Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Antibodies Specific for Methylated Lysines in Histories
This application claims priority under 35 U.S.C. ~119(e) to US
Provisional Patent Application No. 60/302,747, filed on July 3, 2001 and US
Provisional Patent Application No 60/227,767, filed on August 25, 2000, the
disclosures of which are incorporated herein by reference in their entirety.
US Government Rights
This invention was made with United States Government support
under Grant Nos. ROl GM40922 and RO 1 GM53512, awarded by the National
Institutes of Health. The United States Government has certain rights in the
invention.
Field of the Invention
1 S The present invention is directed to antibodies that bind to histone
epitopes created by postranslational modification of the histone protein,
compositions
comprising such antibodies, and the use of such compositions as diagnostic and
screening tools.
Background of the Invention
In eukaryotes, DNA is complexed with histone proteins to form
nucleosomes, the repeating subunits of chromatin. This packaging of DNA
imposes a
severe restriction to proteins seeking access to DNA for ANA-templated
processes
such as transcription or replication. It is becoming increasingly clear that
post-
translational modifications of histone amino-termini play an important role in
,
determining the chromatin structure of the eukaryotic cell genome as well as
regulating the expression of cellular genes.
Posttranslational modifications of histone amino-termini have long
been thought to play a central role in the control of chromatin structure and
function.
A large number of covalent modifications of histories have been documented,
including aceiylation, phosphorylation, methylation, ubiquitination, and ADP
ribosylation, that take place on the amino terminus "tail" domains of
histories. Such
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diversity in the types of modifications and the remarkable specificity for
residues
undergoing these modifications suggest a complex hierarchy of order and
combinatorial function that remains unclear. Of the covalent modifications
known to
take place on histone amino-termini, acetylation is perhaps the best studied
and
appreciated. Recent studies have identified previously characterized
coactivators and
corepressors that acetylate or deacetylate, respectively, specific lysine
residues in
histones in response to their recruitment to target promoters in chromatin
(See Berger
(1999) Curr. Opin. Genet. Dev. 1 l, 336-341). These studies provide compelling
evi-
dence that chromatin remodeling plays a fundamental role in the regulation of
transcription from nucleosomal templates.
Chromosomes in higher eukaryotes have historically been considered
to consist of regions of euchromatin and heterochromatin, which are
distinguished by
the degree of condensation and level of transcriptional activity of the
underlying DNA
sequences. Certain regions of constitutive heterochromatin are found at or
near
specialized structures such as centromeres, and are comprised mostly of
genetically
inert repetitive sequences. In contrast, other regions that have the same
primary DNA
sequences can exhibit characteristics of either type of chromatin, suggesting
that
epigenetic factors, such as packaging of DNA by histones and chromatin
associated
proteins, dictate the heterochromatin status at these loci.
Through the use of antibodies that specifically recognize histones
bearing specific post-translational modifications applicants have been
elucidating a
"histone code." In particular, evidence is emerging that histone proteins, and
their
associated covalent modifications, contribute to a mechanism that can alter
chromatin
structure, thereby leading to inherited differences in transcriptional "on-
off' states or
to.the stable propagation of chromosomes by defining a specialized higher-
order
structure. . '
Histone methylation is one of the least-understood posttranslational
modifications affecting histones. Early work suggests that H3 and H4 are the
primary
histones modified by methylation, and sequencing studies, using bulk histones,
have
shown that several lysines (e.g., 9 and 27 of H3 and 20 of H4) are often
preferred sites
of methylation, although species-specific differences appear to exist.
Interestingly,
each modified lysine has the capacity to be mono-, di-, or trimethylated,
adding yet
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another level of variation to this posttranslational "mark". The present
invention is
directed to antibodies that are specific for histone H3 and H4 that are
methylated at
specific lysines. More particularly, one aspect of the present invention is
directed to
histone H3 lysine 4 and 9. These two lysine residues are found to be
methylated i~
vivo and the methylated forms axe associated with euchromatin and
heterochromatin,
respectively.
Definitions
In describing and claiming the invention, the following terminology
will be used in accordance with the definitions set forth below.
As used herein, the term "nucleic acid" encompasses RNA as well as
single and double-stranded DNA and cDNA. Furthermore, the terms, "nucleic
acid,"
"DNA," "RNA" and similar terms also include nucleic acid analogs, i.e. analogs
having other than a phosphodiester backbone. For example, the so-called
"peptide
nucleic acids," which are lrnown in the art and have peptide bonds instead of
phosphodiester bonds in the backbone, are considered within the scope of the
present
invention.
The term "peptide" encompasses a sequence of 3 or more amino acids
wherein the amino acids are naturally occurring or synthetic (non-naturally
occurring)
amino acids. Peptide mimetics include peptides having one or more of the
following
modifications:
1. peptides wherein one or more of the peptidyl --C(O)NR-- linkages (bonds)
have been replaced by a non-peptidyl linkage such as a --CH2-carbamate linkage
(--CH20C(O)NR--), a phosphonate linkage, a -CH2-sulfonamide (-CH 2--S(O)2NR--
) linkage, a urea (--NHC(O)NH--) linkage, a --CH2 -secondary amine linkage, or
with
an alkylated peptidyl linkage (--C(O)NR--) wherein R; is Cl-C4 alkyl;
2. peptides wherein the N-terminus is derivatized to a --NRRl group, to a
-- NRC(O)R group, to a --NRC(O)OR group, to a --NRS(O)2R group, to a --
NHC(O)NHR group where R and Rl are hydrogen or Cl-C4 alkyl with the proviso
that R and Rl are not both hydrogen;
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3. peptides wherein the C terminus is derivatized to --C(O)R2 where R 2 is
selected from the group consisting of C1-C4 alkoxy, and --NR3R4 where R3 and
R4
are independently selected from the group consisting of hydrogen and C1-C4
alkyl.
Naturally occurring amino acid residues in peptides are abbreviated as
recommended by the IUPAC-ILTB Biochemical Nomenclature Commission as
follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or
I;
Methionine is Met or M; Norleucine is Nle; Valine is Vat or V; Serine is Ser
or S;
Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is
Tyr or Y;
Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine
is Lys
or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or
C;
Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G, and X is
any
amino acid. Other naturally occurring amino acids include, by way of example,
4-
hydroxyproline, 5-hydroxylysine, and the like.
As used herein, the term "conservative amino acid substitution" is
defined herein as exchanges within one of the following five groups:
I. Small aliphatic, nonpolar or slightly polar residues:
Ala, Ser, Thr, Pro, Gly;
II. Polar, negatively charged residues and their amides:
Asp, Asn, Glu, Gln;
III. Polar, positively charged residues:
His, Arg, Lys;
IV. Large, aliphatic, nonpolar residues:
Met Leu, Ile, Val, Cys
V. Large, aromatic residues:
Phe, Tyr, Trp
As used herein, the term "purified" and like terms relate to the isolation
of a molecule or compound in a form that is substantially free of contaminants
normally associated with the molecule or compound in a native or natural
environment.
"Operably linked" refers to a juxtaposition wherein the components are
configured so as to perform their usual function. For example, control
sequences or
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promoters operably linked to a coding sequence are capable of effecting the
expression of the coding sequence.
As used herein, the terms "complementary" or "complementarity" are
used in reference to polynucleotides (i.e., a sequence of nucleotides) related
by the
base-pairing rules. For example, for the sequence "A-G-T," is complementary to
the
sequence"T-C-A."
As used herein, the term "hybridization" is used in reference to the
pairing of complementary nucleic acids. Hybridization and the strength of
hybridization (i.e., the strength of the association between the nucleic
acids) is
impacted by such factors as the degree of complementarity between the nucleic
acids,
stringency of the conditions involved, the length of the formed hybrid, and
the G:C
ratio within the nucleic acids.
"Therapeutic agent," "pharmaceutical agent" or "drug" refers to any
therapeutic or prophylactic agent which may be used in the treatment
(including the
prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease
or injury in
a patient.
As used herein, the term "treating" includes alleviating the symptoms
associated with a specific disorder or condition andlor preventing or
eliminating said
symptoms. For example, treating cancer includes preventing or slowing the
growth
and/or division of cancer cells as well as killing cancer cells.
As used herein, the term "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, water and emulsions such as an oil/water or
water/oil
emulsion, and various types of wetting agents.
As used herein, the term "antigenic fragment of H3 lysine 4" or
"antigenic fragment of H3 lysine 4" encompasses both natural peptide fragments
of
the amino terminus of Histone 3 (including the peptide fragments of SEQ ID NO:
1,
SEQ ID NO: 2 and SEQ ID NO: 3) and synthetic equivalents of those fragments.
As used herein, the term "antibody" refers to a polyclonal or
monoclonal antibody or a binding fragment thereof such as Fab, F(ab')2 and Fv
fragments.
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As used herein, the term "biologically active fragments" of the
Methyl(K4)H3 or Methyl(K9)H3 antibodies encompasses natural or synthetic
portions
of the respective full-length antibody that are capable of specific binding to
the
peptide of SEQ ID NO: 4 or SEQ ID NO: 5, respectively.
As used herein, the term "parenteral" includes administration
subcutaneously, intravenously or intramuscularly.
As used herein the letter K in bold face type (K), when used in the
context of an amino acid sequence, will represent a lysine amino acid that has
been
methylated.
Summary of the Invention
The present invention is directed to antibodies that bind to specific
modifications of the amino terminus of histone H3 and H4 peptides. More
particularly, the present invention is directed to the generation of
methyllysine-
specific histone antibodies. These antibodies recognize lysine residues in
histones H3
and H4 that are specifically methylated and may be linked to human biology and
disease. Compositions comprising these antibodies axe used as diagnostic and
screening tools.
Brief Description of the Drawings
Fig. 1A and 1B represent immunofluorescence patterns of human
metaphase chromosomes from the normal female cell line (HH) stained with
either the
Methyl(K9)H3 antibody (Fig. 1A) or the Methyl(K4)H3 antibody (Fig. 1B).
Localization of the Methyl(K4)H3 and Methyl(K9)H3 antibodies was detected
using
Cy3-conjugated secondary antibody (red). Each of the immunofluorescence
patterns
obtained with the two antibodies revealed one chromosome that is
preferentially
stained compared to the other chromosomes (indicated by large arrow). As shown
in
Fig. 1B only small regions of the inactive X chromosome are enriched for Lys4
methyl H3 staining.
Fig. 2. CHIP analysis of somatic cell hybrid cell lines. Chromatin
from CHO somatic hybrid cells containing the inactive (X;nactive) or active
(active)
human X chromosome was immunoprecipitated using the following antibodies:
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lane 1 = no antibody (control lane); lane 2 = Methyl(K9)H3;
lane 3 = Methyl(K4)H3; lane 4 = Lys9/14-acetylated H3;
lane 5 = no DNA (control); lane 6 = genomic DNA
The presence of Xist and PGKl promoter DNA sequences in the immunoprecipitated
DNA was assayed by PCR. The PCR products were separated on 15%
polyacrylamide gels, imaged by digital camera, and images were electronically
inverted to facilitate visualization of the ethidium bromide-stained bands. As
shown,
from cells containing the inactive X chromosome, the Methyl(K4)H3 and Lys9/14
acetyl H3 antibodies preferentially immunoprecipitated the active Xist gene
whereas
the Methyl(K9)H3 antibody preferentially immunoprecipitated the inactive PGKl
gene. From cells containing the active X chromosome, the exact reverse
immunoprecipitation pattern was observed.
