Note: Descriptions are shown in the official language in which they were submitted.
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UBIQ~ lN CONJUGATING ENZYME'3 HAVING
TRANSCRIPTIONAL REPRESSOR A~:11V1-1Y
The present invention claims priority to
copending United States provisional application Serial
5 No. 60/002,995 and to copending United States provisional
application Serial No. 60/018040. This invention was
developed, in part, through research supported by grants
from the National Institute of Health (2POlCA49712). The
U.S. government may have certain rights in this
invention.
BACKGROUND OF THE INVENTION
The present invention relales to a novel
m~mm~lian ubiquitin conjugating enzyme, and more
particularly, to the identification, isolation, and
purification of a human ubiquitin conjugating enzyme, the
complete amino acid sequence of which has been
elucidated, and to nucleotide sequences encoding the
enzyme. The invention further relates to novel methods
of using the enzyme and similar enzyrnes to regulate gene
transcription and, particularly to suppress transcription
of a target gene in a human and non-human host cells. In
a preferred application, the invention relates to methods
for enhancing the repressor activity of WT1, Wilm's tumor
suppressor gene product.
Ubiquitin has been identified as playing a
central role in tagging proteins for degradation, and
thus in modulating their life-span in the cell. For
example, nuclear proteins that are known to be regulated
by ubiquitination include NFxb, cyclin B, c-jun, p53 and
~ 30 histones. Ubiquitin conjugating enzymes (UBCs) activate
and attach ubiquitin to a protein targeted for
degradation in the proteolytic proteosome pathway by
transferring activated ubiquitin in t:hioester linkage.
At least twelve separate yeast ubiquitin conjugating
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enzymes have been identified and sequenced. Prior to the
present invention, however, only two mammalian UBCs have
been identified and sequenced, and the human counterparts
of yUBC-9 and other yeast UBCs have not been identified.
While two yeast ubiquitin conjugating enzymes have been
reported to mediate cell cycle progression, yUBC 3
(Goeble, M.G., et al. 1988) and yUBC 9 (Seufert, W. et
al. 1995), ubiquitin conjugating enzymes have not
heretofore been characterized as having other activities
independent of their conjugating activities.
Tumor suppressor genes, such as the p53 gene,
the retinoblastoma (Rb) gene and the Wilm's tumor
suppressor gene, encode proteins which inhibit cell
reproduction and/or transcription in various ways. For
example, p53 gene protein is believed to bind to DNA and
induce transcription of another regulatory gene, the
product of which blocks the kinase activity of proteins
important for normal cell cycle progression, thereby
precluding cell replication. The Rb gene protein is
thought to act by masking the activation domain of an
activator protein. A Rb gene product protein and a
method therapeutic use thereof are disclosed in U.S.
Patent No. 5,496,731 to Benedict et al. Gene suppressor
proteins may also act in other ways, including, for
example, by competing with activator proteins for
specific DNA binding sites, and/or by direct or indirect
interaction with the general transcription factors.
Other tumor suppressor genes and gene products are
disclosed in U.S. Patent No. 5,491,064 to Howley et al.
(HTS-1 gene).
The Wilm's Tumor ~WT) suppressor gene product
(WT1) is a bifunctional transcription factor of the
Kruppel zinc-finger family. Loss of function of both
alleles of the WT1 gene (llpl3) is associated with some
Wilm's tumors and associated syndromes. WT1 is a 52~57
kd nuclear protein which contains a glutamine/proline-
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rich N-terminal region and four zinc-fingers of the C2-H2
subclass in the C-terminal region. WT1 is a potent
~ repressor of the promoter activity of several growth
related genes, including the IGF-II, PDGF A-chain, CSF-1
and IGF-R promoters. WT1 has an independent repressor
domain which is active when WT1 interacts with DNA
through the zinc-finger domains. While the activity of
repressor gene products such as WT1 is known to affect
transcription, control over the biochemical mechanism by
which transcriptional repression is effected is not
thoroughly understood and higher lev,els of repression are
desirable for commercial application~.
SUMMARY OF THE INVENTION
It is therefore an object of the present
invention to provide a means for regulating transcription
in cells, and particularly, a means for suppressing
transcription of a target gene in a host cell. It is
also an object of the invention to enhance the repressor
activity of known repressor proteins such as WT1, Wilm's
tumor suppressor gene product.
The present invention, therefore, is directed
to a novel, isolated and substantially purified m~mm~l ian
ubiquitin conjugating enzyme, hUBC-9, having a molecular
weight of about 16 kilodaltons to about 18 kilodaltons,
preferably about 17 kilodaltons, a sequence length of
from about 150 to 165 amino acid residues, preferably 158
amino acid residues, and having conjugating activity
and/or transcriptional repressor activity. The present
invention is also directed to a protein having an amino
acid sequence which includes the amino acid sequence of
hUBC-9. The invention is directed as well to a protein
which has ubiquitin conjugating activity or
transcriptional repressor activity and which includes a
portion of the amino acid sequence of hUBC-9 at least
about 12 amino acid residues in length. The included
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portion of the hUBC-9 sequence confers the conjugating
activity or the repressor activity on the protein. The
invention is directed to proteins which have
transcriptional repressor activity and have at least
about 60% sequence identity to h~3C-9, more preferably at
least about 65% sequence identity, more preferably at
least about 75% sequence identity, more preferably at
least about 85% sequence identity and most preferably at
least about 95% sequence identity to h~3C-9. A C93 mutant
of hUBC-9, which does not have ubiquitin conjugating
activity, but retains its transcriptional repressor
activity, is a particularly preferred protein.
The invention is directed, moreover, to
substantially isolated nucleic acid polymers encoding
hUBC-9. The nucleic acid polymer preferably has a
nucleic acid sequence selected from the group consisting
of: (a) Nucleotide Seq. 1, Fig. lA; (b) Nucleotide Seq.
2, Fig. lA; (c) a nucleic acid sequence which includes
the nucleic acid residues defined by the sequence from
position 88 to position 564 of Nucleotide Seq. 2, Fig.
lA. The invention is also directed to a substantially
isolated nucleic acid polymer which encodes a protein
having ubiquitin conjugating activity or transcriptional
repressor activity and having an amino acid sequence
which includes a portion of the amino acid sequence of
hUBC-9. The included portion is at least about a 12
amino acid residues in length and confers the conjugating
activity or the repressor activity on the protein. The
invention is directed to a nucleic acid polymer which is
at least about 36 nucleic acid residues in length and
which encodes a protein which has transcriptional
repressor activity. Such a nucleic acid fragment can
encode a protein which has at least about 60% sequence
identity to hUBC-9, or alternatively, can hybridize to a
nucleic acid polymer which is complementary to the
aforementioned nucleic acid polymers which constitute a
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part of the invention. The inventiorL is also directed to
nucleic acid polymers which are complementary to the
r aforementioned nucleic acid polymers of the invention.
The invention is directed as well to methods
5 for producing hUBC-9 or a segment thereof using a host
cell transfected with a vector having a DNA which encodes
hUBC-9 or a segment thereof. The method preferably
comprises producing a plasmid vector having DNA
(including genomic DNA and/or genomic DNA). The DNA
10 encodes the aforementioned hUBC-9 protein or a segment or
homolog thereof. The plasmid vector is transfected into
a host cell and hUBC-9 is expressed :i.n the host cell. If
desired, the expressed hUBC-9 may be purified from the
host cell. The invention is also directed to the vector
15 and to the host cell transfected therewith.
The invention is further di.rected to a host
cell co-transfected with first and second plasmid vectors
each comprising DNA. The DNA of the first vector
comprises a nucleic acid polymer which encodes a
20 transcriptional repressor protein other than a UBC-9
protein, including for example, WT1. The DNA of the
second vector comprises a nucleic acid polymer which
encodes an adapter protein having transcriptional
repressor activity which is preferab:Ly independent of the
25 transcriptional repressor activity of the transcriptional
repressor protein. The adapter protein associates or
interacts with the transcriptional repressor protein
after both are co-expressed in the host cell. The
adapter protein has an amino acid sequence which includes
30 a portion of the amino acid sequence of a ubiquitin
conjugating enzyme that has transcriptional repressor
activity. Exemplary ubiquitin conjugating enzymes
include hUBC-9, yUBC-9, other members of the UBC-9 family
and other ubic~itin conjugating enzyrnes. The included
portion of the amino acid sec~uence is at least about 12
amino acid residues in length.
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The lnvention is directed, moreover, to a
fusion protein comprising a transcriptional repressor
domain and a DNA binding domain. The transcriptional
repressor domain has an amino acid sequence which
includes at least a 12 amino acid residue portion of the
amino acid sequence of a ubiquitin conjugating enzyme
that has transcriptional repressor activity. The DNA
binding domain is preferably a domain which binds
sufficiently close to a promoter region of a target gene
to allow the ubiquitin conjugating enzyme to repress
transcription. Exemplary DNA binding domains include
Gal4, LexA and any of the zinc-finger domains. The
invention also relates to nucleic acid polymers encoding
such a fusion protein, plasmid vectors comprising such
nucleic acid polymers and to host cells transformed
therewith. The invention is directed as well to a method
for producing a fusion protein having a transcriptional
repressor domain and a DNA-binding domain. The method
comprises: producing a plasmid vector comprising DNA
which encodes the fusion protein described above,
transfecting the plasmid vector into a host cell,
expressing the fusion protein in the host cell, and, if
desired, purifying the expressed fusion protein ~rom the
host cell.
In another aspect, the invention is directed to
a composition comprising a protein having transcriptional
repressor activity and an acceptable carrier, diluent or
biochemical delivery agent suitable for introducing the
protein into a target cell. The protein has
transcriptional repressor activity and has an amino acid
sequence which includes at least a 12 amino-acid residue
long portion of a ubiquitin conjugating enzyme which has
transcriptional repressor activity. The protein derives
its transcriptional repressor activity from the included
portion of the enzyme. The composition can be used for
non-pharmaceutical (ie, non-human) uses, but can also be
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a pharmaceutical composition, in which the aforementioned
protein is combined with a pharmaceut:ically acceptable
carrier, diluent and/or gene therapy delivery agent.
