (1997) Science, 277, 1310-1313). As well, at least one Ets
factor (ERM) is
known to recruit a TAF via its a-helical acidic activation domain (Defossez et
al. (1997)
Nucleic Acids Res., 2S: 44SS-4463). Therefore, to assess the ability of the a-
helical acidic
activation domain of ESX to bind and recruit such factors, we constructed a
GST-exon 4
fusion protein for in vitro pull-down assays. Unmodified GST and a GST-exon 4
fission
protein containing the helix-destabilizing L143P mutation were used as
negative controls.
Recombinant TBP or TFlIB were applied in binding buffer to glutathione-
Sepharose beads
pre-bound with equivalent amounts of either GST-exon 4, GST-exon 4 (L143P), or
GST.
Extensive washing was followed by elution with either 1 M NaCI for TF)ZB or.
successive
elutions with 0.8, 1.3 and 2.0 M NaCI for TBP. Equal portions of the eluates
were loaded
1S onto SDS acrylamide gels, electrophoresed, blotted onto membranes and
probed with
antibodies to detect TBP or TF11B in the eluates. TF1TB did not bind to
unmodified GST or
to either. of the GST-exon 4 fusion proteins. In contrast, TBP, which showed
no binding to
unmodified GST, bound quantitatively to the GST-exon 4 fusion protein.
Interestingly, the
mutation-bearing GST-exon 4(L143P) fusion protein retained some capacity to
bind TBP
although with substantially reduced affinity relative to the GST-exon 4 fusion
protein
(Figure 7). In this regard, the GST-exon 4(LI43P) protein with its proline
substitution and
otherwise unaltered exon 4 charge distribution, migrated 10- 1 S kDa slower on
SDS-PAGE
relative to the GST-exon 4 fusion protein indicating the disruptive influence
of proline on
the helical secondary structure of exon 4. Therefore, these GST pull-down
results support
2S the likelihood that TBP recruitment by exon 4 in vivo accounts for much of
the
transactivating capacity observed with GAL4(DBD)-exon 4.
Squelching mediated by the ESX transactivation domain.
Squelching occurs when a potent transactivator reduces the expression of a
co-transfected reporter plasmid in a transient transfection assay, with the
resulting decline in
reporter activity thought to be due to sequestration of GTFs and reduction in
their effective
concentration (Natesan et al. (1997) Nature, 390: 349-3S0). As ESX exon 4
exhibited a
potent in vivo transactivating capacity in the GAL4(DBD) assay and bound TBP
in the
context of an in vitro GST-exon 4 pull-down assay, we tested the ability of
the
GAL4(DBD)-exon 4 expression construct to influence two different promoters in
vivo, the
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SV40 early promoter and a synthetic promoter containing three tandem copies of
the HER2
promoter's Ets response element positioned just upstream of a mininnal
thymidine kinase (tk)
promoter (Chang et al. ( 1997) supra. ). As shown in Figure 16A, GAL4{DBD)-
exon 4
equally smpressed the activity of these reporters lacking any GAL4 DNA-binding
sites,
following co-transfection into SKBr3 (or COS-7) cells. The squelching of these
different
reporters by GAL4(DBD)-exon 4 amounted to a 4- 5 fold suppression of reporter
activity as
compared to co-transfection with the control GAL4(DBD) construct. In parallel
experiments, we observed a similar degree of reporter squelching by GAlr4(DBD)-
VP16
(Figure 16A). Interest in the squelching aspects of exon 4 arose following
initial co-
transfection studies using full-length ESX and a HER2 promoter-driven reporter
(Chang et
al. (1997) supra.). In subsequent experiments (Figure 16B), this Ets element-
containing
reporter was up-regulated ~4 fold by co-transfection of ESX into COS-7 cells
but was down-
regulated -y4 fold upon co-transfection into the breast cancer cell lines
SKBr3 or ZR-75-1.
As both SKBr3 and ZRf75-1 cells express relatively high levels of endogenous
ESX
transcripts and protein, we suspected that the exogenously introduced ESX
served to titrate
down TBP levels available for reporter transcription. To examine the potential
role of ESX
exon 4 in.mediating reporter suppression in the breast cancer cell. Iines, the
exon 4 double
mutation (E 144A/D 146A) previously shown to eliminate GAL4(DBD)-exon 4
transactivation was introduced into full-length ESX and compared to an exon 4
deleted
construct. Following co-transfection into SKBr3 cells, reporter activity was 4
fold higher
with both the exon 4 mutated (E 144A/D 146A) and the exon 4 deleted ESX
constructs as
compared to the wild type ESX expression construct (Figure 16B). Conversely,
in COS-7
cells where ESX expression had produced 4 fold upregulation of the Ets-driven
reporter,
both the exon 4 mutated and deleted constructs produced neither activation nor
suppression
of reporter activity relative to transfection of the empty pcDNAI vector
(Figure 16B). These
results support the explanation that squelching observed with ESX under
certain
experimental conditions is mediated by the same exon 4 elements that mediate
ESX
transactivation and binding to TBP.
