U.S. patent application number 10/323968 was filed with the patent office on 2003-08-21 for method for identifying compounds altering higher-order chromatin-dependent chromosome stability.
This patent application is currently assigned to Boehringer Ingelheim International GmbH. Invention is credited to Eisenhaber, Frank, Jenuwein, Thomas, O'Carroll, Donal, Rea, Stephen.
Application Number | 20030157532 10/323968 |
Document ID | / |
Family ID | 27439960 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157532 |
Kind Code |
A1 |
Jenuwein, Thomas ; et
al. |
August 21, 2003 |
Method for identifying compounds altering higher-order
chromatin-dependent chromosome stability
Abstract
A method for identifying compounds that alter higher order
chromatin dependent chromosome stability is based on determining
the compounds' ability to modify a methyltransferase with
Suv39h-like methyltransferase activity. The identified compounds
are useful in therapy, in particular the therapy of human cancer
and for contraception.
Inventors: |
Jenuwein, Thomas; (Wien,
AT) ; Rea, Stephen; (Headford, IE) ;
Eisenhaber, Frank; (Wien, AT) ; O'Carroll, Donal;
(Greystones, IE) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Boehringer Ingelheim International
GmbH
|
Family ID: |
27439960 |
Appl. No.: |
10/323968 |
Filed: |
December 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10323968 |
Dec 20, 2002 |
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09876221 |
Jun 8, 2001 |
|
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6555329 |
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60224220 |
Aug 9, 2000 |
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Current U.S.
Class: |
435/6.12 ;
435/7.23 |
Current CPC
Class: |
C12Q 1/48 20130101; C07K
16/40 20130101; C12N 9/1007 20130101; G01N 33/6875 20130101; G01N
2333/91011 20130101; G01N 2500/00 20130101 |
Class at
Publication: |
435/6 ;
435/7.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 2000 |
EP |
EP 00 112 345.4 |
Jun 9, 2000 |
EP |
EP 00 112 479.1 |
Claims
What is claimed is:
1. A method for identifying a compound that alters higher order
chromatin dependent chromosome stability in mitosis and meiosis,
said method comprising incubating a substrate for a
methyltransferase, in the presence of a methyl donor, with a
methyltransferase with Suv39h-like methyltransferase activity, in
the presence or absence of a test compound and determining whether
the compound modulates the methyltransferase activity.
2. The method of claim 1, wherein the methyltransferase with
Suv39h-like activity methylates histone H3 at lysine 9.
3. The method of claim 2, wherein the methyltransferase with
Suv39h-like activity is murine Suv39h1 or human SUV39H1.
4. The method of claim 2, wherein the methyltransferase with
Suv39h-like activity is murine Suv39h2 or human SUV39H2.
5. The method of any one of claims 1 to 4, wherein the substrate is
histone H3 or an N-terminal fragment thereof that contains the
methylation site at lysine 9.
6. The method of claim 5, wherein the histone H3 N-terminal
fragment has the amino acid sequence as set forth in SEQ ID
NO:7.
7. The method of any one of claims 1 to 6, wherein the methyl donor
is methionine or S-adenosyl-L-methionine.
8. The method of any one of claims 1 to 7, wherein the methyl group
of the methyl donor carries a detectable label.
9. The method of claim 8, wherein the methyl donor carries a
chromogenic label and the methyltransferase activity is determined
by measuring the change in colour upon transfer of the methyl group
to the substrate.
10. The method of claim 8, wherein the methyl donor carries a
radioactive label and the methyltransferase activity is determined
by measuring the radioactivity transferred to the substrate upon
transfer of the methyl group.
11. The method of any one of claims 1 to 7, wherein the
methyltransferase activity is determined immunologically by
quantifying the binding of an antibody specific for the methylation
site of the substrate.
12. The method of claim 11 wherein the substrate carries a
detectable label.
13. An antibody that specifically recognises methylated lysine 9 in
histone H3.
14. The use of the H3-K9 methyl-specific antibody of claim 13 for
diagnosis of a human disease associated with aberrant gene
expression and genomic instability through chromosome
mis-segregation.
15. A compound identified in a method defined in any one of claims
1 to 12 for use in the the therapy of cancer.
16. A compound identified in a method defined in any one of claims
1 to 12 for contraception.
17. The compound of claim 15 for use in temporary male conception.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. Provisional
Application No. 60/224,220, filed Aug. 9, 2000, which is hereby
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a method for identifying compounds
influencing chromosome dynamics in eukaryotic cells. In particular,
the invention relates to the treatment and prevention of human
conditions by modulating higher order chromatin dependent
chromosome stability during mitosis and meiosis.
[0004] 2. Related Art
[0005] Higher-order chromatin is essential for epigenetic gene
control and for the functional organisation of chromosomes.
Differences in higher-order chromatin structure have been linked
with distinct covalent modifications of histone tails which
regulate transcriptional `on` or `off` states (Grunstein, 1998;
Turner, 1998; Strahl and Allis, 2000) and influence chromosome
condensation and segregation (Karpen and Allshire, 1997; Wei et
al., 1999).
[0006] Histones constitute a highly conserved family of proteins
(H3, H4, H2A, H2B, H1) which are the major components of eukaryotic
chromatin structure. Histones compact genomic DNA into basic
repeating structural units, the nucleosomes. In addition to their
DNA packaging function, histones have been proven to be integral
components of the molecular machinery that regulates gene
expression.
[0007] Post-translational modifications of histone N-termini,
particularly of H4 and H3, are well documented and have
functionally been characterised as changes in acetylation
(Grunstein, 1998; Turner, 1998; Strahl and Allis, 2000),
phosphorylation (Wei et al., 1999) and, most recently, methylation
(Chen et al., 1999; Strahl et al., 1999). In contrast to the large
number of described histone acetyltransferases (HATs) and histone
deacetylases (HDACs), genes encoding enzymatic activities that
regulate phosphorylation (Sassone-Corsi et al., 1999; Hsu et al.,
2000) or methylation (Chen et al., 1999) of histone N-termini are
only beginning to be identified. Moreover, the interdependence of
the different histone tail modifications for the integration of
transcriptional output or higher-order chromatin organisation is
currently not understood.
[0008] Overall, there is increasing evidence that the regulation of
normal and aberrant cellular proliferation is not only affected on
the transcriptional level, but that also a higher level of
regulation is involved, i.e. the organisation of chromatin
structure through the modification of histone molecules. The
determination of the proteins and the molecular mechanisms involved
in histone modification will contribute to the understanding of the
cellular proliferation program and will thus shed led light on the
mechanisms involved in aberrant proliferation occurring in tumour
formation and progression (Jacobson and Pillus, 1999).
[0009] Genetic screens for suppressors of position effect
variegation (PEV) in Drosophila (Reuter and Spierer, 1992) and
S.pombe (Allshire et al., 1995) have identified a subfamily of
approximately 30-40 loci which are referred to as Su(var)-group
(Wallrath, 1998) genes. Interestingly, several histone deacetylases
(De Rubertis et al., 1996), protein phosphatase type 1 (Baksa et
al., 1993) and S-adenosyl methionine synthetase (Larsson et al.,
1996) have been classified as Su(var)s. In contrast, Su(var)2-5
(which is allelic to HP1) (Eissenberg et al., 1992), Su(var)3-7
(Clard et al., 1997) and Su(var)3-9 (Tschiersch et al., 1994;
Schotta and Reuter, 2000) encode heterochromatin-associated
proteins. Su(var) gene function thus suggests a model, in which
modifications at the nucleosomal level may initiate the formation
of defined chromosomal subdomains that are then stabilised and
propagated by heterochromatic SU(VAR) proteins (Henikoff,
1997).
[0010] Su(var)3-9 is dominant over most PEV modifier mutations
(Tschiersch et al., 1994), and mutants in the corresponding S.pombe
clr4 gene (Ivanova et al., 1998) disrupt heterochromatin
association of other modifying factors and result in chromosome
segregation defects (Ekwall et al., 1996). Recently, human
(SUV39H1) and murine (Suv39h1 and Suv39h2) Su(var)3-9 homologues
have been isolated (Aagaard et al., 1999). It has been shown that
they encode heterochromatic proteins which associate with mammalian
HP1 (Aagaard et al., 1999). The SU(VAR)3-9 protein family combines
two of the most evolutionarily conserved domains of `chromatin
regulators`: the chromo (Aasland and Stewart, 1995; Koonin et al.,
1995) and the SET (Tschiersch et al., 1994; Jenuwein et al., 1998)
domain. Whereas the 60 amino acids chromo domain represents an
ancient histone-like fold (Ball et al., 1997) that directs eu- or
heterochromatic localisations (Platero et al., 1995), the molecular
role of the 130 amino acids SET domain has remained enigmatic.
Overexpression studies with human SUV39H1 mutants indicated a
dominant interference with higher-order chromatin organisation
that, surprisingly, suggested a functional relationship between the
SET domain and the distribution of phosphorylated (at serine 10)
histone H3 (Melcher et al., 2000).
SUMMARY OF THE INVENTION
[0011] It was an object of the invention to gain further insight
into the molecular pathways leading to histone modifications and
higher-order chromatin organisation in order to harness these
findings for interfering with aberrant gene expression and genomic
instability through chromosome mis-segregation and thus provide new
cancer therapies.
[0012] In particular, it was an object of the invention to
investigate the function of members of the SU(VAR)3-9 protein
family with the view to develop novel strategies to affect
higher-order chromatin dependent chromosome stability. Such
strategies can be employed in therapies for the treatment of
conditions in which aberrant gene expression and genomic
instability through chromosome mis-segregation are causally
involved. (The term "chromosome stability" implies successful
segregation of chromosomes resulting in the maintenance of a stable
karyotype).
[0013] Examples 1 to 7 of the present invention show that mammalian
SU(VAR)3-9 related proteins (human SUV39H1, murine Suv39h1 and
murine Suv39h2) are SET domain-dependent H3-specific histone
methyltransferases which selectively methylate lysine 9 ("K9") of
the H3 N-terminus. Methylation of K9 negatively regulates
phosphorylation of adjacent serine 10 and reveals a `histone code`
that appears intrinsically linked to the organisation of
higher-order chromatin. (In the following, histone
methyltransferases are termed "HMTases" or, more generally,
"MTases").
[0014] After having identified Suv39h1 and Suv39h2 as mammalian
histone H3 lysine 9 specific histone methyltransferases (Suv39h
HMTases), it was shown that these HMTases are
heterochromatin-enriched enzymes which transiently accumulate at
centromeres during mitosis (Aagaard et al., 1999; Aagaard et al.,
2000). Moreover, it was shown that methylation of histone H3 at
lysine 9 (H3-K9) creates a high-affinity binding site for H1
proteins (Lachner et al., 2001; Bannister et al., 2001), thereby
defining the SWV39H1-HP1 methylation system as a crucial regulatory
mechanism for the assembly and propagation of heterochromatin
(Jenuwein, 2001). Overexpression of human SUV39H1 induces ectopic
heterochromatin and results in chromosome nus-segregation in
mammalian cell lines (Melcher et al., 2000). In addition to the
essential mitotic functions described above, heterochromatin is
also crucial for the dynamic reorganization of meiotic chromosomes.
Meiosis is initiated by chromosomal movements from the nuclear
lumen to the nuclear envelope, where chromosomes cluster via their
pericentric satellite sequences (Hawley et al., 1992; Scherthan et
al., 1996). At meiotic prophase, chromosomes condense, followed by
homolog pairing and recombination (at pachytene) between maternal
and paternal chromosomes. The onset of the meiotic divisions is
preceded by desynapsis, further chromosome condensation and histone
H3 phosphorylation at pericentric heterochromatin (Cobb et al.,
1999). In particular for male germ cells, the haploid genome
content is finally organized into one heterochromatic block in
elongating spermatids. In Drosophila, heterochromatin and its
associated satellite sequences have been proposed to assist in the
initial meiotic chromosome movements and in homolog pairing by
orienting chromosomes along a similar higher-order structure
(Hawley et al., 1992; Karpen et al., 1996; Dernburg et al., 1996b).
In germ cells of mammals, a pachytene checkpoint (de Vries et al.,
1999) monitors mis-aligned and unpaired chromosomes and arrests
cells in melotic prophase, thereby preventing the production of
aneuploid gametes.
[0015] It was a further object of the invention to analyse the role
of Suv39h1 and Suv39h2 in embryonic development and in
spermatogenesis in view of utilizing these proteins as drug targets
for conditions involving fertility, in particular male
fertility.
[0016] To solve the problems underlying the present invention, in a
first step bioinformatics techniques were applied. Using the SET
domains of the SU(VAR)3-9 protein family as a starting alignment,
significant sequence and secondary structure similarities (see
Methods) to six plant protein methyltransferases were detected.