The invention is further directed to a
composition suitable for use in introducing a nucleic
acid polymer to a cell, whereby the expression product of
the nucleic acid polymer is exposed t:o and/or contacts a
target gene therein. The composition can be used for
pharmaceutical or non-pharmaceutical applications to
regulate transcription. The composition comprises a
nucleic acid polymer and a gene therapy delivery agent.
When used in a pharmaceutical application, the amount of
the nucleic acid polymer or construct: containing the same
is su~ficient to, upon expression in a host cell, express
a pharmaceutically effective amount of the protein to
regulate a target gene in the cell. The nucleic acid
polymer is used in conjunction with a pharmaceutically
acceptable gene therapy delivery agent. The nucleic acid
polymer encodes a protein having transcriptional
repressor activity and having an amino acid secluence
which includes at least a 12 amino-acid residue long
portion of a ubiquitin conjugating enzyme which has
transcriptional repressor activity. The included portion
of the enzyme confers the repressor activity on the
protein. The nucleic acid polymer can, alternatively,
have a nucleic acid sequence which i~> complementary to
the aforementioned nucleic acid polymers of the
composition. The nucleic acid composition can be a virus
which has a viral genome that includes the nucleic acid
polymer being delivered to the target: gene. The
ubiquitin conjugating enzyme used in such a composition
is preferably a UBC-9 protein such as, hUBC-9, yUBC-9 or
yUBC-9-m. Another pharmaceutical composition comprises a
pharmaceutically active amount of the fusion protein set
out above and a pharmaceutically acceptable carrier.
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The invention is also directed to a method of
regulating transcription of a target gene in a cell. The
method comprises exposing the target gene to and/or
contacting the target gene with a protein having
transcriptional repressor activity. The protein has an
amino acid sequence which includes at least a 12 amino
acid residue portion of the amino acid sequence of a
ubiquitin conjugating enzyme having transcriptional
repressor activity, such as a UBC-9. A composition which
includes the protein or which includes a nucleic acid
polymer encoding the protein may be introduced into the
cells in a number of ways, including by contacting,
infecting or transfecting the target cells with a gene
therapy delivery agent such as a virus. The amount of
protein to which the gene is exposed is more than the
endogenous amount normally present within the cell. The
cell can be an eukaryotic cell such as a fungal cell
(e.g. a yeast cell), a plant cell, a non-human animal or
m~mm~l ian cell or a human cell. The cell can also be a
cell which has been infected with a virus wherein the
viral genome is exposed to the transcription regulating
protein. The invention is also directed to method of
modulating neoplastic tissue growth. In the method,
neoplastic tissue cells are contacted with a neoplastic-
tissue-growth-modulating amount of one of the
pharmaceutical compositions set forth above, thereby
modulating the growth of the neoplastic tissue. The
invention relates as well to a method of inhibiting the
proliferation of Wilm's tumor cells. The method
comprises introducing into Wilm's tumor cells a Wilm's-
tumor-inhibiting amount of a ubiquitin conjugating enzyme
or a segment thereof having transcriptional repressor
activity, or alternatively, introducing a nucleic acid
polymer which encodes such an amount of the enzyme, and
preferably co-expressing WT1 therewith.
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The discoveries described herein provide an
important analytical tool for, and a critical link in the
- development of methods by which transcriptional
repressors or promoters ~ay be made t:o either inhibit or
enhance a variety of cell activities in a desired manner.
hUBC-9 and other members or the UBC-9 family can be used,
~or example, in chemotherapy, gene therapy and drug
development. Other uses of the invention include its use
to regulate the rate of both specific and general gene
transcription and the cell cycle, including the control
of abnormal expression of genes associated with hl~m~n
disease such as those caused by virus, or associated with
yeast infections. The invention has use in non-
pharmaceutical applications in the yeast, baking and
brewing industries, and also in conjunction with
enzymatic conversion methods for producing valuable
chemical commodities such as essential amino acids. The
enzyme, its activities and other features, methods of
expressing the enzyme, and methods for its use are
described in greater detail below.
Other features and objects of the present
invention will be in part apparent to those skilled in
the art and in part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is further disclosed and
illustrated by the accompanying figures. In the earlier
U.S. provisional applications from which the present
invention claims priority, and in the figures therein,
other designations were used to refer to the enzyme hUBC-
9. Specifically, the enzyme has been heretofore referred
to using the acronym "TRA", "human UBC," and/or hUBC-h.
The present terminology, hUBC-9, has been incorporated to
be consistent with the literature. However, each of
these designations refers to the same enzyme, hUBC-9,
shown in Fig. lB and also depicted in Fig. lA as
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corresponding to certain nucleotide sequences shown in
Fig. lA.
FIG. lA and lB show nucleotide and amino acid
sequences for hUBC-9. Figure lA shows the full length
5 nucleotide sequence and the predicted amino acid sequence
for two cDNA clones encoding hUBC-9. The vertical line
connecting the cytosine at position 792 of the longer
form and at position 73 of the shorter form indicates the
splice site and origin of common nucleotide sequences of
10 the two alternative spliced mRNA. Figure lB shows a
comparison of predicted amino acid sequences of hUBC-9
and yUBC-9.
FIG. 2A and 2B show the results of Northern and
Southern blot analyses, respectively. Figure 2A shows
15 Northern blot of hUBC-9 in different human tissues.
Figure 2B shows Southern blot analysis of the hUBC-9
gene.
FIG. 3A and 3B show in vitro binding of WTl and
hUBC-9, in Western blot analyses of associated WTl and
20 hUBC-9 proteins. Figure 3A shows blots of eluates taken
from matrix-coupled GST-hUBC-9 which had been passed over
and incubated with WTl from 293 cell extracts. Figure 3B
shows the results of co-immunoprecipitation of WTl and
hUBC-9 from 293 cells co-transfected with WTl and HA-
25 tagged hUBC-9 expression vectors.
FIG. 4A and 4B show temperature-sensitive yeast
cell cultures at permissive and restrictive temperatures,
respectively.
FIG. 5A and 5B show how hUBC-9 enhances the
30 transcriptional repressor activity of WTl in human
embryonic kidney cells (293). Figure 5A shows the
relative CAT activity when various amounts of WTl and
hUBC-9 expression vectors are co-transfected. Figure 5B
illustrates the relative CAT activity of independent
35 assays at different times ~ standard deviation of each.
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FIG. 6A and 6B show the transcriptional
repressor activity of a hUBC-9/Gal4 DNA binding domain
fu~ion protein. Figure 6A shows the expression and
reporter vector constructs. Figure 6B shows the relative
CAT activity.
FIG. 7A through 7C show the transcriptional
repressor activity of hUBC-9, yUBC-9, and yUBC-9-m /Gal4
DNA binding domain fusion proteins. Figure 7A shows the
expression and reporter vector constructs. Figure 7B
shows the relative CAT activity for hUBC-9/Gal4 fusion
proteins. Figure 7C shows the relati.ve CAT activity for
yUBC-9/Gal4 and yUBC-9-m/Gal4 fusion proteins.
FIG. 8 shows the results of GST/hUBC-9 capture
assays for TATA binding protein (TBP), transcription
factor IIB (TFIIB) and Wilm's tumor ~uppressor gene
product, WT1.
FIG. 9A and 9B relate to GST/hUBC-9 capture
assays with wild type hTBP and with ~everal mutant TATA
binding proteins, mTBP. In the schematic representations
in Fig. 9A, the shaded area of the wild-type hTBP
represents the highly conserved region between species.
The shaded area for the mutant TBP's represents the
portion of the TBP deleted. The term " dl x-y" indicates
that in the mTBP, residue~ x through y were deleted.
Fig. 9B shows the blots resulting from the various
GST/hUBC-9 capture assays.
FIG. lOA through lOD show the results of WT1
turnover experiments done when WT1 is expressed by itself
(Fig. lOA), when WT1 is co-expressed with hUBC-9 (Fig.
lOB), when WT1 is expressed in the presence of
lactocysteine, a known inhibitor of the proteolytic
degradation system (Fig. lOC) and when WT1 is co-
expressed with mUBC-9, a C93S mutant of hUBC-9 (Fig. lOD).
FIG. llA and llB relate to gel mobility shift
assays that show the interaction between hUBC-9 and the
TATA binding protein (TBP). Fig. llA shows the results
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12
of assays using hUBC-9 and an end-labeled DNA probe
containing the TATA box either (a) without TBP present
(columns A1-A3), (b) with TBP present but without TFIIB
present (columns Bl-B3) and (c) with both TBP and TFIIB
present (columns Cl-C3). Fig. llB shows the results of
similar assays in which TBP was present with varying
amounts of hUBC-9.
FIG. 12A and 12B show the results of transient
co-transfection assays using a 5xUAS pSV CAT reporter
vector. Fig. 12A shows the relative level of expression
of the reporter vector for assays where a GAL4/hUBC-9
fusion protein (pSGhUBC-9) (0 or 10 ~g), TBP (0, 0.5 or
2.5 ~g) and/or TFIIB (5 ~g) were co-expressed in 293
cells in varying combinations. Fig. 12B shows the
relative level of expression of the reporter vector where
a mutant TBP, TBP~1-138 (0, 2.5 and 5 ~g), and the hUBC-
9/Gal4 fusion protein (0, 10 ~g) were co-expressed in
various combinations.
FIG. 13 shows the results of transient co-
transfection assays using a 5xUAS pSV CAT reporter vectorwhere TBP~1-138 (0, 2.5 and 5 ~g) was co-expressed with
WTl (10 ~g) in various combinations.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the various symbols for amino
acids are as set forth in Table 1.