Discussion.
A number of groups have identified ESX as a structurally unique epithelium-
restricted member of the Ets family of transcription factors (Andreoli et al.
(1997) Nucleic
Acids Res., 25, 4287-4.295; Chang et al. (1997) Oncogene, 14: 1617-1622; Choi
et al.
(1998) J. Biol. Chem., 273: 110-117; Oettgen et al. (1997a) Mol. Cell. Biol.,
17: 4419-4433;
Tymms et al. (1997) Oncogene, 15, 24.49-2462). To study transcriptional
expression and
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the functional domain organization of the predicted 42 kDa ESX protein, we
first isolated
and sequenced ~an 11 kb genomic fragment encompassing the human ESX gene
locus. The 9
exons encoding the previously defined human ESX cDNA sequence span ~S kb of
this
genomic locus, and all exon-intron boundaries are conserved between mouse and
human
S cDNA sequences (Example 6). The C-terminal DNA-binding Ets domain is encoded
by
exons 8 and 9 and the site of insertion of the intron separating these exons
is also precisely
conserved with that of another Ets factor, ERF (Figure 12B). This conservation
of exon-
intron boundary between ESX and ERF is somewhat surprising in that these two
Ets factors
share only 43% amino acid identity within their Ets domains; in contrast, ERF
and ETS-1
which share 6S% sequence identity exhibit different exonic structure in this
domain. As
genomic structures become available for other family members having more
similar
sequence homologies with ESX in the Ets domain, such as EHF (84% identity) and
E74/ELF-1 (49% identity), this apparent discrepancy in family lineage based on
conservation of Ets domain sequence vs. exonic structure will no doubt become
better
1 S understood.
One surprising finding that emerged from an analysis of the most upstream
ESX genomic sequence was the identification of a terminal exon from a new gene
encoding
a putative ubiquitin-conjugating enzyme variant, UEV-1. Located ~S kb upstream
of the
ESX transcription initiation site, this S89 by region mapped identically to
the terminal 3'
sequences of both the human contig, THC213038, and a recently identified human
cDNA
sequence encoding a cell cycle altering UEV-1 gene product (Sancho et al.
(1998) supra.).
Northern analysis using a genomic fragment overlapping this S89 by region
detected
transcripts migrating at 3.S kb and 1.9 kb, consistent with the prior
observation of a multiply
-spliced UEV-1 transcript (Id.). The more ubiquitously expressed UEV-1 gene is
separated
2S from the epithelium-specific ESX gene by two intervening Alu elements,
which may serve
to delineate the S' boundary of the functionally restricted ESX promoter.
Indeed, our recent
ESX promoter studies indicate that conserved elements responsive to both serum
and
epithelial growth factors (neuregulin and epidermal growth factor) reside
within 3S0 by of
the ESX transcription initiation site (Example 6). This proximal promoter
region maintains
a >80% sequence identity to the corresponding region in the mouse proximal
promoter but
the promoter sequence homology is rapidly lost (<SO% identity) from -3S0 by
upstream to -
1 S00 bp, suggesting a very compact ESX promoter (Example 6).
Northern blots of poly(A)-RNA from whole mouse embryos (7-17 days old)
have demonstrated a single 2.2 kb ESX transcript (Example 6); however,
alternate ESX
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transcripts of larger (3.8- 4.1 kb) and smaller (<2, kb) size have been noted
in some adult
organs (rodent and human) and malignant cell lines (Chang et al. (1997)
supra.; Pongubala
et al. (1993) Science, 259: 1622-1625; Tymms et al. (1997) Oncogene, 15, 2449-
2462). In
this regard, a BLAST EST search of the 0.9 kb genomic sequence lying between
the 3'
termination site of the 2.2 kb ESX transcript and the 3' HindBI site
identified two
overlapping contigs (THC209689 and THC203540), one of which extends 300 by
beyond
the HindlTI site and terminates in two adjacent poly(A) signals (Figure 12B).
Northern
analysis using a genomic probe from the 3' HindBI region identified only the
4.1 kb ESX
transcript, thus establishing that this transcript likely arises from
alternate poly(A) site
selection.