[0017] To investigate whether the SET domain of human SUV39H1 has
enzymatic activity, histones were tested as possible substrates for
in vitro methylation. The obtained results demonstrate that SUV39H1
harbors an intrinsic histone methyltransferase activity and suggest
that this HMTase activity resides in the C-terminal SET domain.
[0018] Using recombinant proteins, both murine GST-Suv39h1(82-412)
and the corresponding human SUV39H1 fusion protein
[GST-SUV39H1(82-412)] were shown to be catalytically active. Short
internal deletions were introduced into the two conserved regions
of the SET domain core in GST-SUV39H11(82-412), and additional
mutants lacking the C-terminal tail (.DELTA.C-tail) or the
SET-associated cysteine-rich region (.DELTA.cys) were generated.
All mutant proteins failed to demonstrate HMTase activity.
[0019] Although these results suggest a significant contribution by
the cysteine-rich regions, their apparent absence in the plant
methyltransferases does not prevent catalytic activity. To
investigate enzyme function of the SET domain in more detail, point
mutations were introduced into the most highly conserved motif. In
vitro HMTase assays indicated that all point mutations, with the
exception of one, abolished enzymatic activity. Surprisingly, the
latter mutation resulted in an hyperactive enzyme with
approximately 20-fold increased activity. The data obtained define
the .sub.320 H.phi..phi.NHSC.sub.326 motif in the SET domain as an
important catalytic site.
[0020] Because the SET domain is one of the most conserved protein
motifs in chromatin regulators (Stassen et al., 1995; Jenuwein et
al., 1998), it was next analysed whether SU(VAR)3-9 family members
or other SET domain proteins contain HMTase activity. GST-fusion
products of the extended SET domains of S.pombe CLR4 (Ivanova et
al., 1998), human EZH2 (Laible et al., 1997) and human HRX (Tkachuk
et al., 1992) were generated that would correspond to
GST-SUV39H1(82-412). Interestingly, GST-CLR4(127-490) displayed
pronounced HMTase activity at three- to five-fold increased levels
as compared to the recombinant SUV39H1 product, consistent with
CLR4 carrying an arginine at the hyperactive position. The results
obtained from this analysis show, in agreement with the mutational
analysis of SUV39H1, that HMTase activity towards free histones
appears to require the combination of the SET domain with adjacent
cysteine-rich regions, which is a quality found in only a
restricted number of SET domain containing proteins.
[0021] These experiments indicated that the HMTase activity of
mammalian SU(VAR)3-9 related proteins is selective for histone H3
under the chosen assay conditions. To examine this finding in more
detail, in vitro methylation reactions were performed with
individual histones. It could be shown that H3 is specifically
methylated by GST-Suv39h1(82-412), whereas no signals are detected
with H2A, H2B or H4. Methylation of H3 has been shown to occur
predominantly at lysine 4 in a wide range of organisms, as well as
at lysine 9 in HeLa cells, although the responsible HMTase(s) have
yet to be defined (Strahl et al., 1999). To investigate the site
utilisation profile of Suv39h1, unmodified peptides comprising the
wild-type H3 N-terminus and a mutant K9L peptide were tested as
substrates. Additionally, insulin and peptides comprising the
N-termini of CENP-A (Sullivan et al., 1994), macroH2A (Pehrson and
Fried, 1992) were included. These in vitro assays revealed
selective methylation of the wild-type H3 peptide. The data
obtained also suggested that the H3 N-terminus is a preferred
residue for Suv39h1-dependent HMTase activity.
[0022] To more definitively determine this site preference, the
wild-type H3 N-terminal peptide was in vitro methylated by
GST-Suv39h1(82-412), using
S-adenosyl-[methyl-.sup.3H]-L-methionine. The labelled peptide,
purified by reverse-phase HPLC, was then directly microsequenced,
and .sup.3H-incorporation associated with each individual amino
acid was analysed. The results confirmed selective transfer of
methyl-label to lysine 9, demonstrating that Suv39h1 is a highly
site-specific HMTase for the H3 N-terminus in vitro.
[0023] Murine Suv39h genes are encoded by 2 loci, Suv39h1 and
Suv39h2. To investigate the in vivo significance of Suv39h function
and Suv39h dependent K9 H3 methylation, mouse strains deficient for
both Suv39h1 and Suv39h2 were generated. Suv39h1 and Suv39h2
deficient strains were intercrossed to produce Suv39h double
deficient mice. Double mutant mice were born in sub-Mendelian
ratios. Some double null embryos exhibited severe growth
retardation and exencephaly. In addition surviving double mutants
were growth retarded, suggesting a role for Suv39h in cell
proliferation.
[0024] In order to determine whether the embryonic phenotypes in
Suv39h null mice can be attributed to mitotic defects, PMEFs
(primary mouse embryonic fibroblasts) derived from Suv39h double
mice were analysed. Suv39h double null PMEFs display a reduced
G1-index and an increased proportion of cells with aberrant nuclear
morphologies, reminiscent of division defects during mitosis.
Furthermore, double null cells also show genomic instabilities and
readily become aneuploid. The severity of these aneuploidies
increases with higher passage numbers. The inability of Suv39h
double null cells to maintain a stable karyotype may underlie the
Suv39h embryonic phenotype.
[0025] Phosphorylation at serine 10 (phosH3) in the N-terminal tail
of H3 has been shown to be required for condensation and subsequent
segregation of chromosomes (Wei et al., 1999). During the cell
cycle, phosH3 initiates within pericentric heterochromatin in late
G2 and then progresses along the entire chromosomes during mitosis
(Hendzel et al., 1997). It was found that in wild-type PMEFs,
approximately 7% of the cells stain positive for the
characteristic, heterochromatin-associated phosH3 foci. In
contrast, this number is increased by a factor of about 3-fold in
Suv39h double null PMEFs. This result suggested that the overall
levels of phosH3 may be enhanced in Suv39h double null PMEFs. This
was confirmed biochemically. Together, the obtained data are most
consistent with a model in which Suv39h-mediated methylation of
lysine 9 in H3 negatively regulates phosphorylation of serine
10.
[0026] Together, these data clearly demonstrate crucial roles for
Suv39h during cell division. Loss of Suv39h function impairs K9
histone H3 metylation and induces defective cell division resulting
in genome instabilities. Segregation defects/genome instability
underlies the aetiology of many human cancers (Lengauer et al.,
1997) and are often a prerequisite for tumour progression. These
observations make Suv39h an excellent candidate for novel
therapeutic approaches for tumour therapies.
[0027] In additon, a set of experiments of the present invention
provides in vivo evidence that the absence of Suv39h HMTase
activities impairs development and viability of mutant mice, and
directly correlates with a nearly complete lack of H3-K9
methylation at pericentric heterochromatin. Notably,
Suv39h-deficient mice display chromosomal instabilities in both
somatic and meiotic cells that are further evidenced by an
increased risk for development of B-cell lymphomas and perturbed
chromosome interactions during male meiosis. These in vivo data
assign a fundamental role for H3-K9 methylation at pericentric
heterochromatin and suggest that the Suv39h HMTases regulate a
`heterochromatic competence` which protects chromosome stability
during mitosis and meiosis.
[0028] Single gene disruptions for either Suv39h1 or Suv39h2 allow
for normal mouse development and do not appear to affect viability
and fertility of mutant mice. This apparent redundancy in gene
function would be consistent with the overlapping expression
profile of the two Suv39h genes during mouse embryogenesis
(O'Carroll et al., 2000). By contrast, combined disruption of both
genes in Suv39h double null (dn) mice results in severly impaired
perinatal viability (.apprxeq.33%; Table I), growth retardation and
hypogonadism in males. Both Suv39h dn males and are infertile.
Although Suv39h dn fetuses appear to develop normally until day
E12.5, they then display smaller body sizes and frequently are
resorbed during late gestation. These in vivo analyses indicate an
important role(s) for the Suv39h genes during mammalian development
and for overall viability. Since the absence of Suv39h HMTase
activities induces genomic instabilities, the high lethality of
Suv39h dn fetuses could mainly be a consequence of perturbed
chromosome segregation which would significantly impair the
proliferation and differentiation programmes of the developing
embryo.
[0029] Although Suv39h enzymes are the major HMTases for H3-K9
methylation at pericentric heterochromatin in somatic cells (see
FIG. 12) and in early meiotic cells (see FIG. 13B), there are
.ltoreq.15 unique gene sequences in the mouse genome that contain
the evolutionarily highly conserved SET domain and which are likely
to encode additional enzymes with putative HMTase activity
(Jenuwein, 2001). At least one of these SET domain containing
proteins can indeed also methylate H3 at lysine Tachibana et al.,
2001 Thus, the .apprxeq.33% viability of Suv39h dn mice could be
dependent on the compensating activity of other HMTases that may be
expressed to varying degrees in the mixed genetic background, in
which the Suv39h dn mice have been analyzed.
[0030] Absence of Suv39h HMTase activities triggers genomic
instabilities in a variety of cell types, including mouse embryonic
fibroblasts (PMEFs) (see FIG. 10), fetal liver and bone marrow
cells and in spermatogonia (see FIG. 14C). In agreement with the
aneuploidies observed in these cellular systems, Suv39h-deficient
mice display an increased risk for tumorigenesis, resulting in
late-onset B-cell lymphomas in .apprxeq.33% of Suv39h dn mice (see
FIG. 11). B-cell lymphomas also develop upon reduced Suv39h gene
dosage in compound mutant mice that contain gene disruptions of
Suv39h1 (see Table II). Intriguingly, the Suv39h-induced
aneuploidies are mainly characterized by segregation failure of the
nearly complete set of the chromosomes, resulting in
hypo-tetraploid or even hypo-octaploid cells (see FIG. 10). These
data suggest a general impairment of chromosome segregation,
consistent with the lack of H3-K9 methylation around all
acrocentric centromeres in Suv39h dn cells (see FIG. 12).
[0031] Distinct modifications of histone N-termini, such as
acetylation (Ekwall et al., 1997) and phosphorylation (Wei et al.,
1999) have been shown to be required for correct chromosome
segregation in S.pombe and Tetrahymena, presumably by inducing a
specialised chromatin structure at pericentric heterochromatin that
facilitates the establishment of a functional centromere. Because
H3-K9 methylation restricts H3 phosphorylation mediated by the
Ipl1/aurora kinase (Hsu et al., 2000) and is also interdependent
with histone acetylation (Rea et al., 2000), the absence of Suv39h
HMTase activities is likely to perturb this distinct histone
modification pattern. Second, in addition to altering nucleosome
arrangements, histone modifications can generate specific
interaction affinities for chromatin-associated proteins (Rice and
Allis, 2001). Although the localisation of CENP epitopes appears
unaltered, heterochromatic enrichment of HP1 proteins is largely
lost in Suv39h dn somatic cells (Lachner et al., 2001). Notably,
HP1 interacts in vitro with INCENP (Ainsztein et al., 1998) which
forms a complex with aurora-B (Adams et al., 2000; Kaitna et al.,
2000). Mutation of INCENP induces severe mitotic abnormalities
including macronuclei and internuclear bridges, and results in
nearly complete chromosome mis-segregation and cytokinesis failure
(Cutts et al., 1999; Adams et al., 2000; Kaitna et al., 2000).
These intriguing parallels suggest a possible in vivo link between
Suv39h-mediated H3-K9 methylation and aurora-B dependent
phosphorylation, and could categorize Suv39h genes as novel tumour
suppressor genes.
[0032] The Suv39h-mediated chromosomal instabilities only affect a
sub-population of cells and do not appear to trigger pronounced
apoptosis (see FIG. 10b), consistent with similar analyses of clr4
mutants in S.pombe (Ekwall et al., 1996; Ivanova et al., 1998).
These data suggest that the Suv39h-induced defects in somatic cells
are not under strict surveillance of known checkpoint controls
(Cortez and Elledge, 2000; but see Bernard et al., 1998) and may be
caused by rather late segregation problems during mitosis. Indeed,
a fraction of Suv39h dn cells contain chromosomes that lag at
anaphase. Since Suv39h dn PMEFs are characterized by
hypo-tetraploid and hypo-octaploid karyotypes (see FIG. 10d) and
tumor cells contain `butterfly` chromosomes (see FIG. 11c), a model
is proposed (FIG. 16, top panel), in which the absence of H3-K9
methylation would allow stronger or more persistent pericentric
associations between aligned metaphase chromosomes. Although a role
for the Suv39h HMTases in centromeric cohesion remains to be
determined, it provides an attractive mechanism to explain possible
cytokinesis failure and mis-segregation of the entire chromosome
complement without activating known checkpoint controls.