Fig. 3 Immunoblot analysis of H3 methyiation between simple vs.
complex organisms. Histories were isolated from various sources, and five ug
of total
core histories from each species, along with 1 ug of recombinant Xehopus H3
were
resolved on a 15% SDS-PAGE, transferred to a PVDF membrane support and probed
with either the Methyl(K4)H3 or Methyl(K9)H3 antibody. Lanes 1-5 represent
histories isolated from recombinant Xenopus H3, budding yeast, Tetrahymena,
chicken and the human cell line 293T, respectively. Identical samples were
analyzed
in parallel and examined by Coomassie staining to monitor histone loading.
-Detailed Description of the Invention
Histone methylation is a poorly understood post-translational
modification affecting histories. This modification occurs on selected lysine
residues
in the amino-terminus of histories. It is now becoming apparent that
methylating
histone enzymes are involved in both gene activation and repression. The
present
invention is directed to the generation of methyllysine-specific histone
antibodies.
These antibodies recognize lysine residues in the histories H3 and H4 that are
specifically methylated and may be linked to human biology and disease.
The present invention is directed to post-translational modifications
that occur on the flexible N-terminal tails of the core histone proteins H3
and H4.
More particularly, the invention is directed to methylated lysine residues.
Applicants
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have discovered that methylation of the lysine residues within the first 15
amino acids
of the amino terminus of H3 (SEQ ID NO: 7) and H4 (SEQ ID NO: 8) play an
important role in the regulation of transcription. For example, methylation of
lysine 4
(K4) on histone H3 has been associated with transcriptionally active regions
of
chromatin, whereas methylation of lysine 9 (K9) on histone H3 has been
associated
with gene silencing. Therefore, in accordance with one aspect of the present
invention
methylation of lysine 4 (K4) and lysine 9 (K9) on histone H3 serve as a
markers of
euchromatin and heterochromatin, respectively, and antibodies recognizing
these
modified proteins have use as important diagnostic tools.
One aspect of the present invention is directed to antigens used to
produce antibodies specific to the amino terminus of H3 and H4. In one
embodiment,
the antigen is a purified antigenic fragment of the amino terminus of H3 or H4
methlated at a lysine and selected from the group consisting of ARTKQTARKSTGG
(SEQ ID NO: 10), ARTKQTARKSTGG (SEQ ID NO: 11), ARTKQTARKSTGV
(SEQ ID NO: 12), ARTKQTARKSTGV (SEQ ID NO: 13), SGRGKGGKGLGKG
(SEQ ID NO: 14) and SGRGKGGKGLGKG (SEQ ID NO: 15) or a synthetic
equivalent thereof, wherein the bold K represents a methylated lysine residue.
In one
embodiment the antigen comprises an H3 amino terminal fragment of 20 amino
acids
or less and comprises the sequence ARTKQTAR (SEQ ID NO: 1), QTARKSTGV
(SEQ l~ NO: 2) or QTARKSTGG (SEQ ID NO: 3), and derivatives of these amino
acid sequences wherein the amino acid sequence contains one or more
conservative
amino acid substitutions. In one preferred embodiment the antigen is ARTKQTAR
(SEQ ID NO: 1), QTARKSTGV (SEQ ID NO: 2) or QTARKSTGG (SEQ ID NO: 3),
or a derivative thereof containing additional non-native amino acids added to
either
end of the peptide sequence.
In an alternative embodiment, the purif ed antigen comprises a
polypeptide linked to a suitable carrier, such as bovine serum albumin or
Keyhole
limpet hemocyanin. In one preferred embodiment the antigen consists of an H3
peptide fragment peptide comprising a sequence selected from the group
ARTKQTAR (SEQ ID NO: 1), QTARKSTGV (SEQ II7 NO: 2) or QTARKSTGG
(SEQ ID NO: 3, and derivatives of this amino acid sequence wherein the amino
acid
sequence contains one or more conservative amino acid substitutions, and a
Garner
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protein linked to the peptide. For example, the antigen may comprise a peptide
having the sequence ARTKQTARGC (SEQ ID NO: 4), QTARKSTGVCG (SEQ ID
NO: 5), QTARKSTGGCG (SEQ ID NO: 6), AARKSAPVCG (SEQ ID NO: 16),
SGGVKKPHKCG (SEQ ID NO: 17) or RHRHILRDCG (SEQ ID NO: 18) wherein
the bold K represents the methylated lysine residue and the underlined GC
refers to
artificial amino acids added to the natural histone sequence. Furthermore, the
antigen
can optionally be linked to a carrier protein. .
In addition to the antigens described above, the present invention is
also directed to antibodies that specifically bind to peptide fragments of the
H3 or H4
protein that have been methylated at a lysine residue. Preferably the antibody
will
recognize one or more methylated lysine residues present in the first 20 amino
acid
residues of the amino terminus of the H3 and H4 histones. More particularly,
the
present invention is directed to an antibody that specifically binds to the
peptide
ARTKQTARGC (SEQ ID NO: 4), QTARKSTGGCG (SEQ ID NO: 6),
QTARKSTGVCG (SEQ ID NO: 5), AARKSAPVCG (SEQ m NO: 16),
SGGVKKPHKCG (SEQ ID NO: 17) or RHRKILRDCG (SEQ ID NO: 18), wherein
the bold K represents the methylated lysine residue and the underlined GC
refers to
artificial amino acids added to the natural histone sequence to aid in the
production of
the antibody. In one embodiment the antibody is specific for a peptide
comprising
the amino acid selected from the group consisting of ARTKQTARGC (SEQ ID NO:
4), QTARKSTGVCG (SEQ ID NO: 5), QTARKSTGGCG (SEQ ID NO: 6); and
amino acid sequences that differ from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID or
SEQ
ID NO: 6 by one or more conservative amino acid substitutions. In one
preferred
embodiment the antibody specifically binds to the peptide ARTKQTARGC (SEQ ID
NO: 4) or QTARKSTGVCG (SEQ ID NO: 5).
Antibodies that specifically bind an H3~ peptide that is methylated at
lysine 4 (i.e. the peptide of SEQ ID NO: 4) will be referred to as
Methyl(K4)H3 and
antibodies that specifically bind an H3 peptide that is methylated at lysine 9
(i.e. the
peptide of SEQ ID NO: 5 or SEQ ID NO: 6) will be referred to as Methyl(K9)H3.
These two antibodies do not cross react and will not bind to the non-
methylated
peptide sequences. The present invention also encompasses antibodies that bind
to
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the non-methylated histone peptides. In one embodiment, the antibodies of the
present invention are monoclonal antibodies.
The antibodies or antibody fragments of the present invention can be
combined with a carrier or diluent to form a composition. In one embodiment,
the
carrier is a pharmaceutically acceptable carrier. Such carriers and diluents
include
sterile liquids such as water and oils, with or without the addition of a
surfactant and
other pharmaceutically and physiologically acceptable carrier, including
adjuvants,
excipients or stabilizers. Illustrative oils are those of petroleum, animal,
vegetable, or
synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In
general,
water, saline, aqueous dextrose, and related sugar solution, and glycols such
as,
propylene glycol or polyethylene glycol, are preferred liquid carriers,
particularly for
injectable solutions. The compositions comprising the Methyl(K4)H3 or
Methyl(K9)H3 antibody, or bioactive fragments thereof, and a carrier or
diluent can
be used in conjunction with the method to detect heterochromatin verses
euchromatin.
One method used to generate the antibodies of the present invention
involves administration of an antigen, comprising the sequence ARTKQTAR (SEQ
ID NO: 1), QTARKSTGV (SEQ ID NO: 2) or QTARKSTGG (SEQ ID NO: 3), to a
laboratory animal, typically a rabbit, to trigger production of antibodies
specific for
the antigen. The dose and regiment of antigen administration to trigger
antibody
production as well as the methods for purification of the antibody are well
known to
those skilled in the art. Typically, such antibodies can be raised by
administering the
antigen of interest subcutaneously to New Zealand white rabbits which have
first been
bled to obtain pre-immune serum. The antigens can be injected at a total
volume of
100 u1 per site at six different sites. Each injected material will contain
synthetic
surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing
the
protein or polypeptide after SDS-polyacrylamide gel electrophoresis.
The rabbits are then bled two weeks after the first injection and
periodically boosted with the same antigen three times every six weeks. A
sample of
serum is then collected 10 days after each boost. Polyclonal antibodies are
then
recovered from the serum by aff'mity chromatography using the corresponding
antigen
to capture the antibody. Ultimately, the rabbits are euthenized with
pentobarbital 150
mg/Kg IV. This and other procedures for raising polyclonal antibodies are
disclosed
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in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988), which
is
hereby incorporated by reference. The specificity of antibodies may be
determined by
enzyme-linked immunosorbent assay or immunoblotting, or similar methods known
to
those skilled in the art.
The present invention also encompasses monoclonal antibodies that
specifically bind to the respective antigen ARTKQTAR (SEQ ID NO: 1),
QTARKSTGV (SEQ ID NO: 2) or QTARKSTGG (SEQ ID NO: 3) and their non-
methylated counterpart peptides. Monoclonal antibody production may be
effected
using techniques well-known to those skilled in the art. Basically, the
process
involves first obtaining immune cells (lymphocytes) from the spleen of a
mammal
(e.g., mouse) which has been previously immunized with the antigen of interest
either
i~z vivo or in vitro. The antibody-secreting lymphocytes are then fused with
myeloma
cells or transformed cells, which are capable of replicating indefinitely in
cell culture,
thereby producing an immortal, immunoglobulin-secreting cell line. The
resulting
fused cells, or hybridomas, are cultured, and the resulting colonies screened
for the
production of the desired monoclonal antibodies. Colonies producing such
antibodies
are cloned, and grown either in vivo or i~c vitro to produce large quantities
of antibody.
One embodiment of the invention is directed to a hybridoma cell line which
produces
monoclonal antibodies which bind the methyl-lysine peptides of the present
invention.
A description of the theoretical basis and practical methodology of fusing
such cells is
set forth in I~ohler and Milstein, Nature, 256:495 (1975), which is hereby
incorporated by reference.
In addition to whole antibodies, fragments of antibodies can retain
binding specificity for a particular antigen. Antibody fragments can be
generated by
several methods, including, but not limited to proteolysis or synthesis using
recombinant DNA technology. An example of such an embodiment is selective
proteolysis of an antibody by papain to generate Fab fragments, or by pepsin
to
generate a F(ab')2 fragment. These antibody fragments can be made by
conventional
procedures, as described in T. Goding, Monoclonal Antibodies: Principles and
Practice, pp. 98~118 (N.Y. Academic Press 1983), which is hereby incorporated
by
reference. Other fragments of the present antibodies which retain the specific
binding
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of the whole antibody can be generated by other means known to those skilled
in the
art.
In one embodiment the antibodies are labeled. It is not intended that
the present invention be limited to any particular detection system or label.
The
antibody may be labeled with a fluorophoxe, a radioisotope, ox a non-isotopic
labeling
reagent such as biotin or digoxigenin; antibodies containing biotin may be
detected
using "detection reagents" such as avidin conjugated to any desirable label
such as a
fluorochrome. In one embodiment the histone specific antibodies of the present
invention are detected through the use of a secondary antibody, wherein the
secondary
antibody is labeled and is specific for the primary (histone specific)
antibody.