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Table 1: Amino Acid Abbreviations
A Ala Alanine
B Asx Asparagine or aspartic acid
C Cys Cysteine
5 D Asp Aspartic acid
E Glu Glutamic acid
F Phe Phenylalanine
G Gly Glycine
H His Histidine
10 I Ile Isoleucine
K Lys Lysine
L Leu Leucine
M Met Methionine
N Asn Asparagine
15 P Pro Proline
Q Gln Glutamine
R Arg Arginine
S Ser Serine
T Thr Threonine
20 V Val Valine
W Trp Tryptophan
Y Tyr Tyrosine
Z Glx Glutamine or glutamic acid
As used herein, a "substant:ially purified"
protein means that the protein is separated from a
majority of host cell proteins normal.ly associated with
it or that the protein is synthesized in substantially
purified form, such synthesis including expression of the
protein in a host cell from a nucleic acid polymer
exogenously introduced into the cell by any suitable
gene-therapy delivery means. A "substantially isolated"
nucleic acid polymer means that the mixture which
comprises the nucleic acid polymer of interest is
essentially free of a majority of oth,er nucleic acid
polymers normally associated with it. A "nucleic acid
polymer" includes a polymer of nuclec,tides or nucleotide
derivatives or analogs, including ~or example
deoxyribonucleotides, ribonucleotides, etc. Genomic DNA,
cDNA and mRNA are exemplary nucleic acid polymers. The
term ~regulate transcription" is intended to include
enhancement and/or repression of transcription. The term
"gene" is intended to include both endogenous and
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14
heterologous genes, and specifically, both genomic DNA
which encodes a target protein in a naturally occurring
cell, and also cDNA encoding the target protein, wherein
the cDNA is a part of a nucleic acid construct such as a
plasmid vector or virus which has been introduced into a
cell. The contents of each of the references cited
herein are being incorporated by reference in their
entirety.
The present invention relates to newly
discovered human ubiquitin conjugating enzymes,
designated hUBC-9, which in addition to having a
functional conjugating activity, have an independent
transcriptional repressor activity. Both the conjugating
and the repressor activities have been found to influence
transcription. The conjugating activity of hUBC-9
~nh~nces tran5cription through degradation of
transcription suppressor proteins such as WT1, and
possibly, of hUBC-9 itself. The repressor activity of
hUBC-9 represses transcription independently of the
conjugating activity. For example, hUBC-9 strikingly
enhances the function of WT1 as a repressor of gene
transcription. The enzyme also acts independently of WT1
to suppress gene transcription itself, particularly when
fused to proteins having a DNA binding domain, such as
Gal4.
While not being bound to a particular theory,
UBC-9 acts as a potent repressor by disrupting the
transcriptional initiation complex through specific
interactions with the DNA binding region of the TATA
binding protein (TBP). Such interactions are
concentration dependent and result in destabilized
TPB/DNA interactions and interference with formation of
the TFIIB/TBP transcription initiation complex.
Moreover, hUBC-9 can operate in conjunction with other
proteins having a repressor effect, such as WT1, to
result in a combined repressor effect which is enhanced
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relative to the repressor effect of WTl alone or of hUBC-
9 alone. Hence, the association o~ hUBC-9 with other
repressor proteins, either through protein-protein
interactions as with WTl or by positioning hUBC-9 in the
vicinity of the promoter as a fusion protein comprising
hUBC-9 and a DNA binding domain, such as the domain of
Gal4, Lex A, zinc-fingers or others. DNA binding
proteins which are fused to hUBC-9 or repressor proteins
which specifically interact with hUBC-9 appear to
position human UBC-9 to an appropriate site in relation
to promoter DNA such that hUBC-9 can interact with the
TBP, and thereby reduce transcription initiation.
Moreover, in the combined WT1/hUBC-9 sy~tem, the
conjugating activity of hUBC-9 appears to operate in
conjunction with hUBC-9's repressor activity by
regulating the levels of WTl present in the system. Such
regulation of WT1 levels is accomplished through its
ubiquitin conjugating activity and the associated
ubiquitin-dependent proteolytic pathway. Moreover, the
ubiquitin conjugating activity of hUBC-9 and its
repressor activity may act at the same time, with hUBC-9
interacting simultaneously with WT1 (via its conjugating
activity) and with the TBP (via its repressor activity).
hUBC-9 may also interact with other repressors such as
p53 and Rb in a similar manner. Adva}ltageously,
inhibition of UBC-9's conjugating activity results in
even a greater degree of repression of the transcription
initiative.
Moreover, the homologous ubiquitin conjugating
enzymes of other eukaryotes, such as yeast (yUBC-9),
exhibit the same bifunctional activities as hUBC-9:
transcription repression and ubiquitin conjugation. The
highly conserved nature of UBC-9 among species, both in
amino acid sequence and in function, suggests a universal
role for this family of proteins. Accordingly, many
facets of the invention are directed to the family of
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16
proteins which are structurally homologous and
functionally equivalent to hUBC-9, this family being
collectively referred to herein using the designation
UBC-9. UBC-9 includes any such proteins whether they are
identified herein or discovered in the future. Aspects
of the invention which relate to individual species'
proteins are designated herein as hUBC-9 for hllmAnR and
yUBC-9 for yeast. Many facets of the invention also
relate to other ubiquitin conjugating enzymes other than
the UBC-9 enzymes, provided, that such ubiquitin
conjugating enzymes have transcriptional repressor
activity in addition to their conjugating activity.
The several aspects of the present invention,
including the hUBC-9 protein and nucleic acid polymers
which encode it, the transcriptional repressor activity
of ubiquitin conjugating enzymes such as a UBC-9 enzyme,
and the interaction between UBC-9 enzymes and other
transcription repressor proteins, especially such
proteins having a DNA binding domain, and in particular
tumor suppressor proteins such as WT1, collectively
enable several practical applications, including both
pharmaceutical applications involving hllmAn~ and non-
pharmaceutical uses.
hUBC-9
A yeast two hybrid system was used to identify
clones encoding the human ubiquitin conjugating (UBC)
enzyme of the present invention. (Example 1). Figure lA
shows the complete nucleotide sequences of two
independent cDNA clones, designated as Nucleotide
Sequence No.'s 1 and 2, which were established from two
alternatively spliced mRNAs. The cDNA clones both encode
hUBC-9. The amino acid product resulting from
transcription and translation of these cDNA clones
migrated identically in SDS-containing polyacrylamide gel
as a 17 kilodalton protein.
-
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huBc-s has the amino acid se~uence set forth in
Figure lB. Based on a comparison with data in Genebank,
hUBC-9 is an active human (h) homolo~ of the yeast
ubiquitin conjugating enzyme-9, yUBC-9 or E2, the
intermediate enzyme in the ubiquitin protein degradation
pathway. The hl~m~n U~3C-9 sequence has a 56% amino acid
identity overall with yUBC-9, including identical
sequences of 9 amino acids in separate regions. The 158
amino acid sequence of hUBC-9 also contains a cysteine
residue in precise alignment with the active site
cysteine of yeast UBC-9 (boxed, Fig. lB).
Human UBC was expressed in a all human tissues
tested, including heart, brain, placenta, lung, smooth
muscle, kidney and pancreas tissues. (Example 2).
However, the level of expression varied in different
tissues, as demonstrated by the results of a Northern
blot experiment shown in Figure 2A. Northern blots of
hUBC-9 in different tissues resulted in generally strong
hybridization signals of 2.8 and 1.3 kb. However, heart
and smooth muscle are seen to express significantly
higher levels of transcripts relative to other tissues
analyzed, and kidneys appear to expreAs relatively less
of the 2.8 kb mRNA isoform.
hUBC-9 is encoded by a single gene, as
demonstrated by experiments in which Southern blots of
hllm~n genomic DNA were digested with different
restriction enzymes and probed with the 1.1 kb fragment
of hUBC-g cDNA. (Example 3). Single hybridization
signals were seen in digests of PstI and BamH I (Fig.
2B), suggesting that the human UBC gene exists as a
single copy gene in the human genome.
hUBC-9 is further characterized by its
association with the repressor domain of the Wilm's tumor
suppressor gene product, WT1. Human UBC binds to WT1
both in vivo and in vitro. Protein-protein interactions
between hU~3C-9 and WT1 were initially demonstrated in the
CA 02230689 1998-02-27
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18
yeast two hybrid system by high levels of i~-galactosidase
activity, as shown in Table 1. (Example 1). Additional
experiments were carried out to confirm the WT1-human UBC
protein interactions observed in yeast. Human UBC was
5 expressed as a glutathione S-transferase fusion protein
(GST-human UBC) in E. coli and coupled to a glutathione
matrix. (Example 4). As shown in Figure 3A, when
extracts from 293 cells transfected with WT1 expression
plasmids were incubated with the GST-human UBC
10 glutathione matrix, eluates analyzed by Western blots
probed with an anti-WT1 antibody demonstrated that the
GST-human UBC matrix complexed with WT1, whereas the
matrix of GST alone did not. In another experiment,
extracts of 293 cells transfected with WT1 and a
15 hemagglutinin (HA)-tagged human UBC (HA-human UBC)
expression vector were immunoprecipitated with an anti-
WTl antibody and analyzed with anti-HA antibody in
Western blots. (Example 5). As shown in Figure 3B, HA-
tagged hnmAn UBC was identified in WT1 immune complexes
20 from 293 cells co-transfected with both human UBC and WT1
expression vectors but not in 293 cells transfected with
human UBC alone.
hUBC-9 also interacts directly with the TATA-
binding protein (TBP), as demonstrated by GST capture
25 assays. A GST fusion protein having the full-length
hUBC-9 amino acid sequence captured TBP and WT1
selectively over the transcriptional factor TFIIB, which
was also present in the asssay. (Fig. 8). Further assays
demonstrated that hUBC-9 interacts with the TBP through
30 the highly conserved C-terminal domain of the TBP.
Several mutants of hTBP were constructed. (Fig. 9A). In
GST capture assays, GST/hUBC-9 captured wild-type TBP as
well as several of the mutant TPB's; however, deletion of
the C-terminal region of TBP (amino acid residues 196-
35 335) significantly reduced the capture efficiency, anddeletion of a larger portion thereof (amino acid residues
CA 02230689 1998-02-27
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163-335) resulted in no interactions being detectable in
the assay. (Fig. 9B). Hence, hUBC-9 specifically
interacts with the C-terminal domain which includes amino
acids 163-335 of the TATA binding protein. Gel mobility
shift assays, discussed below, further confirmed the
specificity of the interaction between TBP and hUBC-9.
The hUBC-9 Enzyme has an Active Conjugating ~ctivity
hUBC-9 is an active ubiquitin conjugating
enzyme, and as such, is a member of a family of
ubiquitinating enzymes. Although sorne variation is
believed to exist in the precise amino acid sequence of
m~mm~l ian ubiquitin conjugating enzymes, the active
cysteine residue at position 93 (boxed, Fig. lB) is
characteristic of all of the ubiquitin conjugating
enzymes discovered to date, and it has been determined
that the presence of the active site cysteine is
important to the ubiquitin conjugating activity. This
cysteine is believed to provide the enzyme with its
ability to participate in thioester i-ormation. Hence,
human UBC-9, which has 56% sequence identity to yeast
UBC-9 and shares the same active cyst:eine site, forms an
integral part of the proteolytic proteosome pathway.