[0033] In contrast to somatic cells, Suv39h-mediated defects in
male meiosis induce pronounced apoptosis of stage V-VI
spermatocytes during the transition from mid to late pachytene (see
FIG. 13a). Activation of programmed cell death at this stage has
also been observed in mouse mutants that are impaired in DNA damage
control (Xu et al., 1996), meiotic recombination (Yoshida et al.,
1998; de Vries et al., 1999; Baudat et al., 2000) and synaptonemal
complex formation (Yuan et al., 2000). In Suv39h dn spermatocytes,
pericentric H3-K9 methylation is specifically reduced at the
pre-leptotene stage but, surprisingly, appears as a wild-type
staining during later meiotic stages (see FIG. 13b, bottom panel).
Thus, in analogy to the increased centromeric associations
discussed above, it is proposed that impairment of H3-K9
methylation at the onset of meiosis induces aberrant centromere
clustering that can no longer be `rescued` by the hypothetical
activity of additional H3-K9 HMTases during mid-pachytene. This
model (see FIG. 16, bottom panel) would characterize
Suv39h-dependent H3-K9 methylation as one of the earliest
requirements to ensure successful meiosis and to prevent
illegitimate heterochromatic interactions. Because non-homologous
interactions will result in delayed synapsis or even complete
pairing failure (see FIG. 14a), they trigger apoptosis by
activating the pachytene checkpoint (de Vries et al., 1999),
thereby protecting the male germ line from accumulating
aneuploidies.
[0034] In Suv39h dn mice, spermatogenic failure is promoted by
illegitimate chromosomal interactions, synaptic delay, unpaired sex
chromosomes and bivalent mis-segregation at meiosis I (see FIGS.
14-15). Notably, a major fraction of these `forbidden` interactions
comprises physical contacts between the sex chromosomes and
autosomes that are largely mediated through centromeric regions
(see FIGS. 14D and 14J). These data suggest that the impairment of
H3-K9 methylation may allow pericentric heterochromatin to form a
more relaxed configuration which is prone to become engaged in
random associations. Cytological and genetic studies in Drosophila
demonstrated the intrinsic potential of heterochromatin to restrict
inter- and intrachromosomal interactions (Dernbrug et al., 1996a;
Csink and Henikoff, 1996). Moreover, pericentric heterochromatin
has been shown to initiate and maintain alignment and pairing of
achiasmate chromosomes until meiosis I (Karpen et al.,1996;
Dernburg et al.,1996b), suggesting a role for heterochromatin in
defining a `self-complementary` higher-order chromosome structure
that would ensure partner recognition of homologous chromosomes
(Karpen et al., 1996). The in vivo data on the function of the
Suv39h HMTases would be consistent with these proposed roles of
heterochromatin and reveal the first evidence that impaired
definition of meiotic heterochromatin can affect chromosome
identity in a mammalian organism.
[0035] Finally, Suv39h deficiency induces uni-valency of the sex
chromosomes at pachytene and at diakinesis (see FIG. 15).
Intriguingly, HP1.beta. (Motzkus et al., 1999; Turner et al., 2000)
and the Suv39h2 HMTase (O'Carroll et al., 2000) localise to the
specialised chromatin structure of the sex chromosomes in the XY
body. Although XY body formation appears normal in early/mid
pachytene of Suv39h dn spermatocytes, Suv39h deficiency prolonges
H3-K9 methylation (see arrows in FIG. 13B) and induces
hypo-condensation of the Y chromosome (see FIG. 15E). These results
involve the Suv39h HMTase activities in the definition of the
heterochromatic identity of the Y chromosome and suggest that
Suv39h-mediated H3-K9 methylation may indirectly promote or
stabilise homolog pairing of the heteromorphic sex chromosomes.
[0036] Heterochromatin has been first described more than 70 years
agao (Heitz, 1928). Because of its stable appearance in the cell
nucleus, it has been proposed to serve crucial functions for the
inheritance of cell type identities and the fidelity of chromosome
segregation. The discoveries of the first HMTases (Rea et al.,
2000; O'Carroll et al., 2000) and their mechanistic link to
generate a heterochromatic affinity through H3-K9 methylation and
recruitment of HP1 proteins (Lachner et al., 2001; Bannister et
al., 2001; Nakayama et al., 2001) has now defined an entry point to
start dissecting some of the basic roles of heterochromatin.
[0037] The experiments of the present invention have provided in
vivo evidence that H3-K9 methylation at pericentric heterochromatin
is indeed a crucial requirement to ensure mammalian development and
to protect chromosome stability in both somatic cells and male germ
cells. Because Suv39h deficiency impairs chromosome function in
mitosis and meiosis, the data assign a fundamental role for H3-K9
methylation in directing a `heterochromatic competence` for overall
chromosome dynamics and identity--and reveal some of the direct
biological functions of the enigmatic entity called
heterochromatin.
[0038] In a first aspect, the results obtained in the experiments
of the present invention show that members of the SU(VAR)3-9
protein family have HMTase activity which identifies them as novel
targets for the therapy of proliferative disorders, in particular
cancer.
[0039] Furthermore, the experiments of the invention demonstrate
that the Suv39h HMTases are important for embryonic development and
spermatogenesis.
[0040] Combined disruption of both Suv39h HMTase genes abolishes
H3-K9 methylation at pericentric heterochromatin and induces
chromosomal instabilities with an increased risk for
tumorigenesis.
[0041] In addition, Suv39h double null male mice display complete
spermatogenic failure that is largely caused by non-homologous
chromosome associations and delayed synapsis, resulting in
apoptosis of meiotic prophase cells. Together, these results
establish histone H3-K9 methylation as a crucial determinant for
pericentric heterochromatin and provide a direct role for the
Suv39h HMTases in maintaining a `heterochromatic competence` that
protects chromosome stability during mitosis and meiosis.
[0042] The identification of members of the SU(VAR)3-9 protein
family, exemplified by human SUV39H1, murine Suv39h1 and murine
Suv39h2, as K9 specific histone H3 MTases is the prerequisite for
designing assay methods that allow for finding compounds altering,
in particular interfering with, higher order chromatin dependent
chromosome stability, which is the basis for novel approaches in
cancer therapy. (In the following, if not otherwise stated, the
term "Suv39h" refers to both the murine and the human protein).
[0043] Due to the role of Suv39h1 or Suv39h2 in spermatogenesis,
compounds modulating the MTase activity of these proteins and thus
modulating spermatogenesis may also be used in the treatment of
male infertility (using compounds that enhance Suv39h MTase
activity) and for reversible male contraception (using compounds
that inhibit Suv39h MTase activity).
[0044] The present invention relates to a method for identifying a
compound that alters higher order chromatin dependent chromosome
stability during mitosis and meiosis, said method comprising
incubating a substrate for a methyltransferase, in the presence of
a methyl donor, with a MTase with Suv39h-like MTase activity, in
the presence or absence of a test compound and determining whether
the compound modulates the MTase activity.
[0045] The group of MTases with Suv39h-like MTase activity (in the
following also termed "Suv39h-like MTases") encompasses enzymes
which display histone H3 K9 MTase activity or methyltransferase
activity for other yet to be identified substrates.
[0046] The term "histone H3 K9" is not limited to the human SUV39H
or mouse Suv39h substrate (i.e. the methylation site of histone H3
at lysine 9), but is meant to encompass any substrate of the
histone or histone variant-type of protein, the methylation of
which results in the below-defined epigenetic signal.
[0047] Additional members of the group of MTases can be identified
by bioinformatic/biochemical techniques and tested biochemically.
By way of example, in a first step, by searching data bases for
similarities, as described in Example 1. Next, an identified
candidate can be verified as a MTase with Suv39h-like MTase
activity in biochemical assays similar to or identical with those
described in the Examples.
[0048] This group of Suv39h-like MTases also encompasses MTases
with specificities for other histone H3 residues than K9 or for
substrates other than histone H3, which are, like the Suv39h K9
histone H3 HMTase activity observed in the present invention,
required for higher order chromatin dependent chromosome stability.
This epigenetic signal may be a consequence of histone methylation
at lysine 9 on H3 alone; however, it cannot be excluded that MTase
activity on undefined substrates or a combination of substrate
methylation and other covalent modifications, such as
phosphorylation or acetylation, at other histone residues are
involved.
[0049] In the experiments of the present invention, Suv39h variants
with point mutations in the SET domain were shown to confer
hyperactive HMTase activity to the protein, these Suv39h variants
may be advantageously used in the method of the invention.
[0050] In a preferred embodiment, the MTase is mouse Suv39h1 or
Suv39h2, most preferably, the MTase is human SUV39H1 or
SUV39H2.
[0051] Since it has been shown in the present invention that
recombinant Suv39h retains HMTase activity, most preferably, a
recombinant MTase is employed. Suv39h or Suv39h variants can be
produced recombinantly according to standard methods by expression
in suitable hosts, e.g. bacteria, yeast, insect or eukaryotic cells
and purified, e.g. on glutathione-agarose columns if it has been
tagged with GST.
[0052] The Suv39h1 and SUV39H1 cDNA sequences are known from the
literature (Aagaard et al., 1999), the Suv39h2 cDNA sequence is
shown in SEQ ID NO: 1; the human SUV39H2 cDNA is defined by the
ESTs as shown in SEQ ID NO: 3-6.
[0053] In the case of testing the compounds for their effect on
Suv39h activity, the assay comprises, as its essential features,
incubating a histone H3 protein or a histone H3 N-terminal fragment
including K9, a methyl donor, e.g. methionine or
S-adenosyl-L-methionine, with a preparation containing a Suv39h
MTase activity and determining MTase activity in the presence or
absence of a test substance.
[0054] MTase substrates useful in the method of the invention may
be those equivalent to or mimicking the naturally occurring
substrates, e.g. biochemically purified histone H3, recombinantly
produced histone H3, or a histone H3 peptide that contains the K9
methylation site, or other yet to be identified proteins which act
as substrates for Suv39h MTases. Novel Suv39h substrates can be
identified by bioinformatic and biochemical techniques and tested
using the biochemical assays described in the Examples of the
present invention. For example, novel Suv39h substrates can be
identified by co-immunoprecipitation techniques. Suv39h proteins or
tagged versions of Suv39h proteins can be immunoprecipitated with
specific antisera and interacting proteins identified by mass
spectroscopy techniques. A yeast two hybrid screen using Suv39h
proteins or portions of Suv39h proteins as a bait can also be
employed to identify novel interacting protein from a variety of
cDNA libraries.
[0055] In a preferred embodiment, the histone H3 fragment
ARTKQTARKSTGGKAPRKQL (SEQ ID NO:7) is employed. Alternatively, a
modified peptide may be used for which the MTase has increased
affinity/activity. Such peptides can be designed by exchanging
and/or adding and/or deleting amino acids and testing the substrate
in serial experiments for MTase affinity/activity.
[0056] The methyl group of the methyl donor preferably carries a
detectable label, e.g. a radioactive or a chromogenic label, which
can be quantified upon transfer to the substrate.
[0057] Preferably, the methyl donor is radioactively labelled
methionine or S-adenosyl-L-methionine.
[0058] Alternatively to using a labelled methyl donor, the
substrate, upon methylation by the enzyme, is used to serve as an
epitope which can be recognised by a specific antibody and hence be
quantified by standard immunoassay techniques, e.g. ELISAs.
Antibodies useful in this type of assay can be obtained by using
the methylated substrate, preferably a small peptide, e.g. the
peptide with the sequence shown in SEQ ID NO:7, as an antigen and
obtaining polyclonal or monoclonal antibodies according to standard
techniques. The generation and purification of a methyl-specific
antibody against the histone H3 lysine 9 position is described in
the Materials and Methods section. A suitable H3-K9 methyl antibody
was also described by Nakayama et al., 2001.
[0059] For small scale applications, the screening method can be
based on an assay as described in Example 2, 3 or 4.
[0060] In an alternative embodiment, the screening method of the
invention utilizes the fact that the methylation of histone H3 at
lysine 9 (H3-K9) creates a high-affinity binding site for HP1
proteins. In this embodiment, the substrate, upon methylation, is
allowed to bind to HP1 and then incubated with a labelled anti-HP1
antibody. The difference in label intensity between the reaction in
the absence or presence of the test compound is indicative for the
compound's modulating effect on MTase activity.
[0061] HP1 is preferably used in recombinant form. Based on the
information of the HP1 cDNA sequence (Jones et al., 2000; Accession
No. BC006821), HP1 is produced recombinantly according to standard
technology. The recombinant protein or fragments thereof are used
to generate polyclonal or monoclonal antibodies that are employed
in this assay format.
[0062] In a preferred embodiment, the method of the invention is
performed on a high-throughput scale. For this embodiment, the
major assay components, in particular Suv39h, are employed in
recombinant form.