Of the several ESX structural domains initially postulated on the basis of
sequence homologies (Andreoli et al. (1997) supra.; Chang et al. (1997)
supra.; Oettgen et
al. (1997) supra.), the A/T hook homologous region adjacent to the Ets domain
is
provocative as a possible supplementary DNA recognition element that could
facilitate
chromatin association by minor groove binding within A/T-rich DNA stretches.
One report
has described weak ESX binding in vitro to a consensus A!T response element
(Andreoli et
al. (1997)-supra.). Moreover, the identification of an A/T hook motif from an
HMG1
architectural factor being chromosomally disrupted and chimerically fused to
acidic
transactivation domains in human lipomas underscores the potential biological
relevance of
. this A/T hook homology in ESX (Ashar et al. (1995) Cell, 82, 57-65). To look
for enhanced
chromatin association by ESX an antibody to the C-terminal portion of ESX was
used to
Western blot nuclear fractions from the ESX overexpressing cell line, SKBr3
(Chang et al.
( 1997) supra. ). Nuclei were fractionated into a standard Dignam-type extract
(NE) and a
fully solubilized chromatin-nuclear matrix fraction (NP). The potential
influence of the Ets
DNA-binding domain in directing preferential nuclear association of ESX was
assessed by
comparing NP/NE partitioning of ESX with another Ets factor, ELF-l, given its
sequence
homology in the Ets domain. Additionally, other SKBr3 expressed transcription
factors AP-
2, Spl, and NF-xB were also assessed as controls, since the latter two are
known to be
components of the nuclear matrix Raziuddin et al. (1997) supra.; Van Wijnen et
al. (1993)
supra.). AP-2 was found most highly associated with the NE fraction. In
contrast, ESXwas
found to have a ~5 fold greater affinity than ELF-1 for the NP fraction and
showed an
NP/NE ratio comparable to that of the known nuclear matrix components, Sp I
and NF-xB.
While not directly implicating the A/T hook region in ESX for this chromatin-
nuclear matrix
association, the much greater affinity of ESX over ELF-1 for this association
suggests that it
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may have been mediated by a functional element in ESX outside of the Ets
domain. Another
candidate element in ESX under study in this regard is the putative bipartite
nuclear
localization sequence (NLS) that partially overlaps the A/T hook region in
exon 7 (Bobbins
et al. ( 1991 ) Cell, 64: 615-623).
Since all initial reports documented the capacity of ESX to transactivate an
epithelial promoter harboring an appropriate Ets response element (Andreoli et
al. (1997)
Nucleic Acids Res., 25, 4287-4295; Chang et al. (1997) Oncogene, 14: 1617-
1622; Chen et
all (1998) Gene, 207: 209-218; Choi et al. (1998) J. Biol. Chem., 273: 110-
117; Tymms et
al. (1997) Oncogene, 15, 2449-2462; Oettgen et al. (1997) Genomics, 45: 456-
457), a series
of exon-based C-terminal deletions of ESX fused 3' to GAL4(DBD) were
constructed and
assayed for their transactivating capacity on a GAL4(DBD) responsive reporter.
Transactivation to the level established by a GAL4(DBD)-VP16 positive control
was
achieved with all deletion constructs that included exon 4, which is located
in the N-terminal
region of ESX and spans amino acids 129-159. All GAL~4(DBD)-ESX fusion
constructs
missing exon 4 exhibited virtually no transactivating capacity. One recent
study exploring
the transactivation capacity of ESX had shown that large N-terminal deletions
abolished this
activity;.however, without full knowledge of ESX genomic organization this
study was
limited in its ability to map the ESX transactivation domain (Choi ef al.
(1998) supra.).
To establish that the ESX domain encoded by exon 4 could function
autonomously as a transactivator, various contiguous exonic groupings were
fused to
GAL4(DBD) and examined for their transactivating capacity. In all cases
transactivation
was absolutely dependent upon exon 4, and maximal reporter activity was
achievable with
exon 4 alone or any combination of exons with exon 4, except those containing
the A/T hook
encoding exon 7 (aa 236-268). GAL4(DBD)-ESX fusion constructs containing both
exon 4
and exon 7 upreguiated the GAL4(DBD) responsive reporter but with aniy ~10% of
the
maximal transactivating capacity of all other exon 4 containing constructs,
consistent with an
earlier observation (Choi et al. (1998) supra.) and suggesting that exon 7
provides an
attenuating influence on ESA transactivation. While the mechanism by which
exon 7
attenuates the transactivating capacity of exon 4 in these transient
transfection assays
remains unclear, it is not affected by the presence of the Ets domain encoded
by exons 8 and
9 but may relate to the chromatin-nuclear matrix sequestering property of ESX
and/or the
presence of an A/T hook and candidate bipartite nuclear localization sequence
in exon 7,
effectively reducing the distribution of soluble transactivator seen by the co-
transfected
reporter gene.