To demonstrate that the conserved cysteine
residue of hUBC-9 is involved in mono-ubiquitin thioester
formation, we used in vitro transcription/translation of
the hl~m~n UBC cDNA in the rabbit reticulocyte system,
which contains the three coupled enz~es [El E2 E3]
required for protein ubiquitination. Upon testing the
product with lM neutral hydroxylamine, a slower migrating
protein band was detected, suggesting that human UBC
contains a thioester that was hydrolyzed by
hydroxylamine. In another experiment, an anti-ubiquitin
antibody recognized the slower migrat:ing band that was
sensitive to hydroxylamine in Western blots. In
further experiment, we expressed the full-length hUBC-9
CA 02230689 1998-02-27
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cDNA in a yeast carrying a temperature sensitive mutant
form of yUBC-9. (Example 6). As shown in Figures 4A and
4B, growth was fully restored to the temperature
sensitive (ts) yUBC-9 yeast at the otherwise
nonpermissive temperature. The results of these
experiments independently and cumulatively establish that
the hUBC-9 cDNA encodes an active ubiquitin conjugating
enzyme.
The Conjugating Activity of hUBC-9 Regulate~
Transcription.
The human ubiquitin conjugating protein of the
present invention is a member of a family of en~ymes
which, via their conjugating activity, function to
regulate the cell cycle and duplication o~ DNA. It has
now been determined, moreover, that the ubiquitin-
dependent protease degradation system i5 directly
involved in transcriptional regulation. The conjugating
activity of hUBC-9 appears to modulate gene transcription
by contributing to the degradation of repressor proteins
such as WTl, thereby regulating the level of repressor
activity.
WTl was shown to be rapidly degraded by the
ubiquitin proteosome proteolysis pathway when expressed
in rabbit reticulocyte lysates containing the enzymes
required for transiting the proteolysis pathway. In a
control experiment, cDNA of WTl was added to rabbit
reticulocyte lysates that contained El, E2 and E3
enzymes, ubiquitin and the 26S proteosome complex
required for protein ubiquitination and degradation. A
distinct band that migrated identically to WTl was
observed upon analysis in SDS gels. In an experiment in
which cDNAs of both WTl and hUBC-9 were added to the
rabbit reticulocyte system, one protein band had a
greatly diminished intensity relative to the band
observed in the control experiment. Moreover, lower
=~
-
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molecular weight immunoreactive species were found and a
single, higher apparent molecular weight species was
observed which migrated consistent w:Lth monoubiquitin-
WT1. These results further confirm t;he interaction
between hUBC-9 and WT1, and significantly, demonstrate
that WT1 is degraded in a hUB~-9-dependent m~nne~
The effect of the conjugating activity is al~o
demonstrated by inhibition of the proteolytic pathway
with lactocysteine, a specific inhibitor of protease
activity associated with the 26S proteosome complex, with
such inhibition causing the half-life of WT1 to increase.
The turnover of WTl was tested in a ~3eries of experiments
in which WT1 was (a) expressed in 293 cells alone, (b)
co-expressed with hUBC-9, (c) expressed in the presence
of lactocysteine, a known inhibitor of the proteolytic
degradation system or (d) co-expressed with mUBC-9, a C93S
mutant of hUBC-9. In each case, the cells were treated
with cycloheximide, a protein synthe~is inhibitor. Cells
harvested at different times were lysed and analyzed by
Western blot~ with anti-WT1 antibody. In the control
experiment (WT1 alone), the steady st:ate levels of WT1
decreased dramatically upon the addit:ion of the
cycloheximide and the half-life of Wl'1 was determined to
be about 1 1/2 hours. (Fig. lOA). The half-life of WT1
decreased when co-expressed with hUBC-9. (Fig. lOB).
However, co-treatment of the cells with lactocysteine
resulted in a rise in the steady state levels of WT1 by a
factor of about 5. (Fig. lOC). Moreover, a similar
increase in the amount of WT1 was observed when a WT1 was
co-expressed with a mutant hU~3C-9 which lacked ubiquitin
conjugating activity due to substitution of serine at the
active site cysteine. (Fig. lOD).
WTl-dependent transcription.al repression is
in~luenced by the proteosome degradation system. When
WT1 was expressed in 293 cells co-transfected with a
reporter plasmid and cultured with lactocysteine (50 ~M),
CA 02230689 1998-02-27
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a two-fold to three-fold enhancement of repressor
activity was observed. Furthermore, the enhancement
effect of lactocysteine progressively decreased as the
level of expression of WT1 was increased. Lactocysteine
was without effect at an upper limiting level of WT1.
The conjugating activity of hUBC-9 and its effect on
repression is independently demonstrated by removal of
the conjugating activity. In a cotransfection experiment
detailed below, it was shown that removal of the
ubiquitin conjugating activity by mutation of yUBC-9 at
the active cysteine site results in a greater degree of
repression activity than yUBC-9 having the active site
cysteine. (Example 9).
These experiments demonstrate, individually and
cumulatively, that h~3C-9 has conjugating activity which
is specific to the WT1 and possibly to other suppressor
proteins. The conjugating activity of hU~3C-9 positively
influences transcription through degradation of
repressors such as WTl, and possibly, of hUBC-9 itself.
UBC's have a Repressor Activity which Suppresses
Transcription.
Ubiquitin conjugating enzymes such as hUBC-9
and yUBC-9 have a transcriptional repression activity.
The repression activity of hU~3C-9 enhances the existing
repressor activity of WT1 and perhaps other repressor
gene products. Moreover, the suppression of gene
transcription by human and yeast UBC's themselves become~
significant when these enzymes are fused to or associate
via protein-protein interactions with proteins having a
DNA binding domain, such as Gal4 or WT1.
The repression activity of hUBC-9 enzyme
strikingly enhances the repressor activity of Wilm's
tumor suppressor gene product, WT1. The ability of human
U~3C to modulate the transcriptional regulatory activity
of WT1 was analyzed in cotransfection experiments
CA 02230689 1998-02-27
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(Bxample 7). As shown in Figure 5A, when human UBC was
expressed alone at 15 ~g, without WTI and without being
fused to a DNA binding domain, expression of the reporter
gene was reduced slightly more than 2-fold (42% relative
- 5 activity). When WT1 was expressed a:lone at 10 ~g,
expression of the reporter gene was reduced by about a
factor of ten (8% relative activity). However, when
human UBC was expressed together with WT1, the repressor
activity of WT1 was increased by an additional four-fold
factor, resulting in a total transcr:iptional repression
of about 50-fold (2% relative activity). In additional
experiments, essentially identical results were obtained
(Fig. 5B). Human UBC significantly enhances the
repressor activity of WT1 in vivo when both are expressed
at high levels together.
Furthermore, hUBC-9 is itself a potent
transcriptional repressor when it is coupled to a
functional DNA binding domain recognized by an
appropriate promoter element. Human UBC was coupled to
the Gal4 DNA binding domain and tested with a promoter
containing five upstream Gal4 DNA binding sequences (5 X
UAS) in co-transfection experiments. (Example 8). As
shown in Figure 6B, when human UBC WclS tested with the
control promoter (lacking 5 X UAS) reporter plasmid to
which human UBC was unable to bind, t:he influence of
hl]m~n UBC was minimal (66% relative activity). However,
human UBC was a powerful transcriptional repressor when
it was able to directly bind, via the Gal4 binding
domain, to the promoter/reporter construct containing the
Gal4 DNA binding sequences. In this case, human UBC
repressed promoter activity by about eight-fold (12%
relative activity), establishing that: human UBC alone is
an effective repressor when it binds to an appropriate
promoter element. When the above experiment was repeated
(Example 9), the h-UBC-9/Gal4 fusion protein again
CA 02230689 1998-02-27
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24
demonstrated significant repression activity (15%
relative activity), as shown in Figure 7B.
These results strongly support the conclusion
that human UBC has transcriptional repressor activity.
The repressor activity of hUBC is particularly
significant when hUBC is positioned near the promoter
regions, either through protein-protein interactions with
other proteins, such as WT1, or through fusion with DNA
binding domains, such as Gal4, both of which appear to
tether hllm~n UBC to gene-specific promoter sites.
Other ubiquitin conjugating enzymes, such as
yUBC-9, also function efficiently as transcription
repressors when fused to a DNA binding domain. Yeast
UBC-9 was coupled to the Gal4 DNA binding domain and co-
expressed with a reporter vector having five upstreamGal4 DNA binding sequences. (Example 9). As shown in
Figure 7C, when co-expressed by itself at 20 ~g, the
yUBC-9/Gal4 fusion protein repressed transcription by
about three-fold (0.35 relative activity).
The transcriptional repression activity of UBC'~ is
independent o~ their conjugating activity.
As noted above, UBC's have, in addition to
their ubiquitin conjugating activity, a transcriptional
repression activity. Significantly, the repression
activity is independent of the conjugating activity, as
demonstrated by data showing that yUBC-9-m, a yUBC-9 C93S
mutant which lacks ubiquitin conjugating activity,
functions efficiently as transcription repressor when
fused to a DNA binding domain. Briefly, a mutant form of
yUBC-9 lacking the active site cysteine at position 93
(designated yUBC-9-m), was coupled to the Gal4 DNA
binding domain and co-expressed with a reporter vector
having five upstream Gal4 DNA binding sequences.
(Example 9). As shown in Figure 7C, when co-expressed by
itself at 20 ~g, the yUBC-9-m/Gal4 fusion protein
=
CA 02230689 1998-02-27
WO 97/0$19~ ~nUS96~ 13
repressed transcription by about five-fold (20% relative
activity).
~ Moreover, the independence of U3C repression
activity is supported by data showin~ a higher degree of
- 5 transcriptional repression for mutant yUBC-9-m than for
yUBC-9. Referring to Figure 7C, whereas yUBC-9 expressed
alone showed a 35% relative activity, yUBC-9-m expressed
alone showed a 20% relative activity --- an increase in
repression activity of about 15%. Consistently, when
both normal yUBC-9 and mutant yUBC-9-m were co-expressed
with the reporter vector in equal (10 ~g) amounts, the
degree of transcriptional repression was intermediate
(28% relative activity) between the ~alues for the normal
or mutant strain alone.