[0063] For the high throughput format, the screening methods of the
invention to identify MTase inhibitors, are carried out according
to standard assay procedures. Such assays are based on the
catalytic transfer, mediated by Suv39h or a Suv39h variant, of a
methyl group from a donor to a substrate, e.g. a histone H3
peptide. To achieve this, the substrate, e.g. histone H3 or a
variant or fragment thereof, is immobilised on a carrier, usually a
microtiter plate, and incubated with recombinant Suv39h or a Suv39h
variant and a methyl donor.
[0064] The methyl group of the methyl donor carries a label,
preferably a chromogenic or radioactive label.
[0065] Fluorescent or radioactive labels and the other reagents for
carrying out the enzymatic reaction on a high-throughput scale are
commercially available and can be employed according to the
supplier's instructions (e.g. Molecular Probes, Wallac). Examples
for suitable fluorescent labels are coumarin derivatives, e.g.,
7-amino-4-methylcoumarin or 7-amino-4-trifluoromethylcoumarin. The
radioactive label may be a .sup.14C or a .sup.3H atom. Upon
transfer of the methyl group to the substrate by Suv39h, in the
case of a chromogenic reagent, the methyl donor changes colour
which can be quantified. In the case of using a radioactive methyl
donor, the methyl group is transferred to the substrate and can be
directly quantified.
[0066] The specific assay design depends on various parameters,
e.g. on the size of the substrate used. In the the case of using a
short peptide, the fluorescence quenching or the fluorescence
resonance energy transfer methods are examples for suitable assay
technologies, as described below.
[0067] The substrate may be tagged, e.g. with biotin, the reaction
is then carried out in solution and then transferred to
streptavidin coated microtiter plates, e.g. in the case of a
radioactive methyl group, "flash" plates, the material of which
contains the scintillant, or plates which are coated with
scintillant. Thus the level of methylation of the substrate can be
quantified in a suitable scintillation machine/reader.
Alternatively, the assay can be carried out in the streptavidin
coated "flash" plates with the biotinylated substrate already bound
to the plates. This type of assay may also be conducted in the form
of a so-called "homogenous assay" (an assay type which does not
require intermediate transfer and washing steps) e.g. by using
microbeads that are coated with scintillant and streptavidin, to
which the biotinylated substrate is bound.
[0068] Similarly to biotin, other commonly used tags, e.g. Flag,
Myc, HA, GST, that are suitable to immobilize the substrate to the
plate that is coated with the tag-specific antibody, may be used in
the above-described assays.
[0069] In a variant, this assay is conducted in the format ELISA
type assay; in this case, a methyl-specific antibody is used to
detect the amount of methylated substrate bound to the plate.
[0070] Alternatively, the plate is coated with an antibody against
the methylated substrate to capture the methylated substrate; the
substrate is also either tagged or chromogenically labeled and the
amount of bound methylated tagged/labeled substrate can be
quantified either by a tag-specific antibody or by measuring the
level of chromogenic label. By way of example, the substrate is a
linear or a branched peptide, e.g. [TARKST].sub.4-K.sub.2-K-cys)
that is labeled with a chromogenic label, e.g. europium, and upon
methylation by a Suv39h-like MTase becomes an epitope for a
Lys9-methyl specific antibody (see materials and methods)
immobilised on a carrier (e.g. microtiter plate). The non-captured
substrate is washed away, the europium label is then cleaved and
its fluorescence enhanced and the level of fluorescence is
calculated by time resolved fluorescence. The level of fluorescence
is directly related to the level of methylated substrate (FIG.
17).
[0071] An alternative embodiment is based on the principle that
methylation of the peptide may alter its sensitivity to cleavage by
a protease. Utilizing this principle, the fluorescence quenching
(Resonance Energy Transfer "RET") assay may be employed to
determine the amount of methylation of peptidic substrates. In a
first step, a Suv39h peptidic substrate, which contains the
methylation site and a recognition/cleavage site for a defined
protease, that is sensitive to modification (in the particular
case, methylation of the lysine) of the recognition/cleavage site,
e.g . trypsin or LysC. The peptide carries a fluorescent donor near
one end and an acceptor near the other end. In the uncleaved
substrate, the fluorescence of the substrate is quenched by the
persisting intramolecular RET between donor and acceptor. Upon
cleavage of the (unmethylated) substrate by the protease, the
cleavage products are released from RET quenching and a
fluorescence signal is generated. Methylation of the substrate
abolishes the ability of the protease to cleave the substrate.
Thus, abolishment of the protease activity (which is proportional
to methylation) is reflected by signal repression, in case of total
protease inhibtion, total signal repression to the basal level.
[0072] An assay of this type may be carried out as follows: the
solution of the labeled substrate (e.g. the peptide labeled with
4-[[4'-(dimethylamino)phenyl]azo]benzoic acid (DABCYL) at the one
end and with 5-[(2'-aminoethyl)amino]naphtalenesulfonic acid
(EDANS) at the other end or labeled with benzyloxycarbonyl at the
one end and with 4-aminomethylcoumarin at the other end) in assay
buffer is transferred into each well of black 96-well microtiter
plates. After addition of the test substances in the defined
concentration, the MTase and the methyldonor are added to the
wells. After incubation under reaction conditions and for a period
of time sufficient for the methylation reaction, e.g. for 40 min at
room temperature, the protease, e.g. trypsin, is added and allowed
to react under suitable conditions, finally, the fluorescence is
measured in a fluorometer at the excitation wavelength, e.g. at 340
nm, and at the emission wavelength, e.g. at 485 nm.
[0073] In the case of using the FRET assay, the following
commercially availabe labeling pairs are suitable for the method of
the invention: Europium (Eu) and Allophycocyanin (APC), Eu and Cy5,
Eu and PE (Wallac, Turku, Finland). If a test substance is a
modulator of the MTase activity, there will be, depending on the
detection system and depending on whether the test substance has an
inhibiting or an activating effect, a decrease or an increase in
the detectable signal as compared to a control sample in the
absence of a test substance. In the high-throughput format,
compounds with a modulating effect Suv39h MTase activity can be
identified by screening test substances from compound libraries
according to known assay principles, e.g. in an automated system on
microtiter plates.
[0074] By providing a method to identify compounds which exert
their effect by directly modulating, in particular by inhibiting, a
Suv39h-like MTase, the present invention provides a basis for
inhibiting the proliferation of rapidly dividing animal cells, in
particular tumour cells.
[0075] The compounds identified in the above methods, which are
also subject of the invention, have the ability to interfere with
chromosome stability and high fidelity chromosome segregation by
modulating the MTase activity of Suv39h.
[0076] In a preferred embodiment, the compounds of the invention
are inhibitors of Suv39h HMTase activity.
[0077] Preferably, the compounds are specific modulators of Suv39h,
in particular Suv39h1 or Suv39h2.
[0078] The present invention also relates to compounds, which act
as modulators of a Suv39h-like MTase activity, in particular
modulators of Suv39h, for use in human therapy, in particular
cancer therapy.
[0079] Compounds inhibiting Suv39h HMTase activity result in
decreased genome stability and can be used in therapy for targeting
dividing cells, in particular highly proliferative tumour cells.
They are preferably administered in combination with other genome
destabilising agents, e.g. mitose inhibitors like tubulin binders
(taxanes, e.g. taxol, Paclitaxel; or epithelones). SUV39H
inhibitors may also be used jointly with or before the application
of conventional tumour therapies, e.g. radiotherapy or
chemotherapy, in particular DNA damaging agents, in order to
pre-sensitize the tumour cells. By destabilizing the cell's genome,
the SUV39H inhibitors make the cell more susceptible to the
parallel/subsequent treatment.
[0080] The SUV39H inhibitors will preferably be used in a
combination therapy and applied in consecutive and transient
treatments. Since the development of B-cell lymphomas in Suv39h
double null mice only occurs with a late onset (i.e. after 9 months
of age), transient treatments with SUV39H inhibitors should not
induce an immediate increase in tumor risk but rather weaken
overall genomic stabilities of highly proliferating cells.
[0081] Likewise, agents which enhance Suv39h HMTase activity can be
used to stabilise the genome of inherently unstable cells,
rendering them less prone to acquiring proliferation promoting
mutations. A model for Suv39h function and effects of inhibition or
enhancement of Suv39h enzymes is shown in FIG. 8.
[0082] The efficacy of compounds identified as Suv39h modulators
can be tested for in vivo efficacy in mammalian cells with Suv39h
double null cells serving as a positive control. Compounds
effective in cancer therapy should interfere with chromosome
stability and segregation, which can be measured by karyotyping,
e.g. by analysing the DNA content by FACS or standard cytological
techniques. Substances whose potential for therapeutic use has been
confirmed in such secondary screens can be further tested for their
effect on tumour cells. To test the inhibition of tumour cell
proliferation, primary human tumour cells are incubated with the
compound identified in the screen and the inhibition of tumour cell
proliferation is tested by conventional methods, e.g.
bromo-desoxy-uridine or .sup.3H thymidine incorporation. Compounds
that exhibit an anti-proliferative effect in these assays may be
further tested in tumour animal models and used for the therapy of
tumours.
[0083] Compounds intended for male fertility applications can be
tested in animal models described by Vigil et al., 1985, in animal
models developed for experimental studies of human spermatogenesis,
as described by Weinbauer et al., 2001, or in animal models that
mimic human male reproductive defects, as described by Lamb and
Niederberger (1994). Guidance for a valid application of animal
data to the assessment of human reproductive disorders is given by
Working, 1988.
[0084] Toxicity and therapeutic efficacy of the compounds
identified as drug candidates by the method of the invention can be
determined by standard pharmaceutical procedures, which include
conducting cell culture and animal experiments to determine the
IC.sub.50, LD.sub.50, the ED.sub.50. The data obtained are used for
determining the human dose range, which will also depend on the
dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and
the administration route (oral, buccal, nasal, paterental, rectal
or, in the case of temporary male contraceptive applications, local
sustained release form applications, e. g. slow-releasing
micropellets that are implanted into or adjacent to the gonads). A
pharmaceutical composition containing the compound as the active
ingredient can be formulated in conventional manner using one or
more physologically active carriers and excipients. Methods for
making such formulations can be found in manuals, e.g. "Remington
Pharmaceutical Sciences".
[0085] As Suv39h is required to maintain a stable karyotype, it can
be considered as a tumour suppressor gene. If SUV39H mutations also
prove to be a factor underlying cellular transformation events in
humans, which is strongly indicated by the analysis of Suv39h
double null mice in developing B-cell lymphomas, it can be expected
that the re-introduction of a wild type Suv39h gene by gene therapy
results in increased genomic stability delaying or inhibiting
cancer progression.
[0086] For gene therapy, the Suv39h DNA molecules may be
administered, preferably contained on a plasmid in recombinant
form, directly or as part of a recombinant virus or bacterium. In
principle, any method of gene therapy may be used for applying
Suv39h recombinant DNA, both in vivo and ex vivo.
[0087] Examples of in vivo administration are the direct injection
of "naked" DNA, either by intramuscular route or using gene guns.
Examples of recombinant organisms are vaccinia virus or adenovirus.
Moreover, synthetic carriers for nucleic acids such as cationic
lipids, microspheres, micropellets or liposomes may be used for in
vivo administration of nucleic acid molecules coding for the Suv39h
polypeptide.
[0088] Since it has been shown in the present invention that Suv39h
mediates dynamic transitions in higher-order mammalian chromatin
largely through its intrinsic HMTase activity, histone H3-K9
methylation (H3-K9 Me) represents an important epigenetic imprint
for chromosome dynamics during cell division. Hence, antibodies
specific for H3-K9 Me can be used to screen cells/patients for
heterochromatin based genome instabilities. In essence, H3-K9
methylation specific antibodies can be used as a diagnostic tool
for human diseases associated with aberrant gene expression and
genomic instability through chromosome mis-segregation or with
aberrant definition or organisation of heterochromatin.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0089] FIG. 1: HMTase activity of transfected and recombinant
SUV39H1 and Suv39h1 proteins.
[0090] FIG. 2: Specific HMTase activity of the SET domain of
mammalian SU(VAR)3-9 related proteins.
[0091] FIG. 3: H istone H3 lysine 9 is the major site for in vitro
methylation by recombinant Suv39h1.
[0092] FIG. 4: Targeting of Suv39h1 and Suv39h2 in the mouse
germline.
[0093] FIG. 5: Analys is of Suv39h double null PMEFs.
[0094] FIG. 6: Aberrant mitoses in Suv39h double null PMEFs.
[0095] FIG. 7: Increased phosH3 phosphorylation in Suv39h double
null PMEFs.
[0096] FIG. 8: Model for Suv39h HMTase function.
[0097] FIG. 9: Generation and genotyping of Suv39h1- and
Suv39h2-deficient mice.