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With 22% of its 31 amino acids encoding either aspartic acid (D) or glutamic
acid (E), the exon 4 domain of ESX may be classified as an acidic activation
domain
although it lacks sequence homology with other well studied acidic
transactivation domains
such as those found in the yeast transcription factor, GAL4, and the Herpes
Simplex
transcription factor, VP16 (Chang and Gralla (1993) supra.' Ptashne and Gann
(1997)
supra.; Uesugi et al. (1997) supra.). To define those amino acids essential
for ESX
transacdvation, an array of single and double alanine (A) substitutions within
exon 4 were
introduced into the GAL4(DBD)-ESX(129-159) fusion construct co-transfected
into SKBr3
cells. Results from these experiments were typical of similar mutagenesis
mapping studies
performed on VP 16-like acidic transactivators in so much as they demonstrated
the presence
of both dispersible and indispensible acidic residues without revealing the
underlying
mechanism of transactivation by such acidic domains (Uesugi et al. (1997)
supra.). Some
mutations (E135A and E152A/D155A) had virtually no impact on transactivating
capacity
relative to the unmodified exon 4 construct, one (E141A) reduced activity by
nearly 60%,
and others (D134A, D134A/E135A, and E144A/D146A) completely crippled the
activating
function of this domain.
Two provocative motifs are contained within exon 4 and their roles in the
transactivating mechanism used by this domain were also assessed by
mutagenesis. Two
overlapping casein kinase (CK) LI elements (129-TSSSED-134) in the N-terminus
of exon 4
are potential targets for phosphorylation that could affect ESX
transactivation, as has been
shown for the CK II element present in another Ets family member, PU.1
(Pongubala et al.
(i993) supra.). To test the relevance of these overlapping CK II sites, the
double mutation
S131R/S132A was introduced into exon 4 and compared to the unmodified
GAL4(DBD)-
ESX(129-159) control construct. Upon transfection into SKBr3 cells the mutated
construct
activated the GAL4(DBD) reporter as well as the unmodified control construct,
demonstrating that CK IT phosphorylation plays little if any role in exon 4
transactivation.
Another motif appearing in the C-terminal portion of exon 4 (150-FXX~~-154)
has not only
been shown to be critical for VP16 transactivation and its interaction with
TAFII31, but is
also thought to fimctiowas a general recognition element for acidic activators
(Uesugi et al.
(1997) supra.). Alanine substitutions in this motif, including a double
mutation that in the
context of VP 16 (479- FXX~~-483) completely abrogated its transactivating
capacity, had
little effect on the transactivating capacity of the GAL4(DBD)-ESX(129-159)
construct. To
confirm that elements residing in either terminus of this domain are not
required for exon 4
transactivation, a 13 residue acidic core domain (DELSW1IELLKDG) expressed as
a
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GAL4(DBD)-ESX(134-147) fusion construct was shown to retain nearly 50% of the
full
exon 4 transactivating capacity.
While structural themes mediating the function of acidic transactivators are
not fully understood, the acidic transactivating domains of GCN4 and p65NF-~B
are known
to assume, respectively, a (3 sheet or a-helical structure that depends on pH
or local
hydrophobic environment (Schmitz et al. (1994) J. Biol. Chem., 269: 25613-
25620; Van
Hoy et al. (1993) Cell, 72: 587-594). The 13 amino acid (aa 134-147) core
domain from
exon 4 was predicted to form a amphipathic a-helical structure. In agreement
with this
prediction, a helix-destabilizing proline mutation introduced into the middle
of exon 4
(L143P) completely abolished its transactivating capacity. To confirm the
predicted
secondary structure of this domain, CD analysis of a synthetic 25 residue ESX
exon 4
peptide demonstrated a-helical structure that was completely dependent on its
hydrophobic
(50% methanol) environment.