Alternatively, the histone specific antibody may be directly labeled with a
radioisotope or fluorochrome such as FITC ox rhodamine; in such cases
secondary
detection reagents may not be required for the detection of the labeled probe.
In accordance with one embodiment of the present invention a method
of detecting the presence of methylated lysine residues in the H3 and H4
histones is
provided. The method comprises the steps of contacting histone proteins with a
labeled antibody, wherein the antibody specifically binds only to H3 that is
methylated
at lysine 4 or H3 methylated at lysine 9.
In accordance with one embodiment the antibodies of the present
invention are labeled for use in diagnostic imaging. Examples of labels useful
for
diagnostic imaging in accordance with the present invention are radiolabels
such as
131h 111~~ 123h 99mTc~ 32p~ 125h 3H~ 14C~ ~d 188' fluorescent labels such as
fluorescein and rhodamine, nuclear magnetic resonance active labels, electron
dense
or radiopaque materials, positron emitting isotopes detectable by a positron
emission
tomography ("pET") scanner, chemilluminescers such as luciferin, and enzymatic
markers such as peroxidase or phosphatase. Short-range radiation emitters,
such as
isotopes detectable by short-range detector probes, such as a transrectal
probe, can
also be employed. These isotopes and transrectal detector probes, when used in
combination, are especially useful in detecting prostatic fossa recurrences
and pelvic
nodal disease. The antibodies of the present invention can be labeled with
such
reagents using techniques known in the art. For example, see Wensel and
Meaxes,
Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York (1983), which
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is hereby incorporated by reference, for techniques relating to the
radiolabeling of
antibodies. See also, D. Colcher et al., "Use of Monoclonal Antibodies as
Radiopharmaceuticals for the Localization of Human Carcinoma Xenografts in
Athymic Mice," Meth. EnzvmQL, 121: 802-816 (1986), which is hereby
incorporated
by reference. In accordance with one preferred embodiment the antibody is
labeled
with a fluorophore or chromophore using standard moieties known in the art.
Applicants have discovered that methylation of histone H3 at lysines 4
and 9 (K4 and K9), correlates, respectively, with activation and inactivation
of
expression of genes in proximity to the modified histones. Histone H3 when
methylated at K9 is a preferred binding site for the heterochromatin protein
HP 1,
which in turn can recruit the enzyme Suv39h responsible for K9 methylation,
creating
a mechanism by which the inactivation signal can be propagated. Methylation of
K9
also precludes acetylation at that site, further contributing to repression.
In contrast to
the reaction at K9, methylation at histone H3 K4 is correlated with
transcriptional
activity. Therefore in accordance with one embodiment of the present
invention,
antibodies specific for the K4 methylated histone H3 can be used to detect
transcriptionally active regions of chromatin and antibodies specific for K9
methylated histone H3 transcriptionally can be used to detect inactive regions
of
chromatin. In fact, in situ staining of chromosomes reveals that the staining
patterns
generated by the Methyl(K4)H3 and Methyl(K9)H3 antibodies produce mirror
images
of one another.
Because the Methyl(K4)H3. and Methyl(K9)H3 antibodies have the
potential for use in humans as diagnostic and therapeutic agents, one
embodiment of
the present invention is directed to humanized versions of the Methyl(K4)H3
and
Methyl(K9)H3 antibodies. Humanized versions of the antibodies are needed for
therapeutic applications because antibodies from non=human species may be
recognized as foreign substances by the human immune system and neutralized
such
that they are less useful. Humanized antibodies are immunoglobulin molecules
comprising a human and non-human portion. More specifically, the antigen
combining region (variable region) of a humanized antibody is derived from a
non-
human source (e.g. marine) and the constant region of the humanized antibody
is
derived from a human source. The humanized antibody should have the antigen
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binding specificity of the non-human antibody molecule and the effector
function
conferred by the human antibody molecule. Typically, creation of a humanized
antibody involves the use of recombinant DNA techniques.
The-antibodies of the present invention can also be linked to an
insoluble support to provide a means of isolating euchromatin or
heterochromatin
from cells. The support may be in particulate or solid form and could include,
but is
not limited to: a plate, a test tube, beads, a-ball, a filter or a membrane.
Methods for
fixing antibodies to insoluble supports are known to those skilled in the art.
In one
embodiment an antibody of the current invention is fixed to an insoluble
support that
is suitable for use in affinity chromatography.
The Methyl(K4)H3 and Methyl(K9)H3 antibodies are large-scale or
domain-sensitive chromatin marks that are somehow set up by boundary elements.
In
particular, chromatin that is associated with histones that include H3
methylated at
Lsy4 represent an "on" domain, or at least a domain that is competent fox
transcriptional activity. Alternatively, chromatin that is associated with
histones that
include H3 methylated at Lsy9 represent an "off" domain, that is not competent
for
transcriptional activity. This pattern is conserved across a diverse range of
species.
Zoo blots with these antibodies suggest that most of the H3 histones from
'simple'
organisms (budding yeast) and Tetrahymena, contain a methylated Lys4 (ON),
whereas in striking contrast, most of the H3 histones present in 'complex'
organisms
have a methylated Lys9 (OFF) (see Fig. 3). This agrees well with the fording
that
most of the genomic DNA in yeast.and in Tetrahymena is expressed (ON) while
most
of the DNA in humans, mice, etc. is OFF. Thus, it may well be that the
'default' or
ground state in more complex eukaryotes is OFF. Knowing how to identify ON
chromatin through use of the Lys4 methyl mark may prove invaluable in
developing
strategies for better targeting of transgene to more 'friendly' chromatin.
Chromatin immunoprecipitation data supports the above 'ONlOFF'
marking system model. In particular, S. pombe chromatin IP data, published
(Science
Nakayama et al., 2001) and the work on the inactive X chromosome in humans
(see
Example 2) supports this model. Furthermore there are 'hot spots' of Lys4 H3
methylation on the 'inactive' X chromosome and in some cases tumor supressor
genes
have been mapped into this chromosomal region. Loss of heterozygosity at this
gene
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correlates well with a significant number of advanced cases of ovarian
cancers.
Therefore the present antibodies can also be used as diagnostics for detecting
cancer
and for determining therapy strategies. In accordance with one embodiment a
method
is provided for detecting chromatin alterations that are associated with a
disease state.
The term "disease state" is intended to encompass any condition that is
associated
with an impairment of the normal state of a living animal or plant including
congenital defects, pathological conditions such as cancer, and responses to
environmental factors and infectious agents (bacterial, viral, etc.). The
method
comprises the steps of isolating chromatin from both normal and diseased
tissue,
contacting the two pools of chromatin with either the Methyl(K4)H3 or
Methyl(K9)H3 antibody and comparing the staining pattern of the chromatin
isolated
from normal tissue to that of the diseased tissue. Furthermore, using
chromatin
immunoprecipitation, unique tumor suppressor genes could be isolation by
differential
screening using the antibodies of the present invention.
In accordance with one embodiment of the present invention the
Methyl(K4)H3 and Methyl(K9)H3 antibodies are used to identify heterochromatin
and
euchromatin regions and thus detect transcriptionally active and inactive
regions of
chromatin. More particularly, the antibodies can be used to detect changes in
chromatin structure that are associated with a given disease state. Therefore
the
~0 antibodies can be used as a diagnostic to detect alterations of chromatin
structure that
are associated alterations in expression patterns (i.e. differences in
heterochromatin vs
euchromatin patterns relative to predominant native patterns). Alterations in
chromatin structure for a specific region of chromatin may be diagnostic of a
particular disease state. For example, conversion of a normally euchromatic
region of
the genome to heterochromatin may represent the suppression of a tumor
suppressor
gene that is indicative of cancer or a pre-cancer state. 'Similarly the
conversion of a
region of heterochromatin to euchromatin may be associated with the
inappropriate or
overexpression of a gene that has deleterious effects on the host
cell/organism.
The present invention is also directed to a method of using broad-based
differential screening techniques to isolate nucleic acid regions that have
altered
expression patterns in diseased tissues. For example, chromatin can be
isolated from
diseased tissues and compared to chromatin isolated from healthy tissues to
determine
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if there are any differences in the chromatin structure (i.e. changes in
heterochromatin
vs. euchromatin) that are associated with the disease state. Such differences
in
chromatin structure may represent suppression or overexpression of genes that
play a
direct or indirect role in the disease. The anti-methyl(Lys 9) H3 and anti-
methyl(Lys
4) H3 antibodies can be used to detect such changes in chromatin structure and
help
identify genes that are associated with the disease state. The identification
of such
genes will assist in designing more effective therapies for treating the
disease.
In one embodiment the method for detecting alterations in chromatin
structure associated with a particular disease comprises chromatin
immunoprecipitation assays, using modification-specific histone antobodies.
This
process allows for the analysis of a wide range of DNA-templated processes
that are
governed by the chromatin environment. More particularly, the method comprises
the
steps of isolating chromatin from both diseased tissue and healthy tissue,
fragmenting
the DNA (preferably by sonification), and immunoprecipitating chromatin using
an
antibody that specifically binds to the amino acid sequence of ARTKQTAR (SEQ
ID
NO: 1) or QTARKSTGV (SEQ ID NO: 2), wherein the bold I~ represents a
methylated lysine residue, and comparing the chromatin (and the associated DNA
sequences) immunoprecipitated from the healthy tissue relative to the diseased
tissue.
Comparison of the two pools of immunoprecipitated chromatin will allow for the
~ identification of differences between diseased and healthy tissues.
In one embodiment, comparison of the two pools of
immunoprecipitated chromatin comprises the steps of isolating the nucleic acid
sequences associated with the two pools of immunoprecipitated chromatin and
comparing the resulting two pools of nucleic acid sequences. Comparison of the
two
pools of nucleic acid sequences can be conducted using any of the standard
molecular
techniques, including PCR, gel electrophoresis, nucleic acid sequencing and
nucleic
acid hybridization analysis. Those nucleic acid sequences that are present in
only one
of the two pools of nucleic acid sequences are then recovered. These nucleic
acid
sequences represent expressed/suppressed genes that are associated with either
the
normal or diseased tissue. In one embodiment the antibodies used to
immunoprecipitate the chromatin are selected from the group consisting of
Methyl(K4)H3 and Methyl(K9)H3 antibody.
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The use of methyl Lys4/Lys9 histone H3 antibodies in chromatin
immunoprecipitation (chromatin IP) assays is one way to enrich for genomic DNA
corresponding to the epigenetic 'ON/OFF' state of the human genome (and other
genomes as well). By combining chromatin immunoprecipitated DNA with current
genomic microarray technology (on chips), one has the potential to survey any
portion
of the human (or other) genome as to their on/off state through the 'histone
code'. For
example, DNA immunoprecipitated using the Methyl(K4)H3 antibody can be
immobilized on a solid surface or "chip" and thus represent all the nucleic
acid
sequences of a given cell that is competent for transcription. Similarly, DNA
immunoprecipitated using the Methyl(K9)H3 antibody can be immobilized on a
solid
surface or "chip" and thus represent all the nucleic acid sequences of a given
cell that
is not competent for transcription. Harvesting mRNA or preparing cDNA from a
target cell, labeling the target nucleic acids and then hybridizing the target
DNA with
the immobilized DNA will reveal abnormal expression of genes.
Knowing this information may prove invaluable in determining the
on/off state of key tumor suppressor or oncogenic proteins in various human
cancers.