[0098] FIG. 10: Chromosomal instabilities in Suv39h dn PMEFs.
[0099] FIG. 11: Development of B-cell lymphomas in Suv39h mutant
mice.
[0100] FIG. 12: Suv39h-dependent H3-K9 methylation at pericentric
heterochromatin.
[0101] FIG. 13: Spermatogenic failure and H3-K9 methylation in germ
cells of Suv39h dn mice.
[0102] FIG. 14: Illegitimate associations and delayed synaps is of
Suv39h dn meiotic chromosomes.
[0103] FIG. 15: Aberrant function of the Y chromosome during meios
is of Suv39h dn spermatocytes.
[0104] FIG. 16: Model for a `heterochromatic competence` in
protecting chromosome stability.
[0105] FIG. 17: Schematic illustration of a screening method for
identifying Suv 39h modulators.
DETAILED DESCRIPTION OF THE INVENTION
[0106] Materials and Methods
[0107] a) Sequence Alignments and Secondary Structure
Predictions
[0108] The SET domains of human SUV39H1 (Aagaard et al., 1999),
Drosophila SU(VAR)3-9 (Tschiersch et al., 1994) and S.pombe CLR4
(Ivanova et al., 1998) were used as a multiple starting alignment
for database similarity searches using Profile (Birney et al.,
1996), hidden Markov (Eddy, 1998) and position-specific iterative
BLAST (Altschul et al., 1997) methods (representative listings are
available from the SET domain page of the SMART WWW-server (Schultz
et al., 2000). These searches revealed significant similarities to
six plant proteins (accession numbers Q43088, O65218, P94026,
O80013, AAC29137 and AC007576.sub.--12) described as putative
lysine N-methyltransferases. For example, a PSI-BLAST search with
the S.pombe hypothetical protein SPAC3c7.09 as query identified
these plant sequences and well-known SET domain sequences within
ten rounds using an E-value inclusion threshold of 0.001. The same
search also revealed the presence of a SET domain in YHR109w, which
is known to encode a cytochrome c MTase (Martzen et al., 1999),
within three rounds. Consensus secondary structures were predicted
by described algorithms (Frishman and Argos, 1997).
[0109] b) Epitope-Tagged SUV39H1 Proteins in HeLa Cells
[0110] The HeLa cell lines overexpressing full-length
(myc).sub.3-SUV39H1 (aa 3-412) or (myc).sub.3-Nchromo (aa 3-118)
have been described (Aagaard et al., 1999; Melcher et al., 2000).
Nuclear extracts were immunoprecipitated with anti-myc antibody
beads (Aagaard et al., 1999), and approximately 1-3 .mu.g of
matrix-bound (myc).sub.3-tagged SUV39H1 proteins were used for in
vitro HMTase assays.
[0111] c) Generation and Purification of GST-Fusion Proteins
[0112] The GST-Suv1(82-412) product expressed from the pGEX-2T
vector (Pharmacia) as a glutathione-S-transferase (GST) fusion
protein has been described (Aagaard et al., 1999). Additional GST
constructs were generated by transferring BamIH-EcoRI PCR amplicons
into pGEX-2T, encoding in-frame fusions for Suv39h1(7-221),
SUV39H1(82-412), SUV39H1(82-378) .DELTA.C-tail, SUV39H1(255-412)
.DELTA.cys, Suv39h2(157-477) (O'Carroll et al., 2000),
CLR4(127-490) (Ivanova et al., 1998), EZH2(382-747) (Laible et al.,
1997) and HRX(3643-3969) (Tkachuk et al., 1992). Short internal
deletions (.DELTA.NHSCDPN.sub.323-329 and
.DELTA.GEELTFDY.sub.358-365) or point mutations within the
-.sub.320H.phi..phi.NHSC.sub.326- motif were directly engineered in
the GST-SUV39H1(82-412) plasmid by double PCR mutagenesis. All
constructs were confirmed by sequencing.
[0113] Recombinant proteins were expressed in 11 cultures of E.coli
strain BL21 and solubilized in 10 ml RIPA buffer (20 mM Tris pH
7.5, 500 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate)
containing a full set of protease inhibitors (Boehringer Mannheim)
and lysozyme (5 mg/ml; Sigma) by freeze-thawing in liquid N.sub.2,
followed by sonication. Soluble proteins were cleared by
centrifugation, purified with 800 .mu.l glutathione Sepharose beads
(Pharmacia) and washed twice in RIPA buffer. Protein concentration
was determined by Coomassie staining of SDS-PAGE gels. Matrix-bound
fusion proteins were used immediately for in vitro HMTase assays or
stored at 4.degree. C.
[0114] d) In vitro Histone Methyltransferase (HMTase) Assay
[0115] In vitro HMTase reactions were modified based on described
protocols (Strahl et al., 1999) and carried out in a volume of 50
.mu.l of methylase activity buffer (MAB: 50 mM Tris pH 8.5, 20 mM
KCl, 10 mM MgCl.sub.2, 10 mM .beta.-ME, 250 mM sucrose), containing
10 .mu.g of free histones (mixture of H1, H3, H2B, H2A and H4;
Boehringer Mannheim) as substrates and 300 nCi
S-adenosyl-[methyl-.sup.14C]-L-methionine (25 .mu.Ci/ml) (Amersham)
as methyl donor. 10 .mu.g of matrix-bound GST-fusion proteins were
routinely used to assay for HMTase activity. After incubation for
60 min. at 37.degree. C., reactions were stopped by boiling in SDS
loading buffer, and proteins were separated by 15% or 18% SDS-PAGE
and visualised by Coomassie staining and fluorography.
[0116] HMTase assays with individual histones (Boehringer
Mannheim), insulin (Sigma) or N-terminal peptides were performed
with 5 .mu.g of substrate. The following peptides were used:
wild-type N-terminus of human histone H3 (ARTKQTARKSTGGKAPRKQL)
(SEQ ID NO:7) and mutant peptide which changes lysine 9 (bold) to
leucine; N-terminus of human CENP-A (MGPRRRSRKPEAPRRRSPSP) (SEQ ID
NO:8) (Sullivan et al., 1994); N-terminus of rat macro-H2A
(MSSRGGKKKSTKTSRSAKAG) (SEQ ID NO:9) (Pehrson and Fried, 1992).
[0117] Peptide microsequencing of the in vitro methylated wild-type
H3 N-terminal peptide and determination of .sup.3H-incorporation of
individual amino acids by scintillation counting was done as
described (Strahl et al., 1999). Targeting of the Suv39h1 and
Suv39h2 gene loci in embryonic stem cells
[0118] Partial genomic clones of the Suv39h1 locus (X chromosome)
and of the Suv39h2 locus (chromosome 2) (O'Carroll et al., 2000)
were used to generate short and long arms of homology, in a
strategy to produce in-frame fusion proteins of the first 40 amino
acids of Suv39h1 or of the first 113 amino acids of Suv39h2 with
.beta.-galactosidase (LacZ) modified with a nuclear localization
signal (nls). For targeting, a 1.2 kb Pfu PCR amplicon and a 5.4 kb
SacI DNA fragment were derived from the genomic subclone gSuv39h1
#18, and a 1.3 kb Pfu PCR amplicon and a 5.0 kb MluI/Apal DNA
fragment were prepared from the genomic subclone gSuv39h2 #28 (see
FIG. 9A). The pGNA-derived targeting cassettes contained an
RSV-neomycin (neo) gene for positive selection and two
polyadenylation sites. The diphtheria toxin A (DTA) gene under the
control of the MCI promoter was used to select against random
integration and was inserted 3' of the long arms of homoloy. After
linearisation. with NotI, Suv39h1 and Suv39h2 targeting constructs
were electroporated into feeder-dependent R1 and E14.1 (129/Sv)
embryonic stem (ES) cells.
[0119] After selection, G418-resistant ES cell colonies were
screened for homologous recombination by nested PCR using primers
external to the short arms of Suv39h1 (PCR1:
5'-ATGGGGGCAGGGTTTTCGGGTAGAC, SEQ ID NO:10; PCR2:
5'-AAATGGTATTTGCAGGCCAC-TTCTTG, SEQ ID NO:11) or of Suv39h2 (PCR1:
5'-GAAAAGGTTGTTCTCCAGCTC, SEQ ID NO:12; PCR2:
5'-GGATGGGATGGTGG-AATGGTTTT- AT, SEQ ID NO:13) and primers within
the lacZ gene (lacZ-PCR1: 5'-AACCCGTCGGATTCTCCGTGGGAAC, SEQ ID
NO:14; lacZ-PCR2: 5'-CTCAGGAA-GATCGCACTCCAGCC, SEQ ID NO:15).
[0120] Successful targeting was confirmed by Southern blot analysis
of PvuII-digested ES cell DNA with a .apprxeq.500 bp external
Suv39h1 intron probe, generated with the primers g24r
(5'-GACTGC-CTAGTCTGGCACTGAACT, SEQ ID NO:16) and g13
(5'-GATCACTGCGTACATATAC-ACTGAT, SEQ ID NO:17), or of
HindIII-digested ES cell DNA with a .apprxeq.500 bp external
Suv39h2 exon/intron probe, generated with the primers P1f
(5'-TAGACTT-CTACTACATTA- ACG, SEQ ID NO:18) and P1r
(5'-GATGTCAGTGGCTATGAATG, SEQ ID NO:19). These DNA probes detect a
4.5 kb fragment from the wildtype Suv39h1 allele and a 4.0 kb
fragment from the targeted allele, or 11 kb and 6.1 kb fragments
from the Suv39h2 wildtype and targeted alleles (see FIG. 9B).
[0121] f) Generation and Genotyping of Suv39h1- and
Suv39h2-Deficient Mice
[0122] Several independently targeted ES cell clones gave rise to
chimaeric mice which passed the mutations through the germline.
Suv39h1-/- and Suv39h2-/- mice were intercrossed to produce
compound Suv39h mutant mice (e.g. Suv39h1-/-, Suv39h2+/-;
null1/het2), which were then mated to generate Suv39h double null
(dn) mice. All mice described in this study were maintained on a
mixed genetic background of 129/Sv and C57B1/6J origin.
[0123] Genotyping of mutant mice was done by Southern blot analysis
as described above. Protein blot analysis of nuclear extracts from
mouse testes with .alpha.-Suv39h1 and .alpha.-Suv39h2 antibodies
was performed as described previously (O'Carroll et al., 2000).
[0124] g) Generation and Analysis of Suv39h Double Null Primary
Mouse Embryonic Fibroblasts (PMEFs)
[0125] PMEFs were derived from day E12.5 Suv39h double null embryos
obtained after intercrossing Suv39h1.sup.-/-/Suv39h2.sup.+/-
compound mutant mice. As controls, PMEFs were prepared from
wild-type embryos of the same genetic background. For cell cycle
profiles and growth curve analysis, passage 2 PMEFs were analyzed
as described (Xu et al., 1999). Staining of PMEF interphase
chromatin with .alpha.-phosH3 (Hendzel et al., 1997) antibodies was
done in unpermeabilized cells as described (Melcher et al., 2000).
For the biochemical analysis, total nuclear extracts were
precalibrated by Ponceau staining, immuno-blotted with .alpha.-H3
(Upstate Biotechnology) and .alpha.-phosH3 (Hendzel et al., 1997)
antibodies and visualised by peroxidase staining using Enhanced
ChemiLuminescence (ECL) (Amersham).
[0126] h) Growth Curves and FACS Analyses of PMEFs
[0127] To analyze the proliferative potential of wild-type and
mutant cells, PMEFs were seeded onto 10 cm.sup.2 dishes. Over the
next 30 passages, 3.times.10.sup.5 cells were continually reseeded
every third day onto a new 10 cm.sup.2 dish (3T3 protocol), and
their doubling rates determined. The DNA profiles of passage 3 and
passage 8 PMEF cultures were obtained by FACS of ethanol-fixed and
propidium-iodide stained cells, using chicken erythrocyte nuclei
(Becton Dickinson) as an internal standard.
[0128] i) Bone Marrow Culture and FACS Analysis of B-cell Lymphoma
Cells
[0129] Bone marrow cells from wt and Suv39h dn mice were cultivated
for two weeks in StemPro-34 SFM medium (Life Technologies)
supplemented with IL-3 (10 ng/ml), IL-6 (5 ng/ml), SCF (100 ng/ml),
FLT 3 ligand (20 ng/ml), GM-CSF (1 ng/ml) (all from R&D
Systems), 10 .mu.M dexamethasone (Sigma) and IGF-1 (40 ng/ml)
(Sigma). Cultures were grown at densities of
.apprxeq.3.times.10.sup.6 cells per ml, and purified from
differentiated and dead cells by Ficoll-Paque gradient
centrifugation (Pharmacia).