While not being bound by theory, because the
yUBC-9-m lacking the active site cysteine was active as a
repressor, and further, even more aclive than the normal
yUBC-9 having an active site cysteine, the ubiquitin
conjugating activity does not appear to be required for
the transcription repressor activity. Nonetheless, as
discussed above, the conjugating act:ivity of UBC-9
appears to affect and regulate the level of repressor
activity. The conjugating activity of hUBC-9 facilitates
proteolytic degradation of WT1 and thereby at least
partially relieves the repressor effect of WT1.
hUBC-9 Functions as a Repre~sor Thro~gh its Interactions
with the TATA B; n~; n~ Protein (TBP)
As noted, hUBC-9 interacts with the C-terminal
domain of the TATA binding protein (rrBp) in GST capture
assays. Gel mobility shift assays confirmed this
interaction, and further demonstrated that hUBC-9 appears
to suppress transcription by disrupting the binding of
TBP to DNA and by disrupting the formation of the
transcription initiation complex. This model is
consistent with the understanding that the C-terminal of
CA 02230689 1998-02-27
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26
TBP, with which hUBC-9 was shown to interact, contains a
"face" which contacts the major groove of DNA. In the
assays carried out, an end-labeled DNA probe containing
the TATA box was provided an opportunity to complex with
various combinations of TBP, TFIIB and hUBC-9. In
control experiments in which there was no TBP present, no
detectable complex was formed between hUBC-9 and the DNA
probe, between TFIIB and the probe, or between hUBC-9,
TFIIB and the probe. (Fig. llA, columns A1, A2 and A3,
respectively). In further control experiments, the
combination of purified TBP and the DNA probe containing
the TATA box resulted in a single readily detectable
complex. (Fig. llA, column B1). The addition of TFIIB
strengthened the intensity of the band, as expected based
on the reported ability of TFIIB to enhance the binding
of TBP to the TATA sequence. (Fig. llA, column C1).
However, when the above assays were repeated using
purified hUBC-9 (10 ng, 50 ng) in the systems, hUBC-9
reduced the level of complex formation between TBP and
DNA (Fig. llA, columns B2 and B3), and in subsequent
experiments, between TBP and DNA in the presence of TFIIB
(Fig. llA, columns C2 and C3). Hence, hUBC-9
destabilizes TBP/DNA binding. The effect of hUBC-9 on
the DNA binding ability of TBP was concentration
dependent. (Fig. llB).
To further confirm that hUBC-9 interacts in the
region of the TBP DNA binding domain, co-transfection
assays were performed which demonstrated that high levels
of exogenous TBP overcame the repressor activity of hUBC-
9. A 5xUAS pSV CAT reporter vector was used in transientassays in which a GAL4/hUBC-9 fusion protein (pSGhUBC-9)
(0 or 10 ~g), TBP (0, 0.5 or 2.5 ~g) and/or TFIIB (5 ~g)
were co-expressed in 293 cells in varying combinations.
The repressor activity of hUBC-9 was significantly
reduced in a concentration-dependent m~nner by the
presence of TBP (without TFIIB). (Fig. 12A, columns A,
CA 02230689 1998-02-27
W O 97/08195 PCT~US96/14013
B, C and D). The presence o~ TFIIB in the assay had no
effect on hUBC-9 repression (Fig. 12A., columns E and F).
Further experiments were performed to ensure that the
observed decrease in repressor activity of hUBC-9 was
not, in fact, related to an intrinsic activation of
transcription due to expression of TBP. A mutant TBP was
constructed which had an amino acid sequence which
included the TBP domain which interacted with hUBC-9 in
GST capture assays, but which did not include the amino
acid residues 1-138. The mutant TPB (0, 2,5 and 5 ~g),
referred to herein as TBP~1-138, and the hUBC-9/Gal4
fusion protein (0, 10 ~g) were co-expressed i.n various
combinations in transient co-transfection assays similar
to those immediately aforementioned. TBP~1-138 lacked
the ability to substantially activate the promoter, but
effectively relieved the repressor activity of hUBC-9,
although to a lesser extent than wild-type TBP. (Fig.
12B). Hence, hUBC-9 is shown to interact with TBP to
repress transcription. Moreover, the addition of TBP can
effectively titrate hUBC-9 to relieve its repressor
activity, and in effect, enhance tran~cription.
In similar-co-transfection assays, mutant TBP,
TBP~1-138 effectively relieved the repressor activity of
WT1. (Fig. 13). Cumulatively, these results suggest
that WT1 effects suppression in co~m~bination with hUBC-9
by positioning hUBC-9 through protein-protein
interactions for direct hUBC-9 interaction with the TBP
subunit of the TFIID transcription fartor, thereby
destabilizing the TBP/TATA sequence interaction, and more
generally, disrupting formation of the transcription
initiation complex.
Producing hUBC-9
The nucleotide sequence encoding the m~mm~lian
or yeast or other UBC enzyme, or active portion thereof,
is cloned into an expression vector w3ing known
CA 02230689 1998-02-27
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28
procedures. Briefly, specific nucleotide sequences in
the vector are cleaved by site-specific restriction
enzymes such as NcoI and HindIII. Then, after optional
alkaline phosphatase treatment of the vector, the vector
and a target fragment comprising the nucleotide sequence
of interest are ligated together with the resulting
insertion of the target codons in place adjacent to
desired control and expression sequences. The particular
vector employed will depend in part on the type of host
cell chosen for use in gene expression. Typically, a
host-compatible plasmid will be used containing genes for
markers such as ampicillin or tetracycline resistance,
and also containing suitable promoter and terminator
sequences. A preferred plasmid into which the
recombinant DNA expression sequence of the present
invention may been ligated is plasmid pET. A pET plasmid
which expresses human UBC-9 has been deposited in
GeneBank, Ascession No.'s ~L66818 and ~166867.
The plasmid comprising the DNA expression
sequence for the UBC enzymes of the present invention may
then be expressed in a host cell. Bacteria, e.g.,
various strains of E. coli, and yeast, e.g., Baker's
yeast, are most frequently used as host cells for
expression of mAmm~lian UBC enzymes, although techniques
for using more complex cells are known. See, e.g.,
procedures for using plant cells described by Depicker,
A., et al., 1982. E. coli host strain X7029, wild-type
F-, having deletion X74 covering the lac operon is
utilized in a preferred embodiment of the present inven-
tion. A host cell is transformed using a protocoldesigned specifically for the particular host cell. For
E. coli, a calcium treatment produces the transformation.
(Cohen, S.N., 1972). Alternatively and more efficiently,
electroporation of salt-free E. coli is performed
according to the method of Dower et al., 1988. After
transformation, the transformed hosts are selected from
CA 02230689 l998-02-27
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29
other bacteria based on characterist:ics acq~ired from the
expression vector, such as ampicillin resistance, and
then the transformed colonies of bacteria are further
screened for the ability to give rise to high levels of
isopropylthiogalactoside (IPTG)-induced thermostable DNA
polymerase activity. Colonies of transformed E. coli are
then grown in large quantity and expression of mAmmAlian
UBC enzyme is induced for isolation and purification.
Example 4 details the expression of human ~3C in bacteria
as a GST-fusion protein. Example 6 details the
expression of a temperature-sensiti~e yeast UBC strain in
yeast.
Although a variety of purification technigues
are known, all involve the steps of disruption of the E.
coli cells, inactivation and ,e..l~vdl of native proteins
and precipitation of nucleic acids. The enzyme is
separated by taking advantage of such characteristics as
its weight (centrifugation), size (dialysis,
gel-filtration chromatography), or charge (ion-exchange
chromatography). Generally, combinations of these
techniques are employed together in the purification
process. In a preferred process for purifying mAmmAlian
UBC enzyme, the E. coli cells are weakened using lysozyme
and the cells are lysed and nearly all native proteins
are denatured by heating the cell suspension rapidly to
80 ~C and incubating at 80-81 ~C for 20 minutes. The
suspension is then cooled and centrifuged to precipitate
the denatured proteins. The supernatant (containing
mAmmAlian UBC enzyme) then undergoes a high-salt
polyethylene-imine treatment to precipitate nucleic
acids. Centrifugation of the extract removes the nucleic
acids and mAmmAlian UBC enzyme is concentrated by use of
ammonium sulfate precipitation before chromatography,
preferably on a heparin-agarose colu~mn. Preferably, the
purified enzyme is at least 60% (w/w) of the protein of a
CA 02230689 1998-02-27
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preparation. Even more preferably, the protein is
provided as a homogeneous preparation.
Compositions
The ubiquitin conjugating enzymes disclosed
herein as having transcription repression activity (e.g.
hUBC-9, yUBC-9 and y-UBC9-m), as well as other ubiquitin
conjugating enzymes having such an activity, or segments
thereof, may be combined with an acceptable carrier,
diluent or delivery agent to form a useful composition.
The composition has both pharmaceutical (ie, human) and
non-pharmaceutical applications. In either case, the
protein used in the composition has transcriptional
repressor activity. The amino acid sequence of the
protein includes at least a 12 amino acid portion of a
ubiquitin conjugating protein such as a UBC-9 which has a
transcription repression activity. The amino acid
sequence of the protein preferably includes at least a
segment of hUBC-9 or yUBC-9. An active-site mutant of a
ubiquitin conjugating enzyme, such as a cys93 mutant of
hUBC-9, whereby such a mutant lacks its ubiquitin
conjugating activity, or a segment thereof, can also be
used as the protein. A mutant in which a serine residue
replaces the cysteine residue is preferred. In an
alternative method for removing the ubiquitin conjugating
activity from the composition, the composition can
further include a biochemical inhibitor suitable for
inhibiting the active site cysteine of the ubiquitin
conjugating enzyme. An exemplary suitable inhibitor in
n-ethyl-maleimide. The protein in the composition may
have only transcriptional repressor activity, or have
such an activity as well as ubiquitin conjugating
activity. In use, the composition may further comprise
one or more other proteins, including for example a
second protein having transcriptional repressor activity,
such as WT1. The composition may also comprise other
CA 02230689 1998-02-27
W O 97/08195 PCT~US96/140~3
proteins having a DNA binding domain with which the
ubiquitin conjugating enzyme or segment thereof
interacts. Moreover, the protein used in the composition
may be a fusion protein which has a amino acid sequence
that includes a DNA binding domain and a transcriptional
repressor domain. The repressor domain of the fusion
protein preferably includes at least a 12 amino acid
segment of a ubiquitin conjugating enzyme having
transcriptional repressor activity. The DN~ binding
domain is preferably a domain which binds to or interacts
with or otherwise associates with a region of a gene
which is sufficiently close to the promotor region to
allow the ubiquitin conjugating enzy~e or segment thereof
to interact with the promoter region, and particularly,
with the TATA binding protein at the TATA binding site.