Knowing how this epigenetic marking corresponds to genomic DNA will also guide
the ability to produce transgenic animals and plants where one often finds
that most
transgenic DNA enters a 'bad' (Lys9) chromatin environment and is silenced.
Thus,
the implications for knowing how to better 'guide' DNA into a 'good' (Lys4)
chromatin environment for animal and plant transgenic work are high. In.
humans,
this would impact on gene therapy issues as well. .
In one embbdiment, immunoprecipitation of chromatin will be used to
map the location of active genes at a genome-wide level through the use of
microarrays. For example, in one preferred embodiment the method of comparing
the
two pools of immunoprecipitated chromatin (i.e. the iinmunoprecipitated
chromatin
from diseased vs healthy tissues) comprises the use of a gene chip, DNA
microarray,
or a proteomics chip using standard techniques known to those skilled in the
art. For
example any of the systems described in WO O1/16860,W0 01/16860, WO 01/05935,
WO 00/79326, WO 00/73504, WO 00/71'746 and WO 00/53811 (the disclosures of
which are expressly incorporated herein) are suitable for use in the present
invention.
Preferably the chip will contain an ordered array of known compounds, such as
known
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DNA sequences, so that interaction of the immunoprecipitated chromatin at a
specific
location of the chip will identify, and allow for the isolation of, DNA
sequences
associated with the immunoprecipitated chromatin.
The key to this technology is the use of antibodies specific to various
modification as they relate to the histone code. Applying this to human and
other
genomes would lay the foundation of epigenomics. While the present invention
has
detailed the use of Lys4/Lys9 methyl H3 antibodies as respective ON/OFF
antibodies,
this concept applies more generally to any and all antibodies that are
developed
directed at the 'histone code'. For example, Lys9 methyl vs. SerlO phos H3
antibodies may also be a 'methyl/phos' switch that regulates differentiation
vs.
proliferation. The present invention also encompasses antibodies that are
directed to
other methylated regions of the amino terminus of H3 and H4 histones,
including H3
lysines 27 and 36 and H4 lysine 20. The peptides that will be used to generate
these
antibodies are listed below:
H3 lysine 27: AARKSAPVCG (SEQ ID NO: 16)
H3 lysine 36: SGGVKKPHKCG (SEQ ID NO: 17)
H4 lysine 20: R,HR-KTT.RDCG (SEQ ID NO: 18)
wherein the bold K is the methylated lysine residue and underlined GC refers
to
artificial amino acids added to the H3 sequence to aid in the production of
this
antibody.
The antibodies of the present invention can be used in standard
Molecular Biology techniques such as-Western blot analyses,
immunofluorescence,
and immunoprecipitation. As noted above the presence of methylated H3 at
lysine 4
correlates with transcriptionally active nuclei, and therefore, this H3
antibody may be
a useful in the understanding of gene regulation. In addition it is
anticipated that
microinjection of the Methyl(K4)H3 antibody into cells may interfere with the
activation of specific genes.
In one embodiment of the present invention a kit is provided for
detecting euchromatin and heterochromatin. The kit comprises an antibody that
specifically binds to a lysine methlyated modified peptide selected from the
group
consisting of ARTKQTARGC (SEQ ID NO: 4), QTARKSTGVCG (SEQ ID NO: 5),
ARTKQTAR (SEQ ID NO: 1), QTARSTGV (SEQ ID NO: 2), ARTKQTARKSTGV
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(SEQ ID NO: 9), A.ARKSAPVCG (SEQ ID NO: 16), SGGVKKPHKCG (SEQ ID
NO: 17) and RHRHILRDCG (SEQ ID NO: 18). More particularly, the kit comprises
an antibody that binds to the peptide ARTKQTARGC (SEQ ID NO: 4),
QTARKSTGVCG (SEQ ID NO: 5) or QTARKSTGGCG (SEQ ID NO: 6), wherein
the bold K represents a methylated lysine residue. In one embodiment the
antibodies
are attached to an insoluble support, wherein the support is either a
monolithic solid or
is in particular form. In one preferred embodiment the antibodies are
monoclonal
antibodies and in a further embodiment the antibodies are labeled. To this
end, the
antibodies of the present invention can be packaged in a variety of
containers, e. g. ,
vials, tubes, microtiter well plates, bottles, and the like. Other reagents
can be
included in separate containers and provided with the kit; e.g., positive
control
samples, negative control samples, buffers, cell culture media, etc.
In an another embodiment of the invention a kit is provided for use in
an assay to determine if a sample has methylase activity. The kit comprises a
peptide
selected from the group consisting of ARTKQTARGC (SEQ ID NO: 4),
QTARKSTGVCG (SEQ ID NO: 5), ARTKQTAR (SEQ ID NO: 1), QTARSTGV
(SEQ ID NO: 2) and ARTKQTARKSTGV (SEQ ID NO: 9) and an antibody that
specifically binds to a lysine methlyated modified peptide selected from the
group
consisting of ARTKQTARGC (SEQ ID NO: 4), QTARKSTGVCG (SEQ ID NO: 5),
ARTKQTAR (SEQ ID NO: 1), QTARSTGV (SEQ ID NO: 2) and
ARTKQTARKSTGV (SEQ ID NO: 9). In one embodiment the antibodies are
attached to an insoluble support, wherein the support is either a monolithic
solid or is
in particular form. In another embodiment the kit is further provided with an
antibody
that specifically binds to a non-methylated peptide selected from the group
consisting
of ARTKQTARGC (SEQ ID NO: 4), QTARKSTGVCG (SEQ ID NO: 5),
ARTKQTAR (SEQ ID NO: 1), QTARSTGV (SEQ ID NO: 2) and
ARTKQTARKSTGV (SEQ 117 NO: 9). Such an antibody serves as a negative
control.
In one embodiment, the method for detecting the methylase activity of
a sample comprises contacting a peptide selected from the group consisting of
ARTKQTARGC (SEQ ID NO: 4), QTARKSTGVCG (SEQ ID NO: 5), ARTKQTAR
(SEQ ID NO: 1), QTARSTGV (SEQ ID NO: 2) and ARTKQTARKSTGV (SEQ ID
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NO: 9) for a predetermined length of time with a sample that is suspected of
having
methylase activity. The amount of methylation-specific antibody (i.e.
Methyl(K4)H3
or Methyl(K9)H3) that binds to the substrate is a direct correlation of the
extent the
substrate was methylated during the predetermined time length and thus
indicates the
methylase activity of the sample. This assay can also be used to screen for
potential
inhibitors of a methylase. For example, in one embodiment a method of
screening for
inhibitors of arginine methyl transfer activity comprises the steps of
providing a
sample, wherein the sample comprises a methylase and a substrate that is
methylated
by said methylase, adding a potential inhibitor of the methylase to the
sample, and
contacting the sample with an antibody that binds specifically to the
methylated
substrate, but not the non-methylated substrate. In one embodiment, the
antibody is
specific for the peptide ARTKQTAR (SEQ ID NO: 1) or QTARKSTGV (SEQ ID
NO: 2). Quantifying the amount of antibody bound to the peptide is a direct
correlation of the level activity of the methylase in the sample. In one
preferred
embodiment the methylase activity to be detected is SuVar3-9 (for Lys 9) or
Setl (for
Lys 4).
'Knock-out' strains are available for all of the non-essential genes
present in budding yeast...around 4,800. The antibodies of the present
invention have
such a high degree of specificity, that they only detect one or two major
bands in yeast
whole cell lysates, thus allowing for the development of a robotic screening
method to
look at all of these knock-out strains. Using Lys4 methyl H3 should lead to
the entire
upstream pathway of 'regulators' including the on and off enzymes (provided
the gene
product is non-essential).
Using the above approach in 'old-fashioned' blots, is has been found
that Setl is the enzyme responsible for the Lys4 H3 methyl mark in yeast, and
is also
one of the Lys4 HMTases in humans. Surprisingly, using the methyl Lys4 H3
antibodies as a readout to probe yeast whole cell lysates, Lys4 methylation
was
discovered to be regulated by H2B ubiquitination at a conserved Lysine on the
opposite side of the nucleosome. This is the first example of a 'trans-tail'
effect
meaning that one histone modification on one tail effect another modification
on
another not-so-close tail. In humans and in mice, the enzymes that add
ubiquitin to
proteins is human Rad6 (HR6), and HR6 comes in two isoforms, HR6A and HR6B.
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Mice knockouts of HR6B -/- are male sterile in a pathway that is not known,
but
seems to lead to chromatin defects during spermatogenesis leading to sperm
death.
Accordingly it is anticipated that HR6B is responsible for ubiquitin addition
on H2B
and therefore it is possible that Lys4 H3 methyl antibodies will be a
diagnostic for
male infertility. Furthermore, the possibility exists that defects in Lys9
methylation
could impact X-inactivation and lead to female infertility. Therefore in
accordance
with one embodiment of the present invention the Methyl(K4)H3 and Methyl(K9)H3
antibodies are used as a diagnostic to.screen for male and female infertility
defects.
Current models suggest that Lys9 methylation is catalzyed, at least in
some instances by SuVar3-9. It appears that the Lys9 methyl mark is read by
chromodomains, short protein modules that act as chromatin 'velcro' patches.
Best
documented is the chromodomain of the heterochromatin protein HP 1.
Interestingly,
the chromodomain from HP1, binds well to Lys9 methyl H3 peptides, but binds
much
less well to Lys4 or unmodified peptides. It is interesting also that SuVar3-9
itself, a
catalytic HMTase, also has a chromodomain, a module whose site specificity for
methylated histone peptides has yet to be tested.
It seems likely that the Lys4 methyl mark in H3 will be read by a
distinct chromodomain from that of HP 1. In one embodiment the uniquely-
modified
peptides of the present invention, including ARTKQTAR (SEQ ID NO: 1) or
QTARKSTGV (SEQ ID NO: 2) are used as affinity reagents to look for
polypeptides
that bind Lys4 H3 peptides. Several attractive candidates include two histone
acetyltransferases (HATS), Esal and.CDY, both of whom have chromodomains.
Interestingly CDY is a testis-specific HAT encoded on the male Y chromosomes,
and
somatic histones are well known to be hyperacetylated during a reaction that
leads to
replacement by protamines.
It could be that during spermatogenesis the following series of
concerted reactions occur:
1. H2B ubiquitination happens, catalyzed by HR6B
2. Chromatin opening
3. Lys4 methylation catalyzed by human Setl or another Lys4 HMTase
4. Binding of the HAT CDY to the Lys4 methylation mark though its
chromodomain
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Histone hyperacetylation occurs catalyzed by CDY
6. Somatic histones are displaced, followed by transition proteins,
followed by protamines
Defects in any of the above steps could lead to sperm lethality and male
infertility.
Note that three of the steps require chromatin-modifying enzymes:
i) HR6B a E2 ubiquitin-conjugating enzyme
ii) Setl or a resposible Lys4 HMTase
iii) CDY a male-specific HAT
Antibodies to ubiquitinated H2B and H2A, Lys4 methyl H3 and sites of CDY-
catalyzed acetylation would all be of potential diagnostic value in male
infertility
screens.