[0130] Primary lymphoma cells were obtained from spleen and lymph
nodes using a 70 .mu.m Nylon Cell Strainer (Becton Dickinson), and
cultivated in Iscove's modified Dulbecco's medium (IMDM)
supplemented with 5% heat-inactivated fetal calf serum, 2 mM
glutamine and 1% penicillin-streptomycin (all Gibco-BRL). Single
cells suspensions were grown O/N in medium additionally containing
50 .mu.M .beta.-mercaptoethanol and 5% conditioned supernatant from
rIL-7 producing J558L cells.
[0131] The identity of the tumor cells was determined by FACS
analyses using antibodies (all from Pharmingen) that detect
specific cell surface markers. All tumor cells were double positive
for the B-cell markers B220-low (RA3-6B2) and CD19 (1D3), but
negative for the T-cell markers CD3 (145-2C11), CD4 (RM4-5), CD8
(53-6.7), or for the granulocyte/macrophage markers Gr-1 (RB6-8C5),
Mac-1 (M1/70) and for a marker of the eythroid lineage, Ter-119.
The majority of the B-cell lymphoma cells were also double positive
for CD43 (S7) and IgM (R6-60.2), while some clonal cultures
displayed reactivity towards CD5 (53-7.3). These FACS profiles
characterize the Suv39h-mediated tumors as being similar to chronic
lymphoid leukemia in humans (Foon and Gale, 1995).
[0132] j) Chromosome Spreads and Karyotype Analyses
[0133] PMEF and tumor cell karyotypes were analyzed on
colchicine-arrested and Giemsa-stained metaphase chromosome
spreads.
[0134] Metaphase spreads of spermatogonia and spermatocytes were
prepared from isolated seminiferous tubule fragments which had been
hypotonically swollen with 1% sodium citrate for 10 min. at RT and
fixed O/N at 4.degree. C. with Carnoy's solution (75% methanol, 25%
acetic acid). After incubation of seminiferous fragments in 60%
acetic acid for 2 min., a single cell suspension was generated by
repeated pipetting, transferred onto a pre-heated (60.degree. C.)
glass slide, and cells were spread by mechanical shearing with a
glass hockey stick.
[0135] k) Generation and Purification of .alpha.-methH3-K9
Antibodies
[0136] To generate methyl-specific antibodies against the histone
H3 lysine 9 position, a hexameric peptide was generated,
-TARK(Me).sub.2ST-cys, containing a di-methylated lysine (Bachem)
and a terminal cysteine. To increase the antigenicity and
immunogenicity, a `branched` peptide that consists of four
-TARK(Me).sub.2ST- `fingers` which are linked at their C-termini
via lysine residues was also synthesized. The sequence of this
`branched` peptide is [TARK(Me).sub.2ST].sub.4-K.sub.2-K-cys.
Peptides were coupled to KLH and rabbit polyclonal antisera were
raised, indicating that the `branched` peptide was much more
immunogenic than the linear peptide.
[0137] Crude antisera from two positive rabbits (#2233 and #2236)
were batch-absorbed against a `branched`, but unmodified control
peptide, followed by affinity purification against the
di-methylated `branched` antigen that had been crosslinked to a
Poros.TM. column (Lachner et al., 2001). Bound antibodies were
eluted with 100 mM glycine pH 2.5 and neutralised with {fraction
(1/10)} vol. of 2 M Hepes pH 7.9. The methyl-specificity of the
antibodies was confirmed on slotblots presenting unmodified or
K9-dimethylated histone H3 peptides and on protein blots containing
nuclear extracts from wt or Suv39h dn PMEFs. The affinity-purified
.alpha.-methH3-K9 antibodies (concentration .apprxeq.0.6 mg/ml) can
be used at a 1:1,000 dilution for protein blot analysis or at
1:1,000 to 1:5,000 dilutions for indirect immunofluorescence.
[0138] l) Immunofluorescence of Interphase Chromatin and Metaphase
Chromosomes
[0139] Passage 6 PMEFs were fixed with 2% p-FA for 10 min. on ice,
washed, incubated with blocking solution (PBS, 2.5% BSA, 10% goat
serum and 0.1% Tween20) for 30 min at RT and stained O/N at
4.degree. C. with the .alpha.-methH3-K9 antibodies. After several
washes with PBS containing 0.2% BSA and 0.1% Tween20, the primary
antibodies were detected with Alexa Fluor488-conjugated goat
.alpha.-rabbit antibodies (Molecular Probes). DNA was
counterstained with 4',6'-diamidino-2-phenylindole (DAPI), and
samples were embedded in Vectashield (Vector Laboratories).
[0140] For preparation of metaphase chromosomes, bone marrow cells
or primary tumor cells were arrested by colchicine treatment (0.5
mg/ml) (Sigma) for 2.5 hrs., followed by hypotonic swelling in 0.6%
KCl or RBS buffer (10 mM TrisHCl pH 7.4; 10 mM NaCl; 5 mM
MgCl.sub.2) for 15 min. at 37.degree. C. and centrifugation for 8
min. at 2000 rpm in a Cytospin (Shandon). Spreaded cells were
immediately fixed with icecold 2% p-FA in PBS for 15 min., washed
twice and stained with the .alpha.-methH3-K9 antibodies as
described above.
[0141] m) Testes Histology
[0142] Testes were dissected from adult mice, fixed in Bouins fluid
(75% saturated picric acid, 5% glacial acetic acid, 9.3%
formaldehyde) and stained with haematoxylin/eosine. Staging of the
seminiferous tubules was performed according to Oakberg (1956) and
Russell et al. (1990). FISH analyses with mouse major satellite DNA
probes were done as recently described (Scherthan et al., 1996),
and Tunel assays were performed using the DeadEnd apoptosis
detection system (Promega). In addition, testis cryosections
(O'Carroll et al., 2000) were also analyzed by
immuno-histochemistry with .alpha.-Scp, .alpha.-Hp1.beta.,
.alpha.-phosH3 and .alpha.-meth H3-K9 antibodies.
[0143] n) Immunofluorescence of Germ Cells and Meiotic Chromosome
Spreads
[0144] Chromosome spreads of spermatogenic cells were prepared
according to Peters et al. (1997a) with some minor modifications. A
single germ cell suspension was obtained in DMEM medium by
mechanical disruption of isolated seminiferous tubules. After
serveral washes and hypotonic swelling in hypobuffer (30 mM TrisHCl
pH 8.2, 50 mM sucrose, 17 mM sodium citrate) for 10 min. at RT,
cells were resuspended in 100 mM sucrose, 15 mM TrisHCl pH 8.2 and
spreaded on precleaned slides covered by a thin film of 1% p-FA
containing 5 mM borate pH 9.2 and 0.15% TritonX-100. Slides were
dried slowly in a humid chamber for .apprxeq.2 hrs and stored at
-80.degree. C. Classification of meiotic sub-stages was performed
according to the changing morphology of autosomes and sex
chromosomes as described (Peters et al., 1997b).
[0145] Double-labelling immunofluorescence of these germ cell
preparations was performed by sequential incubation with rabbit
polyclonal .alpha.-methH3-K9 antibodies and with goat
.alpha.-rabbit Alexa568-conjugated secondary antibodies. After a
brief fixation in 1% p-FA, samples were incubated with rabbit
polyclonal .alpha.-Scp3 antibodies (Lammers et al., 1995) that were
visualized with goat .alpha.-rabbit Alexa488-conjugated secondary
antibodies. In addition, co-stainings were also done with
.alpha.-Scp3 and .alpha.-Scp1 (Offenberg et al., 1991) (see FIGS.
14A-C), and .alpha.-Scp3 and .alpha.-HP1.beta. (Wreggett et al.,
1994), and .alpha.-Scp3 and .alpha.-phosH3 (Hendzel et al., 1997)
antibodies.
[0146] o) EM Aanalysis
[0147] Preparation and silver staining of SC complexes from
spreaded germ cells (see above) was performed according to Peters
et al. (1997a), and samples were analyzed on a Jeol 1200 EKII
transmission electron microscope.
EXAMPLE 1
Sequence Similarity of SET Domains with Plant
Methyltransferases
[0148] Using the SET domains of the SU(VAR)3-9 protein family as a
starting alignment, significant sequence and secondary structure
similarities (see Methods) to six plant protein methyltransferases
were detected. Although some of these plant sequences have been
classified as potential histone lysine N-methyltransferases, only
one had been functionally characterised but was found to lack
HMTase activity (Klein and Houtz, 1995; Zheng et al., 1998).
[0149] Detected were amino acid and secondary structure
[.beta.-sheet (b) or .alpha.-helix (h)] similarities of the
C-terminal halves of SET domain sequences from human SUV39H1
(Aagaard et al., 1999) (AF019968), murine Suv39h1 (Aagaard et al.,
1999) (AF019969), murine Suv39h2 (O'Carroll et al., 2000),
(AF149205), Drosophila SU(VAR)3-9 (Tschiersch et al., 1994)
(P45975), a C.elegans SU(VAR)3-9-like ORF C15H11.5 (CAB02737),
S.pombe CLR4 (Ivanova et al., 1998) (O074565), human EZH2 (Laible
et al., 1997) (Q15910), the human trithorax homologue HRX (Tkachuk
et al., 1992) (Q03164), and MTases from P.sativum (rubisco ls-MT;
Q43088) (Klein and Houtz, 1995; Zheng et al., 1998) and A.thaliana
(O65218). The plant MTase sequences contain an insertion of
approximately 100 amino acids in the middle of the SET domain.
EXAMPLE 2
HMTase Activity of Transfected and Recombinant SUV39H1 and Suv39h1
Proteins
[0150] To investigate whether the SET domain of human SUV39H1 has
enzymatic activity, histones were tested as possible substrates for
in vitro methylation. Using HeLa cell lines `stably` expressing
triple myc-tagged full-length SUV39H1 (aa 3-412), the ectopic
protein was enriched from nuclear extracts by immunoprecipitation
with anti-myc beads (see FIG. 1/1A, arrowhead top panel) and probed
for activity to transfer a labelled methyl group from
S-adenosyl-[methyl-.sup.14C]-L-methionine to free histones
according to described conditions (Strahl et al., 1999). Reaction
products were separated by SDS-PAGE and visualised by fluorography,
indicating selective transfer of the methyl-label to H3 (FIG. 1/1A,
lower panel). By contrast, no signals were detected with extracts
from a HeLa cell line that expresses only the N-terminal third of
SUV39H1 (aa 3-118) or with extracts from HeLa control cells. To
confirm that the HMTase activity is an intrinsic property of
SUV39H1 and not mediated by a SUV39H1-associated factor, the in
vitro HMTase reactions was repeated with recombinant products that
were purified as GST-fusion proteins from E.coli (see FIG. 1/2B,
arrowheads top panel). For this analysis, murine Suv39h1, which is
95% identical to human SUV39H1 (Aagaard et al., 1999) was used. A
purified GST-product comprising aa 82-412 maintained HMTase
activity (although at a reduced level as compared to transfected
SUV39H1), whereas a purified GST-product comprising aa 7-221 proved
negative, even at higher protein concentrations (FIG. 1/2B, lower
panel). These results suggest that the HMTase activity resides in
the C-terminal SET domain.
[0151] In FIG. 1A, triple myc-tagged full-length human SUV39H1 (aa
3-412) or a C-terminally truncated SUV39H1 protein (aa 3-118) were
immunoprecipitated from `stably` transfected HeLa cell lines with
anti-myc antibody beads and used in in vitro HMTase reactions with
free histones as substrates and
S-adenosyl-[methyl-.sup.14C]-L-methionine as methyl donor. The
Coomassie stain (top panel) shows purified proteins by arrowheads
and free histones by dots. Fluorography (bottom panel, FIG. 1A)
indicates HMTase activity of (myc).sub.3-SUV1(3-412).
[0152] In the experiments shown in FIG. 1B, recombinant GST-fusion
proteins encoding different domains of murine Suv39h1 were used in
increasing protein concentrations for in vitro HMTase reactions as
described above.
EXAMPLE 3
[0153] a) Definition of a Catalytic Motif in the SET Domain of
Human SUV39H1
[0154] Similar to the recombinant murine GST-Suv39h1(82-412)
product, the corresponding human SUV39H1 fusion protein
[GST-SUV39H1(82-412)] is catalytically active (see FIG. 2). Short
internal deletions (.DELTA.NHSCDPN.sub.323-329; .DELTA.NHSC and
.DELTA.GEELTFDY.sub.358-365; .DELTA.GEEL) were introduced into the
two conserved regions of the SET domain core in
GST-SUV39H11(82-412) and, in addition, mutants that lack the
C-terminal tail (.DELTA.C-tail) or the SET-associated cysteine-rich
region (.DELTA.cys) were also generated. All mutant proteins failed
to demonstrate HMTase activity (see FIG. 2/1A). To investigate
enzyme function of the SET domain in more detail, point mutations
were introduced into the most highly conserved motif. In vitro
HMTase assays indicated that all point mutations, with the
exception of one, abolished enzymatic activity. Surprisingly, the
latter mutation (H320R) resulted in an hyperactive enzyme with
approximately 20-fold increased activity. The data obtained define
the .sub.320H.phi..phi.NHSC.sub.326 motif in the SET domain as an
important catalytic site.