Acidic transactivators are thought to recruit components of the basal
transcription complex into a pol II preinitiation complex, as exemplified by
the specific
binding of VP16 to TBP, TFIIIB and TAFII31 (Chang and Gralla (1993) supra.;
ptashne and
Gann (1997) supra.; Pugh (1997) supra.; Uesugi (1997) supra.). Since the
activation
potency of the acidic exon 4 domain from ESX appeared comparable to that of
the activation
domain from VP16, a GST pull-down assay was employed to assess the potential
affinity of
exon 4 for the VP16 binding factors, TBP and TFIIA. As negative binding
controls in this
assay for column bound protein, the leucine to proline exon 4 mutation, GST-
exon
4(L143P), as well as GST alone were used. In particular, the L143P mutation
was
anticipated to be a particularlly stringent control since it exhibited no
transactivation
capacity but leaves the charge distribution unaltered in the critical acidic
core region of exon
4. By SDS-PAGE, the GST-exon 4{L143P) fusion protein consistently migrated ~10-
15
kDa slower than the unmodified GST-exon 4 protein, indicative of the helix-
distorting
influence of the proline substitution. TFIIB did not bind to either the
negative controls or to
the unmodified GST-exon 4 fusion protein. In contrast, the column bound GST-
exon 4
fusion protein quantitatively bound all of the added recombinant TBP.
Interestingly, while
the GST negative control did not bind TBP, the column bound GST-exon 4(L143P)
fusion
protein exhibited some capacity to bind TPB but with substantially reduced
affinity as
compared to the unmodified GST-exon 4 fusion protein. This was shown using
successive
elutions at increasing salt concentrations, with 1.3 M NaCI sufficient to
remove almost all
TBP from the proline mutated exon 4 while unmodified exon 4 still retained
most of its
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bound TBP following this 1.3 M elution condition, requiring 2.0 M NaCI for
significant
elution. Given the ability of a 13 residue acidic core domain from exon 4 to
induce nearly
50% of the transactivating capacity observed with full-length exon 4, it is
tempting to
speculate that its ability to bind and recruit TBP into a pol II preinitiation
complex may be
. sufficient to explain the potent transactivating capacity of this ESX
domain.
A review of published studies exploring the gene activating potential of full-
length ESX on transiently co-transfected Ets responsive promoters suggests
that this
promoter upregulation is best detected in cell lines expressing minimal levels
of endogenous
ESX (Andreoli et al., supra.; Chang et al. supra.; Choi et al., supra.; Tymms
et al. supra.).
Indeed, we have repeatedly shown that ESX can induce >4 fold upregulation of
an Ets
responsive reporter in transiently co-transfected COS-7 cells which express
little if any
endogenous ESX (Chang et aL supra.). As illustrated here, however, a
comparative
assessment of Ets promoter activation in transiently transfected SKBr3 cells
produced the
opposite result, suppression of an Ets responsive promoter following excess
ectopic
production of ESX. For these SKBr3 cells, as well as for similar cell lines
expressing high
levels of endogenous ESX (e.g. MDA-453, ZR-75-1; data not shown), introduction
of a
reporter.construct bearing a tandemly repeated Ets responsive element from the
erbB2IHER2
promoter that was co-transfected with a vector control resulted in >5 fold
higher reporter
activity than that achieved when the same reporter was co-transfected with an
ESX
expressing vector. The complete lack of suppression in this reporter activity
(relative to
vector control) with co-transfection of an ESX expression construct whose
transactivation
domain had been inactivated by either a double mutation (D134A/E135A) or
complete
deletion of exon 4 sequences (aa 129-159), indicated that this promoter
squelching was
dependent on the ESX transactivation domain and potentially due to TBP binding
and
sequestration. To demonstrate that this full squelching effect required only
the acidic
activation domain of ESX and was not dependent on its promoter binding, a
GAL4(DBD)-
ESX(129-159) fusion protein was co-transfected into SKBr3 cells with two
different
GAI,4(DBD) promoter-independent reporters. The strong squelching effect
observed with
this exon 4-encoding fusion protein on both heterologous reporters was
comparable to that
produced by the GAL4(DBD)-VP16(413-490) positive control.
The results from these transient transfection studies are consistent with the
explanation that squelching and transactivation are both mediated by ESX exon
4 and
depend on high-affinity binding by this domain to a limiting component of the
basic
transcriptional machinery, TBP. When TBP is recruited by promoter-bound ESX,
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transactivation with enhanced formation of a pol iI preinitiation complex
occurs. When TBP
is sequestered ~y excess ESX protein unbound to DNA, squelching of TBP-
dependent gene
expression can occur. Clearly, assessment of the ability of ESX to
transactivate individual
Ets responsive promoters must take into account the cell system being employed
(including
its endogenous level of ESX expression) as well as the format of the
transiently transfected
Ets responsive reporter construct.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims. All publications, patents,
and patent
applications cited herein are hereby incbrporated by reference for all
purposes.
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