Such domains include the amino acid se~uences of the Gal4
domain, the LexA domain, and the many zinc-finger
domains.
For pharmaceutical compositions, the protein is
combined with a pharmaceutically acceptable carrier,
diluent or gene therapy delivery agent, and a
pharmaceutically active amount of the protein is used in
the composition. The amount is preferably an amount that
is effective to achieve modulation or regulation or
suppression of gene transcription of a target gene.
While smaller or larger amounts may be suitable in
particular applications, the pharmaceutically active
amount of the protein is preferably an amount sufficient
to increase the concentration of the protein in the cell
of the target gene being regulated b~y a factor ranging
from about 1% to about 1000% relative to the amount of
the protein which is endogenous to the cell. The
increase in concentration more preferably ranges from
about 10~ to about 100%. Where the protein in the
pharmaceutical composition is a segment of a ubiquitin
conjugating enzyme having transcript:i.onal repressor
CA 02230689 1998-02-27
W O 97/08195 PCT~US96/14013
activity, the amount is taken relative to the endogenous
amount of the ubiquitin conjugating enzyme in its natural
full-sequence state. The particular dosage administered
for a particular pharmaceutical application, while
preferably consistent with the aforementioned amounts,
will be dependent upon the age, health, and weight of the
recipient, type of concurrent treatment, if any,
frequency of treatment, the nature of the effect desired,
and whether a localized tissue or system-wide effect is
being sought. For treatment of Wilm's tumor, a tumor-
inhibiting amount is to be administered. Similarly, for
regulating or modulating or suppressing any particular
neoplastic tissue growth, an effective amount to achieve
such regulation, modulation or suppression determined by
the factors outlined above, is to be applied. The amount
of protein used in a non-pharmaceutical application may
be in a range similar to that for pharmaceutical
compositions, but may also include amounts outside this
range.
The nucleic acid polymers which encode a
ubiquitin conjugating enzyme such as a UBC-9 having
transcriptional repressor activity, or which encode a
segment thereof, can be used in a nucleic acid
composition in combination with a gene therapy delivery
agent. As used herein, the term gene therapy relates to
operations and/or manipulations affecting both human and
non-human genes, whether such operations are in-vivo or
ex-vivo in nature. More specifically, the composition
preferably comprises a nucleic acid polymer that encodes
a protein which has transcriptional repressor activity.
The transcriptional repressor protein has an amino acid
se~uence which includes at least a portion of the amino
acid sequence of a ubiquitin conjugating enzyme having
transcriptional repressor activity, with the included
portion being at least about 12 amino acid residues in
length. Alternatively, the nucleic acid polymer can have
-
CA 02230689 1998-02-27
WO 97/0~195 PC'r~US96/1401
a nucleotide sequence complementary to the nucleic acid
sequence of the immediately aforementioned nucleic acid
polymer. The ubiquitin conjugating enzyme can be a UBC-9
such as hUBC-9, or a segment thereof~ or a mutant thereof
lacking ubiquitin conjugating activity. The composition
may further comprise or be used in conjunction with a
biochemical inhibitor of the ubiquitin conjugating
activity of the ubiquitin conjugating enzyme. The
nucleic acid polymer can also encode a fusion protein
such as the aforementioned fusion protein described in
connection with the above-described protein composition.
For pharmaceutical use, thle nucleic acid
composition compri~es a pharmaceutically effective amount
of the nucleic acid and a pharmaceutically acceptable
gene therapy delivery means. The amount of nucleic acid
required will vary depending on the type of cell, the
effect being sought and on the delivlery system used to
introduce the nucleic acid polymer into a target cell.
In general, the amount of nucleic acid polymer is
preferably an amount sufficient to, upon expression in
the target cell, result in an amount of protein
sufficient to regulate or modulate or repress
transcription of the target gene. Preferably, the amount
is sufficient to increase the concentration of the
protein in the cell of the target ge:ne being regulated by
a factor ranging from about 1% to about 1000% relative to
the amount of the protein which is endogenous to the cell
of the gene being regulated. The increase in
concentration more preferably ranges from about 10% to
about 100%. Where the nucleic acid polymer of the agent
encodes a protein which is a segment of a ubiquitin
conjugating enzyme having transcriptional repressor
activity, the amount is taken relative to the endogenous
amount of the ubiquitin conjugating enzyme in its natural
full-sequence state.
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Gene therapy delivery agents are used to
introduce the nucleic acid polymer into target cells or
to enhance the uptake of the nucleic acid polymer by the
target cells. Several approaches for introducing the
nucleic acid polymer into the cell and effecting
expression thereof are known and practiced by those of
skill in the art. (Mulligan, R., The Basic Science of
Gene Therapy, SCIENCE, Vol. 260, pp.926-32 (1993)). In
one approach, the nucleic acid polymer of the composition
may be combined, complexed, coupled or fused with a
delivery agent which introduces the nucleic acid polymer
into a human cell in vivo. For example, the nucleic acid
may be combined with a lipophilic cationic compound,
which may be in the form of liposomes. The use of
liposomes to introduce genes or other pharmaceutically
active ingredients into cells is taught, for example, in
U.S. Pat. Nos. 4,397,355 and 4,394,448. Alternatively,
said nucleic acid may be combined with a lipophilic
carrier such as any one of a number of sterols including
cholesterol, cholate and deoxycholic acid. A preferred
sterol is cholesterol. Additionally, the nucleic acid
may be conjugated to a peptide that is ingested by cells.
Examples of useful peptides include peptide hormones or
antibodies. By choosing a peptide that is selectively
taken up by Wilm's tumor or other neoplastic cells,
specific delivery of the nucleic acid may be effected.
The nucleic acid may be covalently bound to the peptide
via methods well known in the art. The peptide of choice
may then be attached to the activated enzyme via an amino
and sulfydryl reactive hetero bifunctional reagent. The
latter is bound to a cysteine residue present in the
peptide. Upon exposure of target cells to the nucleic
acid bound to the peptide, the nucleic acid is
endocytosed and is rendered available for modulation of
gene transcription. The nucleic acid polymer of the
present invention can also be delivered to specific
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tissues using a DNA-antibody conjugate, such as is
described in U.S. Patent No. 5,428,132 to Hirsch et al.
Other gene therapy delivery agents used to introduce
sense or antisense nucleic acid polymers such as DNA and
RNA into human cells are disclosed in U.S. Patent No.
5,460,831 to Gelman et al. and U.S. Patent No. 5,433,946
to Allen et al.
In an alternative approach, the gene therapy
delivery agent is a construct having cDNA which includes
the nucleic acid polymer and which can be expressed in a
host cell. Such a construct is infected or transfected
into the cell and expresses the ubiquitin conjugating
enzyme having transcriptional repressor activity in the
cell. For example, the composition can be a virus having
a viral genome which comprises the mlcleic acid polymer
of the agent or which is complexed to the nucleic acid
polymer of the agent. Such methods are taught, for
example, in U.S. Patent No. 5,252,47'3 to Srivastava, U.S.
Patent No.'s 5,521,291 and 5,547,932 to Birnstiel et al.,
U.S. Patent No. 5,512,421 to Burns et al., U.S. Patent
No. 5,240,846 to Collins et al., U.S. Patent No.
5,112,767 to Roy-Burman et al. and U.S. Patent No.
5,543,328 to McClelland et al.
In yet another approach, the gene therapy
delivery agent is a human cell. The nucleic acid polymer
of the composition is inserted into a human cell in vitro
and the cell comprising the nucleic acid polymer is then
introduced into the body. The encoded ubiquitin
conjugating enzyme is then expressed by the cells in
vivo. Such a method is taught for example, in U.S.
Patent No. 5,399,346 to Anderson et al.
The composition comprising the nucleic acid
polymer and the pharmaceutical compositions comprising
the protein of the present invention may be administered
by any means that achieve their intended purpose. For
example, administration may be by parenteral,
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subcutaneous, intravenous, intramuscular,
intraperitoneal, or transdermal routes. Formulations for
parenteral administration can include aqueous solutions
of the composition or pharmaceutical composition in
water-soluble form, for example, water-soluble salts. In
addition, suspensions of the active compounds in oily
injection suspensions may be administered. Suitable
lipophilic solvents or vehicles include fatty oils, for
example, sesame oil, or synthetic fatty acid esters, for
example, sodium carboxymethyl cellulose, sorbitol, and/or
dextran. Optionally, the suspension may also contain
stabilizers. The particular gene therapy delivery agent
used in the composition of the present invention and the
determination of optimal ranges of effective amounts of
each component is within capacities of a person of skill
in the art of the art.
The pharmaceutical compositions of the present
invention can be used in a variety of pharmaceutical and
non-pharmaceutical applications. In general, gene
transcription in cells can be regulated, enhanced or
repressed, by controlling the concentration of UBC-9
and/or of TBP to which a target gene is exposed or in
which a target gene comes in contact. In particular,
repression of transcription can be carried out in a gene-
specific manner by positioning the UBC enzymes near thepromoter regions of various genes, for example, by fusion
of a UBC-9 repressor domain with a gene-specific DNA
binding domain, or alternatively, by protein-protein
interactions between UBC-9 and proteins associated with
the promoter region or involved with transcription
initiation, such as WTl, TBP, or others.