[0155] b) Specific HMTase Activity of the SET Domain of Mammalian
SU(VAR)3-9 Related Proteins
[0156] Because the SET domain is one of the most conserved protein
motifs in chromatin regulators (Stassen et al., 1995; Jenuwein et
al., 1998), it was next analyzed whether SU(VAR)3-9 family members
or other SET domain proteins contain HMTase activity. GST-fusion
products of the extended SET domains of S.pombe CLR4 (Ivanova et
al., 1998), human EZH2 (Laible et al., 1997) and human HRX (Tkachuk
et al., 1992) were generated that would correspond to
GST-SUV39H1(82-412) (see FIG. 2/2B). Interestingly,
GST-CLR4(127-490) displayed pronounced HMTase activity at three- to
five-fold increased levels (see FIG. 2/2C) as compared to the
recombinant SUV39H1 product, consistent with CLR4 carrying an
arginine at the hyperactive position. By contrast, both
GST-EZH2(382-747) and GST-HRX(3643-3966) had undetectable HMTase
activity towards free histones (FIG. 2/2C), whereas a comparable
GST product generated from the recently isolated murine Suv39h2
gene (O'Carroll et al., 2000), GST-Suv39h2(157-477), was as active
as GST-SUV39H1(82-412). EZH2 lacks the C-terminal cysteines, and
HRX does not contain the SET associated cysteine-rich region (FIG.
2B). Both of these cysteine domains are present in CLR4, Suv39h2
and SUV39H1. In agreement with the mutational analysis of SUV39H1,
it thus appears that HMTase activity towards free histones requires
the combination of the SET domain with adjacent cysteine-rich
regions, and is a quality found in only a restricted number of SET
domain containing proteins.
[0157] In FIG. 2A, approximately 10 .mu.g of the indicated fusion
proteins encoding GST-SUV1(82-412) (=human SUV39H1) and seven SET
domain mutants were used in in vitro HMTase reactions with free
histones as outlined in FIG. 1. For the hyperactive H320R mutant,
only 1 .mu.g (10%) of the corresponding fusion product was used.
FIG. 2 shows a diagram representing the domain structures of CLR4,
Suv39h2, SUV39H1, EZH2 and HRX proteins, with the arrowheads
demarcating the N-terminal fusion to GST. Cysteine-rich regions are
indicated by grey stippling.
[0158] In FIG. 2C, approximately 10 .mu.g of the indicated fusion
proteins encoding S.pombe CLR4 [GST-CLR4(127-490)], murine Suv39h2
[GST-Suv2(157-477)], human EZH2 [GST-EZH2(382-747)], human HRX
[GST-HRX(3643-3969)] and human SUV39H1 [GST-SUV1(82-402)] were used
in in vitro HMTase reactions with free histones as outlined in FIG.
1.
EXAMPLE 4
Lysine 9 of the H3 N-terminus is the Major Site for in vitro
Methylation by Recombinant Suv39h1
[0159] The above Examples indicated that the HMTase activity of
mammalian SU(VAR)3-9 related proteins is selective for H3 under the
chosen assay conditions. To examine this finding in more detail, in
vitro methylation reactions were performed with individual
histones, using GST-Suv39h1(82-412) as an enzyme. As shown in FIG.
3/1A, H3 is specifically methylated by GST-Suv39h1(82-412), whereas
no signals are detected with H2A, H2B or H4. A weak signal is
present if H1 was used as the sole substrate; the significance of
H1 methylation remains to be determined. Methylation of H3 has been
shown to occur predominantly at lysine 4 in a wide range of
organisms, as well as at lysine 9 in HeLa cells, although the
responsible HMTase(s) have yet to be defined (Strahl et al., 1999).
To investigate the site utilisation profile of Suv39h1, unmodified
peptides comprising the wild-type H3 N-terminus (aa 1-20) and a
mutant K9L peptide, changing lysine 9 to leucine were tested as
substrates. Additionally, insulin and peptides comprising the
N-termini of CENP-A (Sullivan et al., 1994) and macroH2A (Pehrson
and Fried, 1992) were included. Peptides were in vitro methylated
by GST-Suv39h1(82-412), and reaction products were separated by
high percentage SDS-PAGE and visualised by fluorography. These in
vitro assays revealed selective methylation of the wild-type H3
peptide, whereas no signals were detected with the CENP-A or
macroH2A peptides, or with insulin (see FIG. 3/2B). Importantly,
the mutated H3 (K9L) peptide was not a substrate, suggesting that
lysine 9 of the H3 N-terminus is a preferred residue for
Suv39h1-dependent HMTase activity.
[0160] To more definitively determine this site preference, the
wild-type H3N-terminal peptide was in vitro methylated by
GST-Suv39h1(82-412), using
S-adenosyl-[methyl-.sup.3H]-L-methionine. The labelled peptide,
purified by reverse-phase HPLC, was then directly microsequenced,
and .sup.3H-incorporation associated with each individual amino
acid was analysed by scintillation counting. The results confirmed
selective transfer of methyl-label to lysine 9 (see FIG. 3/2C),
demonstrating that Suv39h 1 is a highly site-specific HMTase for
the H3 N-terminus in vitro.
[0161] In FIG. 3A, approximately 10 .mu.g of murine
GST-Suv39h1(82-412) were used in in vitro HMTase reactions with
individual histones as outlined in FIG. 1.
[0162] FIG. 3B shows the results of in vitro methylation assays
using GST-Suv39h1(82-412) as enzyme and the indicated N-terminal
peptides of wild-type H3, mutated H3 (K9L), CENP-A, macroH2A or
insulin as substrates.
[0163] FIG. 3C shows the result of automated sequencing of the
wild-type H3 N-terminal peptide (aa 1-20) that had been methylated
in vitro by recombinant GST-Suv39h1(82-412). Displayed is the
.sup.3H-incorporation of individual amino acids identified at each
successive round of microsequencing.
EXAMPLE 5
Targeting the Suv39h1 and Suv39h2 Loci in the Mouse Germline
[0164] Murine Suv39h genes are encoded by 2 loci, Suv39h1 and
Suv39h2 (O'Carroll et al., 2000). To investigate the in vivo
significance of Suv39h function and Suv39h-dependent H3-K9
methylation, mouse strains deficient for both Suv39h1 and Suv39h2
were generated according to standard techniques. The targeting
strategies are shown in FIG. 4, as well as demonstrating the
production of null alleles for both Suv39h1 and Suv39h2. Mutation
of either gene results in viable and fertile mice as a consequence
of functional redundancy between both loci. Therefore, Suv39h1 and
Suv39h2 deficient strains were intercrossed to produce Suv39h
double null mice. Suv39h double null mice are born in sub-Mendelian
ratios (see Example 8B, below), where only approximately 30% of the
expected Suv39h double mutants are observed. FIG. 4 shows a
conventional targeting strategy used to inactivate the X-linked
Suv39h1 locus. FIG. 4B shows the Northern blot analysis of Suv39h1
from spleen (Sp), liver (Li), kidney (Kidney), and brain (Br) from
wild-type and Suv39h1 null mice. FIG. 4C shows the conventional
targeting strategy used to inactivate the autosomal Suv39h2 locus.
(Bottom panel) Western blot analysis with .alpha.-Suv39h2
antibodies on protein extracts derived from wild-type and Suv39h2
null testis.
EXAMPLE 6
Aberrant Mitoses in Suv39h Double Null Primary Mouse Fibroblasts
(PMEFs)
[0165] In order to determine whether the embryonic phenotypes in
Suv39h double null mice can be attributed to mitotic defects, PMEFs
derived from Suv39h double mice were analysed. Cell cycle profiles
of wild-type and Suv39h double null PMEFs indicated rather similar
percentages of cells to be in S- and G2/M-phases (see FIG. 5A),
whereas Suv39h double null PMEFs display a reduced G1-index and an
increased proportion of cells with aberrant nuclear morphologies,
reminiscent of division defects during mitosis. For example, Suv39h
double null PMEFs contain approximately two-fold elevated numbers
of cells with micro- and polynuclei, and are further characterised
by cell subpopulations with oversized nuclei or a weak definition
of heterochromatin that appears in only a few unusually condensed
foci (see FIG. 5B). Furthermore, Suv39h double null cells also show
genomic instabilities and readily become aneuploid (see also
Example 9, below). The severity of these aneuploidies increases
with higher passage numbers (see FIG. 6). The inability of Suv39h
double null cells to maintain a stable karyotype may underlie the
Suv39h embryonic phenotype.
[0166] FIG. 5A shows the percentages of cells in various cell cycle
stages of wild-type and Suv39h double null PMEFs.
[0167] FIG. 5B shows representative images (left and middle) of
aberrant mitoses in Suv39h double null PMEFs detected by
.alpha.-tubulin (not shown) and DAPI staining. Also shown (right
image) is a nucleus exemplifying the unusual definition of
heterochromatin in a subpopulation of Suv39h double null PMEFs. All
images were taken at a magnification of 630.times..
[0168] FIG. 6A shows the DNA content profile of wild-type and
Suv39h double null PMEFs at passage 3. FIG. 6B shows the DNA
content profile of wild-type and Suv39h double null PMEFs at
passage 8.
EXAMPLE 7
Increased phosH3 Phosphorylation in Suv39h Double Null PMEFs
[0169] Phosphorylation at serine 10 (phosH3) in the N-terminal tail
of H3 has been shown to be required for condensation and subsequent
segregation of chromosomes (Wei et al., 1999). During the cell
cycle, phosH3 initiates within pericentric heterochromatin in late
G2 and then progresses along the entire chromosomes during mitosis
(Hendzel et al., 1997). In wild-type PMEFs, approximately 7% of the
cells stain positive for the characteristic,
heterochromatin-associated phosH3 foci, as detected by indirect
immunofluorescence with .alpha.-phosH3 antibodies (see FIG. 7A,
right panel). In contrast, this number is increased by a factor of
about 3-fold in Suv39h double null PMEFs, with approximately 22% of
the cells containing phosH3-positive foci (FIG. 7A, left panel),
although their definition appears in many small speckles which do
not always overlap with DAPI-dense material. This result suggested
that the overall levels of phosH3 may be enhanced in Suv39h double
null PMEFs. Therefore, the relative abundance of phosH3 in
precalibrated nuclear extracts was determined with
.alpha.-phosH3-specific antibodies. This quantitation indicated a
significantly higher level of phosH3 in Suv39h double null cells as
compared to wild-type controls (see FIG. 7B). Together, the
obtained data are most consistent with a model in which
Suv39h-mediated methylation of lysine 9 in H3 negatively regulates
phosphorylation of serine 10.
[0170] FIG. 7A shows an interphase chromatin staining with
.alpha.-phosH3 antibodies and CY3-conjugated secondary antibodies.
DNA was counterstained with DAPI. At least 1,000 cells were counted
to evaluate the percentage (indicated in the Figure) of
.alpha.-phosH3 positive cells. FIG. 7B shows a quantitative Western
analysis with 15 .mu.g and 30 .mu.g of total nuclear proteins
immuno-blotted with .alpha.-H3 and .alpha.-phosH3 antibodies.
EXAMPLE 8
[0171] a) Generation of Suv39h Double Deficient Mice
[0172] Murine Suv39h HMTases are encoded by two loci which have
been mapped to centromere-proximal positions in the X chromosome
(Suv39h1) or in chromosome 2 (Suv39h2) (O'Carroll et al., 2000).
Both gene loci were independently disrupted by homologous
recombination in embryonic stem (ES) cells using a conventional
targeting approach that replaces parts of the evolutionarily
conserved chromo domain with the bacterial LacZ gene and an
RSV-neomycin selecion cassette (FIG. 9a). These targeting
strategies produce in-frame fusion proteins of the first 40 amino
acids of Suv39h1 or of the first 113 amino acids of Suv39h2 with
lacZ, which maintain .beta.-galactosidase activities. Successfully
targeted ES cell clones were used to generate chimaeric mice that
transmitted the mutated Suv39h1 or Suv39h2 alleles through the germ
line (FIG. 9b). Protein blot analyses of testis nuclear extracts
from wild-type, Suv39h1- and Suv39h2-deficient mice with
.alpha.-Suv39h1 and .alpha.-Suv39h2 specific antibodies (Aagaard et
al., 1999; O'Carroll et al., 2000) indicated the absence of the
respective proteins, demonstrating that had been generated
loss-of-function alleles for both genes (FIG. 9c).