Example 1
Preparation of the Methyl(K4)H3 and the Methyl(K9)H3 antibodies
To generate the Methyl(K4)H3 and the Methyl(K9)H3 antibody, a
short polypeptide corresponding to the amino-acid sequence of histone H3
surrounding lysine 4 (SEQ ID NO: 4; ARTKQTARGC) or lysine 9 (SEQ ID NO: 5;
QTARKSTGVCG) was first chemically synthesized, wherein the bold K is the
methylated lysine residue and underlined GC refers to artificial amino acids
added to
the H3 sequence to aid in the production of this antibody. This polypeptide
was then
conjugated to cationized bovine serum albumin (BSA), and the conjugated-
peptide
was injected into rabbits. One important aspect to this procedure is the fact
that the
standard technique of conjugating a peptide to Keyhole Limpet Hemocyanin (KLH)
proved ineffective in generating high quality methyllysine 4 (H3)-specific
antisera.
Thus, the use of this specialized cationized BSA is now considered a unique
"method"
in generating methyllysine-specific antibodies.
Rabbit serum was harvested at regular intervals post-immunization,
and the Methyl(K4)H3 and Methyl(K9)H3 antibodies were shown to be present in
the
Serum by standard enzyme-linked immunosorbent assays. These antibodies are
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suitable for use in Western blot, immunofluorescence, and immunoprecipitation
assays.
Example 2
Role of Histone H3 Lysine 9 Methylation in Epigenetic Control of
Heterochromatin Assembly
The assembly of higher order chromatin structures has been linked to
the covalent modifications of histone tails. Ih vivo evidence demonstrates
that lysine
9 of histone H3 (H3 Lys 9) is preferentially methylated by. the Clr4 protein
at
heterochromatin-associated regions in fission yeast. Both the conserved chromo-
and
SET domains of Clr4 are required for H3 Lys 9 methylation i~ vivo.
The organization of the higher order chromatin structure has been
linked to the posttranslational modifications of histone tails, including
acetylation,
phosphorylation, and methylation. It has been suggested that distinct
combinations of
covalent histone modifications, also referred to as the "histone code,"
provide a
"mark" on the histone tails to recruit downstream chromatin-modifying
proteins. This
is best illustrated by recent studies indicating that the conserved bromo-
domain of
several transcriptional coactivators bind specifically to acetylated lysine
residues on
histone tails. The mechanisms responsible for the establishment and
maintenance of
multiple covalent modifications within the same or different histone tail are
not-fully
understood.
Modifications of histone tails have also been linked to heterochromatin
assembly. Histones H3 and H4 are largely hypoacetylated in heterochromatic
chromosomal regions in organisms as diverse as yeast, flies, and mammals. In
fission
yeast, hypoacetylation of histones is associated with the silent mating-type
region and
centromeres, chromosomal domains that share many parallels with
heterochromatic
regions in higher eukaryotes. Centromeric regions comprising a central core of
unique sequences surrounded by inner (imr) and outer (otr) repeats are
assembled into
silenced chromatin structures. Similarly, a laxge ;15-kb chromosomal domain at
the
mating-type (mat2l3) region, including the matt and mat3 loci and an interval
between them, known as the K-region, is maintained in a silent epigenetic
state.
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Among the traps-acting factors that affect silencing at these regions, Clr3
and Clr6
belong to family of histone deacetylases (HDACs). Swi6 and Clr4 proteins
contain a
chromodomain, an evolutionarily conserved motif initially identified in HP 1
and
Polycomb proteins. Recently, both Clr4 and its mammalian counter-part,
SUV39H1,
have been shown to have intrinsic histone H3-specific methyltransferase
(HMTase)
activity ih vitro (S. Rea et al., Nature 406, 593 (2000)). However, it is not
known
whether histones are the physiological targets of these methyltransferases ih
vivo.
Consistent with previous findings, recombinant Clr4 (rClr4) was found
to contain HMTase activity exclusively for histone H3. To identify the
specific
residue of H3 methylated by rClr4, synthetic peptides derived from the NH 2 -
terminus of H3 were used as substrates in an in vitro HMTase assay. In
particular,
five milligrams of HeLa or chicken core histones was incubated with 0.55 mCi
of S-
adenosyl-L-[methyl-3H]methionine (3H-AdoMet; 72 Cilmmol; 1 mM final) and 2 mg
of recombinant Clr4 wild-type or mutant proteins in 25 ml of HMTase buffer [50
mM
tris ( pH 8.0), 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol in
10%
glycerol] for 1 hour at 30°C. SDS loading buffer was added to half of
each sample
and boiled followed by separation on a 15% SDS-polyacrylamide gel
electrophoresis
(PAGE) gel. The resulting histone bands were visualized by Coomassie staining
and
fluorography. For the peptide analysis, 5 mg of each peptide derived from the
NH2-
terminus of human histone H3 containing a COOH-terminal cysteine was used.
Half
of the sample was spotted on Whatman P-81 filter paper and washed four times
for 10
min in 50 mM NaHC02 ( pH 9.0), followed by liquid scintillation counting. (B.
D.
Strahl, R. Ohba, R. G. Cook, C. D. Allis, P~oc. Natl. Acad. Sci. U.S.A. 96,
14967
(1999)). Clr4 preferentially methylated the H3 1-20 unmodified peptide but
failed to
methylate the H3 19-35 unmodified peptide, indicating that the target residue
of Clr4
HMTase resides in the first 20 amino acids of H3.
To determine this target residue, a synthetic H3 1-20 peptide set was
developed that contained covalent modifications on different amino acids. With
these
peptides as substrates, only acetyl or methyl modifications on Lys 9
effectively
blocked rClr4 HMTase activity, indicating that Clr4, like its mammalian
homolog
SUV39H1, selectively methylates Lys 9 of H3. Furthermore, similar to SUV39H1,
rClr4 HMTase activity was inhibited by phosphorylation of serine 10. These
results
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demonstrate that enzymatic features of the Su(var)3-9 protein family are
evolutionarily conserved from fission yeast to humans. A recent study
demonstrated
that the conserved SET domain and two flanking cysteine-rich regions were
required
for SUV39H1 HMTase activity in vitro. To determine whether the conserved
domains, the chromo, SET, and cysteine-rich regions, were also critical for
Clr4
HMTase activity, mutant Clr4 proteins were tested for HMTase activity.
Although
mutations in the chromo-domain [Trp3' to- Gly (W31 G) and Trp4' to Gly (W41
G)] had
little effect on Clr4 HMTase activity, mutations in the SET domain [Gly3z8 to
Ser
(G378S)] and both cysteine-rich regions [Arg3zo to His (R320H) and G1y486 to
Asp
(G486D)] greatly reduced Clr4 HMTase activity, indicating that these three
regions
are critical for Clr4 HMTase activity i~ vitro.
To test the hypothetical correlation between H3 Lys 9 methylation and
silencing, an H3 Lys 9 methyl specific antibody was developed. In an enzyme-
linked
immunosorbent assay, the H3 Lys 9 -methyl antibody specifically recognized the
H3
1-20 Lys 9 -methyl peptide in a wide range of antibody dilution. Moreover, the
H3
Lys 9 -methyl antibody did not detect recombinant histone H3 (rH3) alone
compared
with the HeLa core histone positive control but did detect rH3 selectively
methylated
by rClr4, further demonstrating the specificity of this antibody (see Nakayama
et al.,
(2001) Science, 292, pp 110-113; the disclosure of which is incorporated
herein).
Using this antibody in chromatin immunoprecipitation (ChIP)
experiments (Nakayama et al, (2000) Cell, 101, 307), it was discovered that
the H3
Lys 9 methyl modification is specifically localized at the silenced
chromosomal
regions. H3 Lys 9 methylation and Swi6 were preferentially enriched at a
marker
gene (King: : u~a4 1 ) inserted within the silenced mat2/3 chromosomal domain,
compared with control ura4DSlE locus at the endogenous location. Similarly, H3
Lys
'9 methylation was also preferentially enriched at the ui~a41 marker inserted
within the
highly repressed innermost repeat (imrl R: : ura41 ) and the outer repeat
(otr~1 R: : ura4
1 ), but not at the weakly repressed central core (cntl:: ura4 1 )of cehl. In
addition,
H3 Lys 9 methylation coincided with the presence of Swi6 at these regions
(Partridge
et al, (2000), Genes Dev., 14, 783). These findings suggest that H3 Lys 9
methyl
modification and Swi6 are preferentially localized to silent chromosomal
regions and
that Swi6 localization is functionally dependent on H3 Lys 9 methylation.
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Compared with the relatively high levels of Swi6 and H3 Lys 9
methylation at both Kint2: : ura41 and otrl R: : ura4 1 in wild-type cells,
Swi6 and H3
Lys 9 methylation were absent in a clr4D strain at both loci. This result
suggests that
H3 Lys 9 is the physiological target of Clr4 HMTase activity and that Clr4
appears to
S be the exclusive ivy vivo H3 Lys 9 -specific HMTase at mat and cen loci. In
comparison with the ire vitro result showing that only the SET domain is
required for
Clr4 HMTase activity, both the chromo-and SET domains are required for H3 Lys
9
methylation and Swi6 localization ire vivo. Taken together,~these results
indicate that
the chromodomain is presumably required for targeting Clr4 to the mat2/3
region and
centromeres, whereas the SET domain and associated cysteine-rich regions of
Clr4
constitute the catalytic site. The Swi6 levels at mat and ceh in different
clr4 mutant
backgrounds were directly correlated with H3 Lys 9 methylation levels, further
suggesting that Swi6 localization at silent chromosomal domains is
functionally
dependent on H3 Lys 9 methylation.
The importance of the in vivo analyses was further highlighted by
observations that some mutations in Clr4 that decrease its HMTase activity ih
vitro do
not substantially decrease H3 Lys 9 methylation and Swi6 localization ih vivo.
In
addition, mutations in the SET domain and the NH 2 -terminal cysteine-rich
regions
of Clr4 (G378S and R320H) greatly reduce H3 Lys 9 methylation and Swi6
localization at the mat locus; however, these mutations have moderate or
negligible
effects at cell. These mutations also have weak effects on centromeric
silencing
compared with mating-type silencing. The results are consistent with the
notion that
enzymatic defects displayed by recombinant monomeric proteins in vitro can be
"rescued" by functioning in the context of a multisubunit complex in vivo.
Moreover,
the functional organization of the mat2/3 region and centromeres may differ,
and an
additional factors) may help promote Clr4 activity at centromeres.
Mutations in the clr3 HDAC, which specifically deacetylates H3 Lys
14, affects silencing at mat and cen (S. I. S. Grewal, M. J. Bonaduce, A. J.
S. Klar,
Genetics 150, 563 (1998)). ChIP analysis demonstrated that a clr3-735 mutant
partially defective in H3 Lys 14 HDAC activity displayed a moderate decrease
in H3
Lys 9 methylation and Swi6 localization at otrl: : ura4+, coincident with the
apparent
reduction in its HDAC activity. This result suggests that H3 Lys 14
acetylation
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inhibits Clr4 HMTase in vivo. To further investigate the functional
interaction
between Clr3 and Clr4, a double-mutant strain containing the clr3-735 and
clr4R320H
mutations was created, a clr4 mutation that had the least effect on H3 Lys 9
methylation at otrl R: : ura4+. ChIP analysis of the double mutant
demonstrated that
H3 Lys 9 methylation and Swi6 localization were nearly abolished when compared
with the single mutants. These findings indicate that Clr3 acts
synergistically with
Clr4 to effectively localize Swi6 to heterochromatic domains. In other words,
deacetylation of H3 Lys 14 by Clr3 is required for H3 Lys 9 methylation by
Clr4 and
for Swi6 localization either indirectly, by altering Clr4 activity, or
directly or both.