A variety of cells can be used in the present
invention. Eukaryotic cells are particularly preferred,
as naturally occurring eukaryotic cells contain genes
having promoter regions which include a TATA box. hUBC-9
or other UBC-9's or other ubiquitin conjugating enzymes
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having transcriptional repressor activity or segments
thereof which are at least about 12 amino acid residues
in length can disrupt the TATA binding protein's role in
transcription initiation in such genes. For example, the
cells can be ~ungal cells (e.g. yeast cells), plant
cells, non-human animal cells, non-human m~m~l ian cells
and hllmAn cells. Non-eukaryotic cells such as E. Coli
can also be used where the cells comprise genetically
engineered nucleic acid polymer const:ructs which include
a promoter region which involves TBP for initiation of
transcription.
The function of repressor gene products such as
WT1 can be strikingly enhanced by such an approach,
allowing for control of transcription of genes promoters
on which WT1 is known to operate, such as IGF-II, PDGF A-
chain, CSF-l and IGF-R or others later discovered. The
regulation of transcription can also be controlled by
localized inhibition of the conjugating activity of U~3C-
9, for example, through a 93cys mutant: enzyme lacking such
activity, or through agents which inhibit the active cite
cysteine or which otherwise interrupt: the proteolytic
degradation pathway in a specific manner. The regulation
of transcription is particularly useful in medical
treatment, diagnostic and research applications. For
example, UBC-9 can be used in therapeutic compositions
for inhibiting neoplastic tissue growth by itself, or in
combination with known tumor suppressor proteins such as
WT1. It is particularly suited to treating Wilm's tumors
and to treating the other types of tumors with which WT1
suppressor gene is associated, incluating for example
leukemia and mesothelioma. It can also be useful in
controlling any number of human diseases which are
causally linked to an overabundance of a certain protein.
The gene from which the overabundant protein is expressed
could be exposed to a U~3C-9 or other ubiaruitin
conjugating enzymes which have repressor activity to
-
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38
decrease the amount of overabundant protein expressed.
In certain circumstances, the repressor activity of a
UBC-9 or of other ubiquitin conjugating enzymes could be
applied to effect an increase in the expression of a
particular protein of interest. An increase in a protein
of interest can be effected through a "rebound"
mechanism, where the increase therein is a result of a
natural biochemical mechanism following a decrease in the
amount of a second protein present in the system. The
decrease in the amount of the second protein is
accomplished according to the methods of the present
invention directed to the gene which encodes that
protein. Another significant application of the present
invention includes the treatment of a human viral
infection. This application would include exposing the
viral genome of human virus, and particularly, the
promoter region of the genome, to a ubiquitin conjugating
enzyme having transcriptional repressor activity. By
suppressing transcription of a viral genome, the virus
may be killed or at least controlled. The invention
could also be used to kill or at least help control yeast
infections.
Moreover, transcription regulation is useful in
a variety of non-human, non-pharmaceutical applications.
The invention could, for example, be used for the
treatment of animals or of particular animal diseases
much as described above. The present invention is also
useful for treating plant diseases resulting from
overabundant expression, and may have other plant
applications as well.
In another set of non-therapeutic applications,
hUBC-9 may be used to develop animal-based models or in -
vitro assays. For example, an animal having a selective
protein deficiency can be developed by administering the
pharmaceutical composition or the biochemical agent of
the present invention to an animal whereby transcription
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of a target gene encoding the proteirl of interest is
repressed by the repressor activity of a UBC-9 or other
ubiquitin conjugating enzyme having a repressor activity.
An alternative application could inc:lude an in-vitro
comparative assay in which the effect of hUBC-9 on a
culture of neoplastic cells or other cells of interest
(e.g. 293 cells) is used as a standa:rd against which the
effect of other potential anti-cance:r agents could be
evaluated.
Enzymatic conversion processes, in which
chemicals are commercially produced llsing enzymes
expressed in cells can also take advantage of the present
invention. Exemplary bioconversion processes include the
yeast-catalyzed processes associated with the brewing and
baking industries, and as well as the commercial
production of a variety of carboxylic acids, including
essential amino acids or analogs thereof, from amides or
nitriles. The invention can also be used in
bioconversion processes which are inlegral to
bioremediation measures being carried out to effect
environmental cleanup. The cells used in such enzymatic
conversion processes can be eukaryot:ic cells or non-
eukaryotic cells, such as genetically engineered E. coli
cells. Other uses and applications of the several
aspects of the invention will be apparent to those
skilled in the art.
The following examples illustrate the
principles and advantages of the invention.
EXAMPLES
All molecular biological manipulations used in
carrying out the experiments upon which the following
examples are based were performed using methods known in
the art, as described, for example, in Sambrook et al.,
Molecular Cloninq, A Laboratory Manual, Cold Springs
Harbor Laboratory Press, Cold Springs Harbor, NY (1989).
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Example 1: Isolatlon of hUBC-9
The repressor domain (residues 85-179) within
the N-terminal region of each of the alternative splice
variants of WTl were previously mapped and identified as
functioning independently as a potent repressor when
fused to a Gal4 binding domain. (Wang, Z-Y., et al.
1993). This repressor domain was also shown to block the
repressor function o~ WT1 if expressed independently
without a functional DNA binding domain, suggesting that
the repressor domain lacking DNA binding activity
competed with WT1 for an interactive nuclear factor
needed for WT1 to function as transcription repressor.
(Wang, Z-Y., et al., 1995).
The interactive factor, now identified as hUBC-
9, was isolated by using a yeast two hybrid screen. Avector, LexADB-WT-N, was constructed by coupling residues
85-179 of human WT1 with the Lex DNA binding domain. To
construct pLexADB/WT-N, a cDNA fragment encoding the
negative regulating domain (residues 85-179) of WT1 was
obtained by digestion of the plasmid pSGWT-N with XbaI,
blunt ended with Klenow fragment and the EcoRI digestion,
and cloned into EcoRI and SmaI treated vector pStopll6,
which was modified from plasmid pBTM116 by introducing
stop codons in each of three reading frames within the
polylinker region.
The vector expressed a fusion protein with the
LexA DNA binding (DB) domain and the negative regulatory
domain of WT1 (WTN) as "bait". Yeast strain L40 was used
in library screening. L40 was transformed with
pLexADB/WT-N and then with the Gal4 activation domain
fused with human placenta cDNA library (Clontech, CA) as
recommended by the manufacturer. Two million yeast
transformants were screened. Positive colonies on His-
plates were further tested for ~-Galactosidase activity
with a filter assay. Positive clones were tested for
specificity with a LexADB/lamin C hybrid, LexADB/WT-INS
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(containing residues 250-266 of WT1), LexADB/WT-N, and
the LexADB vector alone. Plasmids reactive only with
plexADB/WT-N were recovered from yeast and used to
transform HB101 via electroporation and selected on leu~,
amp~ minimal media. 65 positive clones were identified in
the initial screening.
The 65 positive plasmids recovered were re-
introduced to yeast to re-check specificity and for
quantitation of ~-gal activity. ~-ga.lactosidase activity
units are shown, in Table 2, for the DNA-binding domain
fusion partner coupled with the vector alone and with the
vector fused with hUBC-9 fused with the Gal4 activation
domain. Table 2 shows the binding specificity of hUBC-9
to the negative regulatory domain of WT1 in the yeast two
hybrid system.
Table 2 - Bindinq Specificity of hUBC-s in Two-Hybrid
sYstem
*-Galactosidase activity
DNA-binding
20 domain fusion partner Vector hUBC-9
Vector alone: N.D. 0.2_0.1
+WT 85-179: 0.3_0.2 57.9_16.2
+WT 250-266: 0.2+0.2 0.3_0.2
+T.~m;n C: 0.3_0.1 1.5_0.5
Eleven positive clones which remained specific
to WT1 were sequenced using dideoxy NTPs and sequenase
2.0 according to the manufacturer's specifications (U.S.
Biochemical). Sequence analysis and homology searches
were performed using GCG program (GCG, Madison, WI).
Each of the inserts was in the same reading frame as the
Gal4 activation domain. Seven of these plasmids encoded
the same protein that we designated human UBC. The
insert of the longest cDNA clone detected transcripts of
approximately 2.8 and 1.3 kb in Northern blot analysis.
To seek full-length cDNAs, we rescreened a
human placenta cDNA library with probes obtained with the
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42
two hybrid screen and isolated eight independent clones
after tertiary screening. Five clones contained a 1.1 kb
DNA fragment, one contained a 1.8 kb DNA fragment and two
clones contained small DNA fragments that were not
further analyzed. The 1.8 kb cDNA and 1.1 kb cDNA were
fully sequenced. The two clones share the same sequences
within the coding region and 3' end but alternative
splicing appears to introduce a long 5' untranslated
region (5' UTR) upstream of the translation initiation
site of the longer cDNA isoform. Multiple start and stop
codons were identified in all three reading frames within
the long 5' UTR, indicating that the protein product of
the longer transcript may be under strict translational
regulation. In vitro transcription/translation of both
mRNAs produced the same size protein but the longer cDNA
clone expressed less than 20% of the protein product than
was expressed from the shorter mRNA (data not shown),
confirming that the long 5' UTR sharply reduces
translation efficiency. Because the 5' UTR of the long
form contains multiple intervening start and stop codons
in all three reading frames (Fig. lA), it seems likely
that the presence of these codons may negatively regulate
the rate of translation in the longer mRNA isoform.
In control experiments, LexADB-WT-N failed to
activate transcription of reporter genes containing LexA
binding sites in yeast when analyzed alone.
ExamPle 2: Northern Blot AnalYsis of Human Tissues
A human tissue Northern blot (Clontech, CA) was
probed with 1.1 kb hUBC-9 cDNA, according to the
manufacturer/s recommendation. Figure 2A shows the
Northern blots of hUBC-9 in different human tissues.
Each lane contained 2 ~g poly A' RNA from heart (H), brain
(B), placenta (Pl), lung (Li), smooth muscle (SM), kidney
(K), pancreas (Pa). The ~-actin cDNA was used to probe
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43
the same blot as control. The size markers are indicated
on the left side of the blot.
ExamPle 3: Analvsis of Human Genomic DNA for hU~3C-9 Gene
10 ~g of human genomic DNA (Promega, Madison
WI) were digested with Hind III (H), EcoRI (E), Bgl II
(Bg), or BamHI (B). The digested DN~ was separated by
electrophoresis on 1% agarose gel and Southern blot was
performed with the full-length 1.1 kb hU~3C-9 CDNA as
probe. Figure 2B shows the Southern blot analysis of the
h~3C-9 gene.