These data also support the theory that residues neighboring Lys 9, and
potentially
their modification states, play an important role in establishment of the
appropriate H3
Lys 9 -methyl mark. Previous studies have shown that rikl + affects silencing
as well
as Swi6 localization at silent loci. Computational analyses revealed that Rik1
contains b-propeller domains typically found within WD-40 repeat proteins and
are
theorized to participate in protein:protein interactions. A mutation in rikl
completely
abolished H3 Lys 9 methylation and Swi6 localization at both mat and ceh
compared
with wild type.
WD-40 proteins are involved in many aspects of chromatin remodeling
and histone metabolism, such as chromatin assembly and acetylation or
deacetylation
of histones. Therefore, the b-propeller domains of Rikl may form a complex
with
Clr4 to recruit its HMTase activity to heterochromatic regions and may play a
role in
coupling other transacting factors, such as Swi6 and histone deacetylases.
The possible role of Swi6 on Clr4-dependent methylation of H3 Lys 9
was also tested. Strains carrying swi6-115 (W269R) mutation that severely
reduced
Swi6 protein levels were used. As expected, Swi6 localization at both mat and
cen
was abolished as demonstrated by ChIP analysis. The'swi6-11 S mutation did not
cause any detectable change in H3 Lys 9 methylation when compared with the
wild-
type strain. These data indicate that Swi6 is dispensable for Clr4 function
and suggest
that Swi6 acts down-stream of Clr4 H3 Lys 9 methylation. Collectively, the
above
results define a temporal sequence of events leading to establishment of the
silenced
chromatin state with regard to the covalent modifications of the H3 NH 2 -
terminal
tail. HDACs and HMTases act cooperatively to establish a "histone code" that
is then
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recognized by Swi6. More specifically, the HDACs (Clr6 and/or Hdal)
deacetylate
H3 Lys 9, whereas Clr3 deacetylates H3 Lys 14 before H3 Lys 9 methylation by
the
Clr4/Rik1 HMTase complex. Swi6 binding to the H3 Lys 9 -methyl modification
would then result in self propagating heterochromatin assembly. Because the
heterochromatin-binding domain of Swi6 was mapped to its chromodomain, it is
most
likely that this protein motif has evolved to recognize the H3 Lys 9 -methyl
modification. -
It was recently shown that Swi6 remains associated with the mat2/3
region throughout the cell cycle where it acts as an important determinant of
the
epigenetic cellular memory, promoting inheritance of the silenced state.
Because the
mouse homolog of Swi6, M31, associates with Su(var)3-9, a similar inter-action
between Clr4 and Swi6 is predicted. The close association of Clr4 enzymatic
HMTase activity, followed by recruitment and binding of Swi6 to Lys 9 methyl
"marks" in H3 through its chromodomain, suggests a pathway of epigenetic
inheritance. The extent to which the chromo-domain of Clr4 recognizes H3 Lys 9
-methyl marks is unknown, but it would provide the enzyme a means to bind
chromatin as it performs subsequent methylation events. On the basis of the
conservation of Clr4/SLTV39H1 and Swi6/HP1 proteins and the presence of H3 Lys
9
methyl modification in higher eukaryotes, a similar mechanism may be
responsible
for higher order chromatin assembly in organisms ranging from fission yeast to
humans. Considering the parallels between transcriptional repression by
Polycomb
group proteins in flies and mammals and silencing in fission yeast, it is
likely that
histone methylation coupled with histone deacetylation may help localize
Polycomb in
pathways that lead to the regulation of homeotic gene expression.
Example 3
Differential Sites of Histone H3 Methylation Marks the Active and Inactive
Genes on the Human X Chromosome
Chromosomes in higher eukaryotes have historically been considered
to consist of regions of euchromatin and heterochromatin, which are
distinguished by
the degree of condensation and level of transcriptional activity of the
underlying DNA
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sequences. Certain regions of constitutive heterochromatin are found at or
near
specialized structures such as centromeres, and are comprised mostly of
genetically
inert repetitive sequences. In contrast, other regions that have the same
primary DNA
sequences can exhibit characteristics of either type of chromatin, suggesting
that
epigenetic factors, such as packaging of DNA by histones and chromatin-
associated
proteins, dictate the heterochromatin status at these loci.
One of the most dramatic examples of epigenetic silencing is the X
chromosome inactivation seen in female cells of mammals. This process allows
for
dosage compensation of X-linked genes whereby one of the two copies of the X
chromosome in female cells is randomly inactivated during embryonic
development.
Current evidence suggests that X inactivation is initiated by the up-
regulation of the
non-coding XIST transcript and its association in cis with the chromosome to
be
inactivated. Following its coating by XIST RNA, the inactive X chromosome
acquires heterochromatic characteristics such as late replication timing, a
condensed
appearance (Barr body) in interphase cells, DNA methylation of CpG islands at
house-keeping genes, and association with altered nucleosomes that are
composed of
hypoaceiylated histones and enriched for the H2A variant MacroH2A. While the
exact roles of these properties in the onset of X inactivation remain unclear,
once the
inactive state has been established, these epigenetic characteristics seem to
act
synergistically to maintain the remarkable stability of the inactive X through
many cell
divisions in the adult soma.
Recently, several publications showed that histone H3 methylation is
important in the assembly of heterochromatin in mouse and S pombe (Lachner et
al.,
Nature 410, 116 (2001); Bannister et al., Nature 410, 120 (2001) and Nakayama
et al
Sciehce 292, 110 (2001)). The chromodoma.in of mouse HP1 (and Swi 6 in S.
pombe)
can bind to H3 methylated at lysine 9, and methylatiori of H3 at this site is
thought to
mark and recruit factors involved in heterochromatin assembly. In addition,
acetylation and methylation of H3 at lysine 9 may be competing events in uivo
(Cheung et al., Cell 103, 263 (2000)). Given that H3 is hypoacetylated on the
inactive
X chromosome, and that H3 lysine 9 methylation is important in heterochromatin
assembly, the inactive X was investigated for enrichment for Lys9-methylated
H3.
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Using an antibody specific for Lys9-methylated H3, female human
metaphase chromosomes were examined from a normal lymphoblast cell line (HH)
by
indirect immunofluorescence. In particular, a normal human female lymphoblast
cell
line (HH) and a female lymphoblast cell line which contains five X chromosomes
(6061B) were grown, harvested and collected onto microscope slides with a
Cytospin
3 centrifuge. Modified histones were detected by indirect immunofluorescence,
essentially as described in detail elsewhere (Costanzi and J. R. Pehrson,
Nature 393,
599 (1998)). Briefly, cells were incubated for one hour at 37 °C in a
humid chamber
with serial dilutions of the primary Lys9 methyl H3 or acetyl H4 antisera and
washed
in KCM (120mM KCI, 20mM NaCI 10 mM TRIS-CL, pH 8.0, 0.5 M EDTA, 0.1%
Triton). The cells were then incubated for 30 min at room temperature with Cy3-
conjugated, affinity-purified, donkey anti-rabbit IgG antibody (Jackson
ImmunoResearch) diluted 1:40 in KCM. Cells were once again washed with KCM
and fixed in 4% formaldehyde for 10 min at room temperature. Following a wash
in
sterile water chromosomes were counterstained with 4',6-diamidino-2-
phenylindole
(DAPI), mounted in antifade (Vectashield) and viewed on a Zeiss Axiophot
fluorescence microscope.
Indirect immunofluorescence using an antibody specific for Lys9-
methylated H3 revealed that while most chromosomes have some regions of Lys9-
methylated-H3, one chromosome in each metaphase spread is consistently more
intensely and uniformly stained (Figure 1A, white arrow). To test whether the
chromosome enriched for Lys9-methylated H3 is the inactive X chromosome, .
metaphase spreads from a cell line that contains five X chromosomes was also
stained. In these cells, four out of the five X chromosomes are known to be
inactivated and indeed four chromosomes of equal size show enriched staining
by the
Lys9-methyl H3 antibody. Together, these data suggest that while the H3
molecules
on the inactive X are mostly hypoacetylated, they are highly methylated at
lysine 9.
The N-terminus of histone H3 is post-translationally modified at
multiple sites and lysine 4 of H3 is another documented site of methylation.
However, in contrast to lysine 9, lysine 4 methylation has been correlated
with active
transcription. To further compare these two sites of H3 methylation on the
inactive X
chromosome, metaphase chromosomes were stained using an antibody specific for
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Lys4-methylated H3. Metaphase chromosomes from female cell lines HH and 6061B
were incubated with Lys4 methyl H3 antiserum and the staining pattern was
analyzed
by indirect immunofluorescence as described earlier. Strikingly, this antibody
intensely stains all chromosomes in the metaphase spread except for a single
chromosome per spread (Fig. 1B). This unique chromosome is almost totally
devoid
of staining except for several 'hot spots' of H3 lysine 4 methylation.
Staining of the
metaphase spreads of the SX cell line shows that four of the chromosomes are
understained using the Lys4 methyl H3 antibody, suggesting that the hypo-H3
Lys4-
methylated-chromosomes) is the inactive X chromosome.
Closer examination.of the inactive X chromosome shows that there are
several distinct regions of this chromosome that exhibit intense staining with
the Lys4
methyl H3 antibody. One region appears to be located at the pseudoautosomal
region
of the distal end of the p arm, another is located near Xq25-26 of the q arm,
and
fainter staining is occasionally seen around Xp 11. With the exception of Xq25-
26,
the other regions of Lys4 methyl H3 staining on the inactive X correspond to
the
location of multiple genes known to escape inactivation (Carrel et al, Proc
Natl Acad
Sci LT SA 96, 14440 (1999)), and these data are therefore consistent with the
idea that
Lys4 methylation of H3 is associated with active gene expression.
The intense staining of the Xq25-26 region by the Lys4 methyl H3
antibody is puzzling since this region does not contain any known genes that
escape X
inactivation. However, it is intriguing that loss of heterozygosity at Xq25-
26.1 is
associated with advanced human ovarian carcinomas (see Choi,et al, Genes
Chromosomes Cancer 20, 234 (1997)). Further studies of the enrichment of Lys4-
methylated H3 at this region on the inactive X chromosome, and determining
whether
H3 Lys4/Lys9 methylation correlate with the expression level of the putative
tumor
suppressor genes) at this location will provide additional information on the
link
between histone H3 methylation and gene expression.
The understaining of the inactive X chromosome by the Lys4 methyl
H3 antibody is similar, but not identical, to the staining pattern of this
chromosome
with antibodies against hyperacetylated form of H4. In this study, some
staining at the
telomeric region of the p arm of the inactive X chromosome was seen using the
antibody against hyperacetylated H4, but not at the Xpl 1 and Xq25-26 regions.
In
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contrast, previously published results showed that three regions on the
inactive X
chromosome (Xpter-22.2, Xp11.3-11.2, and Xq22) were stained with an antibody
against hyperacetylated H4, but this was seen only in sodium butyrate-treated
human
cells. At present, the precise relationship between the regions stained by the
hyperacetylated H4 and the Lys4 methyl H3 antibodies has not been clearly
defined.
Nevertheless, these data suggest that the inactive X chromosome is devoid of
hyperacetylated core histones as well as Lys 4-methylated H3, but is enriched
for H3
methylated at Lys9. Together, these combinations of histone modifications may
be
part of a series of concerted reactions that mediate transcriptional silencing
of the
inactive X.
To further establish the link between H3 Lys9 methylation and the
inactive X chromosome, human female IMR90 interphase cells were examined by
immunofluorescence in combination with Fluorescent In Situ Hybridization
(FISH).
In particular, IMR. 90 (ATCC) cells were grown on coverslips for 24-48 hours
then
fixed in 4% formalehyde for 15 minutes at room temperature; the cells were
then
permeabilised in PBS containing 0.5% Triton-X for 4 min. on ice, washed in PBS
and
then washed in 2xSSC prior to RNA FISH. To preserve nuclear structures, cells
were
kept continously hydrated. RNA FISH hybridization and washes were performed
essentially as described elsewhere (Lachner et al, Nature 410, 116 (2001)).
Briefly,
cells were hybridized with a XIST probe comprising a pool of four exon-derived
DNA
fragments spanning a total of 4.5 kb of sequence, labeled by nick translation
with
Spectrum Red or Green dUTP (Vysis, Downer Grove, IL). Following hybridization
overnight at 37°C, standard washes for RNA FISH (ie 3x in 50% formamide
l 2xSSC
and 3x in 2xSSC) were performed. The cells were then washed in PBS / 0.5% BSA
.
prior to performing immunofluorescence. Primary antibody was incubated for 1
hour
at room temperature, cells were washed four time in PBS and the secondary
antibody.
(Texas red conjugated goat anti-rabbit antibody ) was then applied for 1 hour
at room
temperature, followed by four washes in PBS. Nuclei were counterstained with
DAPI. Images were acquired using a Zeiss Axioplan 2 fluorescence microscope
with
an Orca 2 CCD camera (Hamamatsu) and Improvision software (IPLab).
It is well established that the XIST transcript specifically localizes to
the inactive X chromosome at interphase but not at metaphase, and localization
of the
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Xist transcript can be detected by FISH analysis. The Lys9-methyl-H3 antibody
preferentially stains a region that is heterochromatin-dense as indicated by
co-
localization with DAPI-dense regions. This condensed region also co-localizes
with
the XIST RNA signal, indicating that the chromosome enriched for Lys9-
methylated
H3 is indeed the inactive X. Consistent with the metaphase chromosome results,
staining of human interphase cells with the Lys4 methyl H3 antibody shows that
the
region corresponding to the inactive X (based on DAPI dense staining and
localization
of the Xist RNA) is devoid of Lys4 methyl H3 staining. Intriguingly, a
distinct dot
enriched for Lys4 methyl H3 is seen within the region of negative staining for
some of
the cells. Whether this localized region of Lys4 methyl. H3
staining.corrresponds to
the intensely stained regions seen in the metaphase chromosome is at present
not
known. Side by side comparisons of the Lys9 and Lys4 methyl H3 staining in
interphase and metaphase cells further showed that the respective staining
pattern of
these two antibodies are almost reciprocal images. These results have led to
the
conclusion that lysine 4 and lysine 9 methylation of H3 are 'reciprocal marks'
for
transcriptionally active and inactive regions respectively, and hence the
inactive X
chromosome is hypomethylated at lysine 4 but is hypermethylated at lysine 9.
One resulting prediction is that the Lys4 and Lys9 methyl H3
antibodies would respectively enrich for active and inactive genes on the X
chromosome by chromatin immunoprecipitation (ChIP). On the inactive X
chromosome, the XIST gene is transcriptionally active whereas the PGKl gene is
silenced. Conversely, on the active X chromosome, the XIST gene is silent
whereas
the PGKI gene is actively transcribed. To test the above hypothesis, two CHO
somatic hybrid cell lines were used that contain either a single active or
inactive
human X chromosome in ChIP assays in order to examine the histone
modifications
associated with genes present on the active or inactive X chromosomes.
Chromatin
from these two cell lines were immunoprecipitated using antibodies against
Lys9-
methylated H3, Lys4-methylated H3, or Lys9/14-acetylated H3, and the
immunoprecipitated DNA was PCR amplified using primers specific to the
promoter
regions of the human Xist and PGKl genes.
In particular, Chromatin immunoprecipitation assays were done as
described in Cheung et al, Mol Cell 5, 905 (2000). In this case, formaldehyde-
fixed
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chromatin was harvested from CHO somatic cell hybrids containing either the
active
or inactive human X chromosome. Approximately 3 x 106 cells-worth of sonicated
chromatin were used per immunoprecipitation reaction with the antibodies
indicated
in the text. After extensive washing, reverse cross-linking, RNase A and
proteinase K
digestions, the immunoprecipitated DNA was analyzed by PCR using primers
specific
for the promoter regions of the human JPIST and PGKI genes (primer sequences
and
PCR conditions were derived from Gilbert and P. A. Sharp, Proc Natl Acad Sci U
S A
96, 13825 (1999)), and analyzed by polyacrylamide gel electrophoresis.
Consistent with previously published reports, the acetylated H3
antibody immunoprecipitated XIST DNA only from the inactive X chromosome, and
the PGKI DNA only from the active chromosome. Analogous to these results, the
Lys4 methyl H3 antibody preferentially immunoprecipitated the XIST DNA from
the
cells actively expressing XIST (from the cells containing the inactive X
chromosome)
and the PGKI DNA from the cells containing the active X chromosome. Therefore,
both acetylated H3 and Lys4-methylated H3 are enriched at the actively
transcribing
loci on both the active and inactive X chromosomes. Immunoprecipitation using
the
Lys9 methyl H3 antibody showed reciprocal results to those obtained with the
Lys4
methyl and acetyl H3 antibodies. In this case, XIST DNA.was immunoprecipitated
only from cells containing the active X chromosome whereas the PGKI DNA was
immunoprecipitated only from the inactive X chromosome.
Taken together, the chromatin immunoprecipitation data indicate that
Lys4- or Lys9-methylated H3 are reciprocally associated with active and
inactive
genes irrespective of the chromosomal context (active versus inactive X).
Moreover,
combined with~the immunofluorescence results, these data suggest that the two
distinct methylation sites on H3 may mark regions of active and inactive
chromatin,
respectively.
The inactive X chromosomes is a well-studied paradigm for epigenetic
regulation of gene expression and fox linking specialized nucleosomal
architecture
with transcription silencing. As mentioned earlier, the entire inactive X
chromosome
seems to be largely devoid of hyperacetylated histones, and a core histone
variant,
MacroH2A, has been found to be enriched at the inactive X chromosome.
Interestingly, none of the above modifications alone can account for the
stability
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associated with this form of epigenetic regulation. Histone methylation,
however, has
recently been described as a more 'stable' epigenetic chromatin mark whose
functions
in X inactivation and as a potential cellular 'memory' marker have yet to be
explored.
Very recently, methylation of histone H3 at lysine 9 has been defined
to be an important modification for heterochromatin assembly, and as shown
here, this
modification is also enriched in the facultative heterochromatin of the
inactive X
chromosome. In contrast, methylation of-H3 at lysine 4 is complementarily
absent in
the inactive X chromosome, suggesting that methylation of H3 at these two
distinct
sites may be reciprocal, and that H3 molecules methylated at lysine 4 are
preferentially associated with transcriptionally active genes whereas the
opposite is
true for H3 methylated at lysine 9. These findings provide strong evidence in
support
of the concept that specific modifications at specific sites on the histone
amino-
terminal tails can impart distinct characteristics, and perform different
cellular
functions.
While the precedence for H3 methylated at lysine 9 functioning to
recruit chromatin-binding factors has been shown, it is still not clear how
this
modification may participate in the inactivation of the X chromosome.
Analogously,
H3 methylated at lysine 4 may function to recruit transcription-enhancing
factors or to
block the association of transcription-repressive factors; however, direct
evidence for
either of these possibilities is still lacking. The present ChIP assays only
examined
promoters of X-linked genes, but it is anticipated that H3. methylated at
these two ,
respective sites are genome-wide marks that demarcate chromosome domains.
Active
and inactive regions of the eukaryotic genome not only adopt contrasting
chromatin
structure (euchromatin versus heterochromatin), but also have been shown to
occupy
distinct intranuclear domains. Therefore, methylation of H3 at lysines 4 or 9
may
dictate the spatial distribution of associated chromosome regions in
transcriptionally
permissive versus restrictive environment.
Equally intriguing is the question of which enzyme is responsible for
the methylation reaction. While previous studies have shown that the hSuVax3-9
is a
H3 lysine9-specific histone methyltransferase, fibroblasts derived from SuVar3-
9
knock out embryos retain the enrichment of lysine 9 methylated at the inactive
X
chromosome.
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Example 4
Parent-Specific Complementary Patterns of Histone H3 Lys9 And Lys4
Methylation at The Prader-Willi Imprinting Center
Imprinted genetic loci show differential expression of maternal
compared to paternal alleles in some or all tissues at some or all stages of
development. The functional differences between maternal and paternal alleles
of
imprinted genetic loci must reflect structural differences between maternal
and
paternal chromosomes in the regions containing these loci. The simplest models
for
establishment of these structural differences hold that chromosomal domains
are
marked differentially during oogenesis and spermatogenesis, and that these
gametic
imprinting marks are maintained after fertilization in somatic cells. However,
there is
no a priori reason to assume that the gametic imprinting marks are identical
to the
imprinting marks responsible for differential gene expression after
fertilization. If the
gametic marks are not the same as the somatic marks, there must be a~mechanism
for
reading the gametic marks and using their information to impose somatic
imprinting
marks.
The identity of the imprinting marks in mammals has been the subject
of extensive speculation and experimental analysis. An appealing candidate
imprinting mark is 5-methylcytosine in CpG dinucleotides. Many imprinted loci
show parent-of origin specific DNA methylation of imprinted regions, and some
of
these parent-specific DNA methylation marks are established during
gametogenesis
and maintained in somatic cells. A mechanism for replicating methylated CpG
dinucleotides exists: maintenance DNA methyltransferases (DNMTs) recognize
hemimethylated DNA (DNA methylated on only one strand) and add methyl groups
to
cytosine residues on the complementary strand. A frequently-cited experiment
in
support of cytosine methylation as the imprint is the observation that DNMT1 -
/-
embryos, which die early in embryonic development, show biallelic expression
of
some imprinted loci whose normal monoallelic expression is associated with
cytosine
methylation of the inactive allele.
Although abundant data are consistent with cytosine methylation as the
imprint, several lines of evidence exist for additional or alternative
molecular
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imprints. First, a number of imprinted genes, including mouse Mash2, human
CDKN1C, and human UBE3A, show no regions of parent-specific DNA methylation.
Second, some genes show evolutionary conservation of imprinting without
showing
conservation of differentially methylated regions, suggesting that the
differential
cytosine methylation may be a secondary consequence of a primary imprint.
Third,
Mash2 imprinting is not disturbed in the DNMT1 -l- mouse embryo. Finally, the
promoter region of the imprinted SNRPN-gene in the Prader-Willi syndrome (PWS)
imprinting center (IC) shows differential cytosine methylation in somatic
tissues of
mouse and human, and the region is heavily methylated in mouse oocytes but
unmethylated in mouse sperm; however, El-Maarri et al. (Nature Genet. 27, 341
(2001)) have recently shown that this region is completely unmethylated in
human
oocytes, as in human sperm, so that the differential cytosine methylation must
arise
after fertilization.
These data lead to the conclusion that, at least for some imprinted
genes and some imprinted regions, the structural difference between maternal
and
paternal alleles inherited from the gametes that leads to differential gene
expression
after fertilization must be something other than cytosine methylation. In
principle,
this structural difference might be a heritable covalent modification of DNA
other
than cytosine methylation, a DNA-associated protein that remains stably
associated
with either the maternal or the paternal chromosome from the gamete through
somatic
cell divisions, or a covalent modification of a DNA-associated protein that is
inherited
in a parent-specific fashion.