Exam~le 4: Expression of GST-hUBC-9 Fusion Protein in
E. col i
Briefly, hUBC-9 was fused with glutathione S-
transferase (GST) by expression in bacteria as a GST-
hUBC-9 fusion protein. GST-hUBC-9 and GST were
independently coupled to a reduced g:l.utathione sepharose
matrix and washed extensively. Extracts from 293 cells
which had been transfected with vectors expressing WT1
and various WT1 domains were then passed over the
columns, and after incubating and washing, eluates were
obtained. The eluates were separated by SDS-PAGE,
transferred to nitrocellulose filter for immunoblotting,
and analyzed by Western blot using anti-WT1 (1:500) and
anti-IgG coupled with peroxidase. The blot was
visualized by color fluorography.
To construct GST-hUBC-9, p~3S-hUBC-9 was
digested with EcoRI and the 1.1-kb insert was subcloned
into the EcoRl site of the pGEX-KG vector containing GST
in frame. E.coli strain DH5~ was transformed with GST-
hUBC-9 and GST-hUBC-9 waq extracted and purified on
glutathione-sepharose beads. GST ancl GST-hU.3C-9 fusion
protein were independently bound to glutathione-Sepharose
beads and washed extensively.
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WTl and various domains thereof were expressed
in 293 cells as previously described (Wang et al., 1993).
Extracts were made from 2X106 293 (human embryonic kidney
cell) cells transfected with CMV promoter driven
expression vectors encoding full length and the WT1~1-84,
WT1~1-294, and WT1~297-429 domains of WTl.
In vitro binding assays were performed by
incubating the extracts with the sepharose beads
containing 2-3 ~g of GST and GST-hUBC-9 in lysis buffer
(50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1~ NP-40,
50 mM NaF, 1 mM PMSF, 1 ~g leupeptin/ml, 1 ~g
antipain/ml) for 2-3 hours at room temperature.
Complexes were washed extensively with lysis buffer and
lysis buffer with 0.5M NaCl, boiled in SDS PAGE loading
buffer (1% SDS, 10~ ~-mercaptoethanol), and run on 5%
SDS-polyacylamide gels. Gels were transferred to
nitrocellulose membranes (SOSNC) and immunoblotted with
polyclonal anti-WTl antibodies SC089 and SC189 (Santa
Cruz, CA), which recognize the N-terminal and C-terminal
domains of WTl, respectively. Following the addition of
an alkaline phosphatase-conjugated secondary antibody,
bound WTl protein was visualized with 5-bromo-4-chloro-3-
indolyphosphate toluidinium and nitro blue tetrazolium
(BCIP, NBT; Promega, Madison, WI).
Figure 3A shows the in vitro binding of WTl and
hUBC-9. The left column shows the cell lysate control
results with the arrow indicating WTl at the expected
estimated molecular mass of 14kd. The right column shows
the GST control results. The middle column shows the
results for the GST-hUBC-9 fusion protein with the
associated arrows indicating binding between WTl and the
GST-hUBC-9 matrix. Because no similar binding was
observed between WTl and the GST control matrix, these
results demonstrate that WTl binds to or associates with
hUBC-9.
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Example 5: Cell Cotransfection with WT1 and HA-taqqed
Human UBC
An expression vector encoding the influenza
virus hemagglutinin (HA) tagged hUBC-9 was constructed by
cloning the 1.1 k~ EcoRI fragment of hUBC-9 into the
EcoRI site of expression vector pGCN (REF) in frame with
a cDNA fragment encoding the HA peptide.
293 cells were cotransfected with WT1 and HA-
tagged hUBC-9 expression plasmids. Cellular lysates were
prepared and the extracts were immunoprecipitated with
either anti-WT1 antibody or a nonspecific rabbit
polyclonal antibody (anti-Gal4DB). WT1 associated
proteins were separated on 15% SDS-PA~E and blotted. The
blot was then analyzed by probing with anti-HA monoclonal
antibody which recognized the HA tagged hUBC-9.
Figure 3B shows the results of the co-
immunoprecipitation of WT1 and hUBC-9.
Exam~le 6: Co-ExPression of hUBC-9 with ts YUBC-9
Yeast strain W9432 (MATa, ubc9-~ TRP1,
pSE362[ARS1, CEN4, HIS3]-ubc9-1) is isogenic to W303
except for carrying a replacement of ~he genomic yUBC-9
coding sequence by the TRP1 marker and a plasmid-borne
copy of the temperature ~ensitive yUBC-9-1 allele (1.5 kb
Xbal-Sspl fragment).
hUBC-9 cDNA (1.1 kb EcoRI fragment) and yUBC-9
gene (0.6 kb EcoRI-XbaI fragment) were each fused to the
GAL1 promoter in vectors p416GAL1 (ARSH4, CEN6, URA3) and
pSE936 (ARS1, CEN4, URA3), respectively.
Referring to Figures 4A and 4B, the
temperature-sensitive yeast strain (W9432) was
independently transformed with the hUBC-9 (Row 1) and
yUBC-9 (Row 4) control vectors (p416G~L1 and pSE936,
respectively) and with a construct expressing hUBC-9 cDNA
(Row 2) or the yUBC-9 gene (Row 3). 'To compare growth of
these strains, cells were spotted in a dilution series on
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46
galactose-containing plates and incubated for 3.5 days at
the permissive temperatures (23 ~C) or 2 days at the
restrictive temperature (34 ~C).
Example 7: hU~3C-9 / WT1 Co-transfection Experiments
293 cells were co-transfected by calcium
phosphate/DNA precipitation with hUBC-9 and WT1
expression constructs under the control of the CMV
promoter and with a PDGF A-chain promoter driven CAT
reporter plasmid.
The total amount of CMV promoter sequence
transfected into each dish was equalized in each
transfection by the addition of vector DNA. Transfection
efficiency were standardized by co-transfection of a CMV
promoter driven ~B-galactosidase reporter construct. All
experiments were repeated at least three times.
Figure 5A shows the results of the CAT assay
and ~B-galactosidase assays. CAT activity was quantitated
by scintillation counting of excised sections of TLC
plates. Figure 5B shows the relative CAT activity values
from different assays at different times, including the
standard deviation of each. The experiments demonstrate
that hUBC-9 ~nh~nces the transcriptional repressor
activity of WT1 in human embryonic kidney cell (293
cell).
Exam~le 8: hl~}3C-9-Gal4 Cotransfection Assay
hUBC-9 was coupled to the Gal4 DNA binding
domain and evaluated for its effect on transcription in a
reporter system.
Referring to Figure 6A, a control expression
vector, pSG424, was constructed with a SV40 promoter
driven Gal4 DNA binding domain. A fusion protein
expression vector, pSG-hUBC-9, was constructed with full
length cDNA of hUBC-9 fused with Gal4 DNA binding domain
driven by SV40 promoter. pSG-hUBC-9 was constructed by
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inserting an EcoR1 DNA fragment containing full length of
hUBC-9 cDNA into the EcoRl site of expresslon vector
pSG424. Reporter plasmids pSV CAT and 5XUAS pSV CAT were
provided by Dr. S. Weintraub (Washin~ton University at
St. Louis). The pSV CAT plasmid included a SV40 promoter
fused with CAT reporter gene (Promega, Madison, WI). The
5xUAS pSV CAT plasmid included a pSV CAT plasmid with
additional 5 copies of the Gal4 bind:ing sites upstream of
the SV40 promoter.
Co-transfection experiments were done in which
each of the expression vectors were cotransfected with
each of the reporter plasmids. 5 ~g of reporter plasmid
DNA were used in each transfection w:ith various amounts
of expression plasmids. Figure 6B shows the results of
CAT and ~-galactosidase as~ays, performed as described
above (Example 7), for different amollnts of expression
plasmids, as indicated. CAT activity is shown as CAT
activity relative to the control alone.
Example 9: hUF3C-9, YUBC-9 and YUBC-9-m Gal4 Fusion
Proteins
hUBC-9, yUBC-9 and yUBC-9-m (a mutant yUBC-9
with serine in place of the active site cy~teine) were
independently coupled to a Gal4 DNA binding domain and
evaluated for effect on transcription in a reporter
sy~tem.
Referring to Figure 7A, control expression
vector, pSG424, and hUBC-9/Gal4 fusion protein expression
vector, pSG-hUBC-9, were constructed as described above
(Example 8). A yUBC-9/Gal4 expressic)n vector, pSG-yUBC-
9, was constructed by inserting an EcoR1 DNA fragmentcontaining full length of yUBC-9 cDN~ into the EcoRI site
of expression vector pSG424. A yUBC-9-m/Gal4 expression
vector, pSG-yUBC-9-m, was constructed by digestion of
pUC19-yUBC9-m plasmid with HindIII, blunted with Klenow
fragment, and then digested by EcoRI and cloned into
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48
EcoRI-SmaI digested pSG424 plasmid. The reporter
plasmids, depicted in Figure 7A, were as obtained
described above (Example 8).
The results of CAT and ~-galactosidase assays,
performed as discussed above (Example 7), for
cotransfection experiments are shown in Figure 7B and 7C
for human and yeast UBC's, respectively. 5 ~g of
reporter plasmid DNA were used in each transfection with
various amounts of expression plasmids, as indicated.
In light of the detailed description of the
invention and the examples presented above, it can be
appreciated that the several objects of the invention are
achieved. The explanations and illustrations presented
herein are intended to acquaint others skilled in the art
with the invention, its principles, and its practical
application. Those skilled in the art may adapt and
apply the invention in its numerous forms, as may be best
suited to the requirements of a particular use.
Accordingly, the specific embodiments of the present
invention as set forth are not intended as being
exhaustive or limiting of the invention.
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49
BIBLIOGRAPHY
Cohen, S.N., 1972: Proc. Natl. Acad. Sci.
69:2110 (1972)
Depicker, A., et al., 1982: J. Mol. APpl. Gen.
(1982) 1:561.
Dower et al. (1988), Nucleic Acids Research
16:6127-6145.
Goeble, M.G., et al., 1988: The yeast cell
cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.
Science, 1988. 241 (4871): p. 1331-5
Seufert, W. et. al., 1995: Role of a ubiquitin-
conjugating enzyme in degradation of S- and M-phase
cyclins. Nature, 1995. 373(6509): p. 78-81.
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