Histone modifications, especially acetylation, have previously been
shown to mediate effects of a number of transcriptional regulatory proteins,
presumably by changing chromatin structure to increase accessibility to other
transcriptional factors. Unlike acetylated histones, which are quite labile,
methyl
groups attached to histones show a very low level of turnover, making histone
methylation a good candidate modification in epigenetic processes such as
imprinting. Accordingly, a histone modification that has recently been
associated
with the formation of stable silenced chromatin regions in Drosophila and
fission
yeast, (the methylation of histone H3 on Lys9) as been examined along with the
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methylation of histone H3 on Lys4, which has been correlated with
transcriptional
activity in Tetrahymena.
The Prader-Willi syndrome (PWS)/Angelman syndrome (AS) region in
human chromosome 15q11-q13 contains at least 10 imprinted genes within a ~2 Mb
region (Nicholls et al, Trends Genet. 14, 194 (1998)); 8 of these genes are
expressed
exclusively from the paternal chromosome, and loss of the active paternal
alleles of
these genes causes PWS, characterized by infantile hypotonia, mild
developmental
delay, and later-onset hyperphagia and obesity. Loss of the active maternal
allele of
one gene in this region, UBE3A, causes AS, characterized by severe mental
retardation, lack of speech, seizures, and easily provoked laughter. This
region can
exist in either of two mutually exclusive epigenetic states, the paternal
state and the
maternal state. Establishment of the paternal state requires a DNA segment,
referred
to as the PWS imprinting center (PWS-IC) that includes the SNRPN promoter in
cis;
establishment of the maternal state requires a DNA segment approximately 30 kb
centromeric of the PWS-IC referred to as the AS-IC. The functions of the PWS-
IC
and AS-IC in establishing epigenetic states of this region are not known.
Chromatin prepared from stimulated lymphocytes of controls, PWS
individuals (lacking a paternal copy of 15q11-ql3 through deletion or
imprinting
defect), and AS individuals (lacking a maternal copy of 15q11-q13) was
immunoprecipitated with antibodies specific for either H3 methylated on Lys9
or H3
methylated on Lys4. DNA recovered from the immunoprecipitation was assayed by
PCR for sequences in the PWS-IC, including the SNRPN promoter, and for other
sequences in the region. The maternal copy of the PWS imprinting center
(present in
PWS chromatin) was immunoprecipitated by anti-methyl Lys9 H3 antibody, while
there was dramatically reduced precipitation of this sequence on the paternal
copy
(present in AS chromatin). This result correlates welfwith the observation
that .
maintenance of silenced heterochromatin in both Drosophila and fission yeast
requires the function of Lys9 histone H3 methyltransferases. The region of
maternal-
specific H3 Lys9 methylation extends approximately 0.6 kb 5' and 0.5 kb 3'from
the
SNRPN promoter. Conversely, the paternal copy of the PWS-IC was
immunoprecipitated by anti-methyl Lys4 H3 antibody. This sequence was not
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precipitated on the maternal copy. Previous reports of association of this
modification
with active chromatin are consistent with these findings.
Parent-specific differential association of methyl Lys9 H3 was not
detected with the promoters of other imprinted genes in 15q11-q13, including
ZNF127, NDN, MAGEL2, IPW, which are paternally-expressed, and UBE3A and
ATP10C, which show tissue-specific maternal expression. Methyl Lys9 H3 was
also
not associated with the AS-IC. However; methyl Lys4 H3 was found to be
specifically associated with the promoter region of the paternal allele of the
paternally-active gene NDN. Parent-specific Lys4 methylation in lymphocyte
chromatin was not detected for ZNF127, MAGEL2, IPW, UBE3A, or ATP10C.
Each of the modifications that has been described (cytosine
methylation, histone H3 and H4 acetylation, histone H3 Lys4 methylation, and
histone
H3 Lys9 methylation) shows a distinct pattern of distribution and parent
specificity.
The PWS-IC, which overlaps the SNRPN promoter, shows the most extensive
pattern
of modification, with cytosine methylation and H3 Lys9 methylation on the
maternal
allele, and histone H3 and H4 acetylation as well as histone H3 Lys4
methylation on
the paternal allele. The paternal SNRPN promoter region is also the site of a
very
prominent nuclease hypersensitive site that is not present on the maternal
chromosome. The promoter region of NDN, which shows differential cytosine
methylation, does not show eithex differential histone acetylation or
differential H3
Lys9 methylation.
It is clear that the human PWS imprinting center lacks cytosine
methylation in oocytes; therefore, this modification can not be the gametic
imprint for
the AS/PWS region. Among the parent-specific histone modifications of the PWS
imprinting center, H3 and H4 acetylation, as well as H3 Lys4 methylation, also
can
not be the gametic imprint because sperm lack histones, so a paternal gametic
imprint
can not be a histone modification. Methyl Lys9 H3, however, is a potential
candidate
imprinting mark that could be imposed upon histones in the PWS imprinting
center
during gametogenesis. A maternal histone modification irxiprint would have.the
unique feature of undergoing programmed erasure during spermatogenesis, when
histones are removed from chromatin and replaced by protamines.
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SEQUENCE LISTING
<110> The University of Virginia Patent Foundation
Allis, C. David
Strahl, Brian
<120> Antibodies Specific for Methylated Lysines in Histones
IS <130> 00601-02
<150> US 60/227,767
<151> 2000-08-25
<150> US 60/302,747
<151> 2001-07-03
<160> 18
<170> Patentln version 3.0
<210> 1
<211> 8
<212> PRT
4S <213> Homo Sapiens
<220>
<221> MOD RES
<222> (4) . .
(4)
<223> METHYLATION
<400> 1
Ala Arg Thr Lys Gln Thr Ala Arg
1 5
<210> 2
<211> 9
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_2_
<212> PRT
<213> Tetrahymena~thermophila
<220>
<221> MOD RES
<222> (5) . . (5)
<223> METHYLATION . .-
20
<400> 2
Gln Thr Ala Arg Lys Ser Thr Gly Val
1 5
<210> 3
<211> 9
<212> PRT
<213> Homo sapiens
<220>
<221> MOD RES
<222> (5) . .
(5)
<223> METHYLATION
<400> 3
Gln Thr Ala Arg Lys Ser Thr Gly Gly
1 5
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<210> 4
<211> 10
<212> PRT
<213> Artificial
<220>
<223> synthetic peptide used to raise antibodies against the H3
amino terminus having MeLys at the fourth amino acid position
<220>
<221> MOD RES
<222> (4) . . (4)
0 <223> METHYLATION
<220>
<221> VARIANT
<222> (9) . . (10)
<223> artificial amino acids added to the natural histone sequence
to aid in the production of the antibody
<400>
Ala Arg Thr Lys Gln Thr Ala Arg Gly Cys
1 5 10
<210> 5
<211> 11
<212> PRT
<213> Artificial
<220>
<223> synthetic peptide used to raise antibodies against the H3
amino terminus having MeLys at the fifth amino acid position
<220>
<221> MOD RES
<222> (5) . . (5)
<223> METHYLATION
<220>
<221> VARIANT
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-4-
<222> (10)..(11)
<223> artificial amino acids added to the natural histone sequence
to aid in the production of the antibody
<400> 5
Gln Thr Ala Arg Lys Ser Thr Gly val Cys Gly
1 5 10
<210> 6
<211> 11
0 <212> PRT
<213> Artificial
<220>
<223> synthetic peptide used to raise antibodies agains the H3
amino terminus having MeLys at the fifth amino acid position
<220>
<221> MOD RES
<222> (5) . . (5)
<223> METHYLATION
<220>
. .
<221> VARIANT
<222> (10) . . (11) , . ' ':
<223> artificial amino acids added to the natural histone sequence
to aid in the production of the antibody
$0 <400> 6
Gln Thr Ala Arg Lys Ser Thr Gly Gly Cys Gly
r 1 5 10
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-S-
<210> 7
<211> 15
S <212> PRT
<213> Homo Sapiens
1S
<400> 7
Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala
1 5 - 10 15
<210> 8
<211> 15
<212> PRT
ZS <213> Homo sapiens
<400> 8
Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala
1 5 10 15
3S
<210> 9
<211> 13
<212> PRT
<213> Tetrahymena thermophila
4S
<400> 9
Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Val
S0 1 . 5 10
SS
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<210> 10
<211> 13
S <212> PRT
<213> Homo Sapiens
<220>
<221> MOD RES
<222> (4) . . (4)
IS <223> METHYLATION
<400> 10
Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly
1 5 10
2S
<210> 11
<211> 13
<212> PRT
<213> Homo Sapiens
3S <2zo>
<221> MOD RES
<222> (9) . .
(9)
<223> METHYLATION
4S <400> 11
Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly
1 5 10
S0
CA 02420277 2003-02-20
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<210> 12
<211> 13
<212> PRT
<213> Tetrahymena thermophila
<220>
<221> MOD RES
<222> (4) . . (4)
IS <223> METHYLATION
<400> 12
Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Val
1 5 10
<210> 13
<211> 13
<212> PRT
<213> Tetrahymena thermophila
<z2o>
<221> MOD RES
<222> (9)..(9)
<223> METHYLATION
<400> 13
Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Val
1 5 10
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_$_
<210> 14
<211> 13
<212> PRT
<213> Homo Sapiens
<220>
<221> MOD RES
<222> (5) . .
(5)
<223> METHYLATION
<400> 14
5er Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly
1 5 10
<210> 15
<211> 13
<212> PRT
<213> Homo Sapiens
<220>
<221> MOD RES
<222> (8)..(8)
<223> METHYLATION
<400> 15
Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly
1 5 10
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<210> 16
<211> 10
<212> PRT
<213> Artificial
<220>
<223> synthetic peptide used to raise antibodies against the H3
amino terminus having MeLys at the fourth amino acid position
<220>
<221> MOD RES
<222> (4) . . (4)
<223> METHYLATION
<220>
<221> VARIANT
<222> (9) . . (10)
<223> artificial amino acids added to the natural histone sequence
to aid in the production of the antibody
<400> 16
Ala Ala Arg Lys Ser Ala Pro Val Cys Gly
1 5 10
<z1o> 17
<211> 11
<2l2> PRT
<213> Artificial
<220>
<223> synthetic peptide used to raise antibodies against the H3
SS amino terminus having MeLys at the fifth amino acid position
<220>
<221> MOD RES
<222> (S) . . (5)
<223> METHYLATION
<220>
<221> VARIANT
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<222> (10)..(11)
<223> artificial amino acids added to the natural histone sequence
to aid in the production of the antibody
<400> 17
Ser G1y Gly Val Lys Lys Pro His Lys Cys Gly
1 5 10
<210> 18
<211> 10
<212> PRT
<213> Artificial
<220>
<223> synthetic peptide used to raise antibodies against
the H3
amino terminus having MeLys at the fourth amino acid
position
<220>
<221> MOD
RES
<222> _
(4) .(4)
<223> METHYLATION
<220>
<221> VARIANT
<222> (9) . . (10)
<223> artificial amino acids added to the natural histone
sequence
to aid in the production of the antibody
<400> 18
Arg His Arg Lys Ile Leu Arg Asp Cys Gly
1 5 10