[0173] b) Impaired Viability of Suv39h Double Null Mice
[0174] Mice deficient for either Suv39h1 or Suv39h2 display normal
viability and fertility, and do not exhibit apparent phenotypes,
suggesting that both genes may be functionally redundant during
mouse development (O'Carroll et al., 2000). Therefore, Suv39h1-/-
and Suv39h2-/- mice were intercrossed to generate compound Suv39h
mutants that were then used to derive Suv39h double null (dn) mice.
Suv39h dn mice obtained from several different intercrosses (Table
I) are born at only sub-Mendelian ratios, are growth retarded (FIG.
9d) and are characterized by hypogonadism in males. For example,
from a total of 197 mice, 46 mice would have been expected to be
double null (Table I), but only 15 Suv39h dn mice (.apprxeq.33%)
were born. Analysis of mouse embryogenesis indicated normal
development of Suv39h dn fetuses until day E12.5, whereas at later
stages, Suv39h dn fetuses are smaller and display an increased rate
of resorptions and prenatal lethality. Together, these results
demonstrate that the Suv39h genes are required for normal
viability, and for pre- and postnatal development.
[0175] FIG. 9 shows the targeting and genotyping of Suv39h1- and
Suv39h2-deficient mice as follows: (A) Diagrammatic representation
of the Suv39h1 and Suv39h2 genomic loci, the replacement vectors
and the targeted alleles. Exons are indicated by black boxes with
numbers referring to the starting amino acid positions of the
respective exons (O'Carroll et al., 2000). Also shown are the
diagnostic restriction sites and the external probes used for
Southern blot analyses. pA indicates polyadenylation signals. (B)
Southern blot analyses of PvuII- or HindIII-digested DNA isolated
from offspring of Suv39h1+/- or Suv39h2+/- heterozygous
intercrosses. (C) Protein blot analyses of testis nuclear extracts
from wild-type (wt), Suv39h1-/- (Suv1-/-) and Suv39h2-/- (Suv2-/-)
mice with .alpha.-Suv39h1 and .alpha.-Suv39h2 antibodies. The size
of the Suv39h1 or Suv39h2 proteins is indicated by arrows. (D)
Suv39h double null (dn) mice are growth retarded at birth and
during adulthood.
EXAMPLE 9
Chromosome Mis-Segregation in Suv39h dn Embryonic Fibroblasts
[0176] To examine the Suv39h-dependent defects in more detail,
primary mouse embryonic fibroblasts (PMEFs) were derived from day
E12.5 fetuses. Comparative growth curves between wild-type (wt) and
Suv39h dn PMEFs in a 3T3 protocol over the first 20 passages
indicated that Suv39h dn PMEFs displayed a higher doubling rate
until passage 12 (FIG. 10a). At later passages, the Suv39h dn PMEFs
appear to have a slightly reduced proliferative potential than the
immortalised wt PMEFs which survived the characteristic Hayflick
crisis. It was shown recently (see Example 6) that Suv39h dn PMEFs
contain a significant fraction of cells with aberrant nuclear
morphologies, such as macro- and polynuclei, which are reminiscent
of impaired mitosis and chromosome nms-segregation (Rea et al.,
2000). Therefore the DNA content of passage 3 and passage 8 wt and
Suv39h dn PMEFs was analyzed by FACS. Whereas wt PMEFs appear
genomically stable at passage 3, Suv39h dn PMEFs already contain
cells with a greater than 4N DNA content, as indicated by the
aneuploid shoulder in the FACS profile (FIG. 10B, top panels). At
passage 8, wt PMEFs are largely senesced. By contrast, Suv39h dn
PMEFs continue to proliferate, although many cells display
octaploid DNA contents (FIG. 10B, lower panels).
[0177] To further characterize these genomic instabilities,
karyotype analyses with passage 8 PMEFs were performed (FIG. 10C).
In particular, 45 karyotypes each for two independent wt and two
Suv39h dn PMEF cultures were examined. As shown in FIG. 10D, a
major fraction of the wt karyotypes are non-diploid, with
chromosome numbers ranging from 25 to 82. Aneuploidies were
significantly increased in Suv39h dn karyoptypes and comprised
chromosome numbers from 38 to 162. Notably, whereas wt PMEFs
contain a random array of aneuploid karyotypes, Suv39h dn PMEFs are
largely hypo-tetraploid or hypo-octaploid. Chromosomes in Suv39h dn
PMEFs appear of normal morphology and Robertsonian fusions were not
observed. It was concluded that the absence of Suv39h function
induces genomic instabilities, primarily by impairing segregation
of the entire set of chromosomes.
[0178] FIG. 10 shows the chromosomal instabilities in Suv39h dn
PMEFs as follows: (A) Relative doubling rates of wt and Suv39h dn
PMEFs determined in a 3T3 protocl over the first 20 passages. (B)
DNA contents of wt and Suv39h dn PMEF mass cultures at passage 3
and passage 8. (C) Metaphase spreads showing a diploid number
(n=40) of chromosomes for wt and a hyper-tretraploid number (n=82)
of chromosomes for Suv39h dn PMEFs. (D) Statistical karyotype
analysis with two wt and two Suv39h dn PMEF cultures at passage 8.
For each culture, 45 metaphases were evaluated.
EXAMPLE 10
Development of B-cell Lymphomas in Suv39h Mutant Mice
[0179] Next, Suv39h mutant mice were analyzed for the incidence of
tumorigenesis. Because the majority of Suv39h dn mice are
non-viable, distinct Suv39h genotypes that differ in their gene
dosage for either Suv39h1 or Suv39h2 were examined. For example, it
was expected that random X-inactivation of the X-linked Suv39h1
gene could increase the tumor risk in Suv39h1+/- mice, even in the
presence of a functional copy of Suv39h2 which is significantly
down-regulated in most adult tissues (O'Carroll et al., 2000).
Indeed, examination of 98 mice which are either heterozygous (het)
or null for the Suv39h1 locus indicated an .apprxeq.28% penetrance
of tumor formation with an onset between 9-15 months of age (Table
II). These tumors are predominantly B-cell lymphomas (FIG. 11A)
that resemble by FACS profiling (see Materials and Methods) slowly
progressing non-Hodgin lymphomas in humans (Foon and Gale, 1995).
The tumor incidence for late onset B-cell lymphomas was
.apprxeq.33% in the few viable Suv39h dn mice (n=6). By contrast,
Suv39h2+/- or Suv39h2-/- mice developed B-cell lymphomas at only
.ltoreq.5% penetrance (n=21), and tumor formation in control
wild-type mice was not observed.
[0180] Primary cultures were derived from the lymph nodes of Suv39h
dn and of Suv39h1-/-, Suv39h2+/- (null1/het2) tumor mice, and
analyzed the karyotypes of the B-cell lymphoma cells. Consistent
with the aneuploides described above for Suv39h dn PMEF mass
cultures, these tumor cells were largely hyper-diploid but also
comprised some hyper-tetraploid karotypes (FIG. 11B). Surprisingly,
a fraction of Suv39h dn tumor karyotypes, examined in several
independent B-cell lymphomas, is characterized by non-segregated
chromosomes that remain attached through their acrocentric regions
(FIG. 11C). These `butterfly` chromosomes raise the intriguing
possibility that the absence of Suv39h HMTase activities could
impair the quality and function of pericentric heterochromatin by
increasing more persistent interactions between metaphase
chromosomes. Indeed, analysis of H3-K9 methylation with a newly
developed antibody (see Example 11, below) indicates the absence of
methH3-K9 staining at pericentric heterochromatin of tumor
chromosomes derived from Suv39h null1/het2 B-cell lymphoma
cells.
[0181] FIG. 11 shows the development of B-cell lymphomas in Suv39h
mutant mice as follows: (A) Spleen and lymph nodes of an 11-month
old Suv39h dn tumor mouse and of a wild-type control mouse. (B)
Karyotype analysis of four independent primary cultures derived
from the lymph nodes of tumor-bearing Suv39h dn (null1/null2) and
Suv39h1-/-, Suv39h+/- (null1/het2) mice. (C) Metaphase spread from
a primary Suv39h dn B-cell lymphoma cell showing `butterfly`
chromosomes that remain associated through their acrocentric
regions.
EXAMPLE 11
Absence of H3-K9 methylation at Suv39h dn heterochromatin
[0182] The above karyotype analyses on PMEF and tumor cells
suggested a general mechanism through which segregation of the
entire chromosome complement may be impaired by Suv39h-dependent
defects in pericentric chromatin organization. To assess directly
the role of the Suv39h HMTases in histone methylation and
heterochromatin formation, a rabbit polyclonal antiserum was raised
that specifically recognizes histone H3 when di-methylated at
lysine 9 (.alpha.-methH3-K9). As shown in FIG. 12A, this antiserum
detects a focal staining in wt PMEFs that significantly overlaps
with DAPI-rich heterochromatin. In PMEFs derived from single
Suv39h1- or Suv39h2-deficient mice, .apprxeq.75% of cells stain
positive for heterochromatic foci with these .alpha.-methH3-K9
antibodies. Importantly, heterochromatic staining for methH3-K9 was
abolished in Suv39h dn PMEFs (FIG. 12A, right row).
[0183] Mitotic chromosome spreads from bone marrow cells were also
analyzed with the .alpha.-methH3-K9 antiserum. In wt spreads,
pericentric heterochromatin was selectively visualised (see inserts
in FIG. 12B), whereas only residual staining was detected in Suv39h
dn spreads. Thus, consistent with the localization of SUV39H1 at
active centromeres (Aagaard et al., 2000), these data demonstrate
that both Suv39h enzymes are the major HMTases to methylate H3-K9
in pericentric heterochromatin of somatic cells. Moreover, these
results also characterize the .alpha.-methH3-K9 antibodies as a
novel cytological marker for heterochromatin and corroborate recent
S.pombe studies, in which enrichment of H3-K9 methylation at MAT
and CEN regions was shown to be dependent upon a functional Clr4
enzyme (Nakayama et al., 2001).
[0184] FIG. 12 shows the Suv39h-dependent H3-K9 methylation at
pericentric heterochromatin as follows: (A) DAPI and methH3-K9
staining on interphase chromatin of wild-type (wt), Suv39h1-/-,
Suv39h2-/-, and Suv39h dn PMEFs. Percentages refer to interphase
nuclei displaying H3-K9 methylation at heterochromatic foci. (B)
DAPI and methH3-K9 staining on mitotic chromosomes prepared from in
vitro cultured wt and Suv39h dn bone marrow cells.
EXAMPLE 12
[0185] a) Hypogonadism and Complete Spermatogenic Failure in Suv39h
dn Mice
[0186] The expression pattern of the Suv39h genes suggests an
important role during spermatogenesis (O'Carroll et al., 2000).
Indeed, Suv39h dn males (n=7) are infertile, do not contain mature
sperm and their testis weights are 3-10 fold reduced as compared to
that of wt males (FIG. 13A). To investigate the spermatogenic
failure in more detail, histological sections were performed,
demonstrating normally developed seminiferous tubules in wt testis
which display the characteristic differentiation from the
mitotically proliferating spermatogonia (Sg) to meiotic
spermatocytes (Sc) and the post-meiotic haploid spermatids (St)
(FIG. 13A). By contrast, spermatogenesis was severely impaired in
Suv39h dn mice, with an apparent differentiation arrest at the
transition between early to late spermatocytes, resulting in highly
vacuolarized seminiferous tubules (FIG. 13A).
[0187] FISH analyses with mouse major satellite DNA probes and
TUNEL assays were used to characterize the Suv39h-dependent
spermatogenic defects further. Whereas mitotic proliferation of
spermatogonia appeared normal, a 3 to 10 fold increase in the
percentage of pre-leptotene spermatocytes was observed. These
pre-leptotene spermatocytes often were enlarged. These results
suggest that the entry into meiotic prophase is delayed in the
absence of Suv39h function. Despite this delay, further progression
through meiotic prophase until mid-pachytene appeared normal.
Between mid- to late pachytene, however, most spermatocytes undergo
apoptosis, resulting in stage V-VI tubules (see FIG. 13A) that
largely lack late pachytene spermatocytes and which do not contain
haploid spermatids. It was concluded that the absence of Suv39h
gene function induces delayed entry into meiotic prophase and
triggers pronounced apoptosis of spermatocytes during the mid- to
late pachytene stage.
[0188] b) H3-K9 Methylation at Meiotic Heterochromatin
[0189] To investigate whether the Suv39h-dependent spermatogenic
failure could be correlated with a distinct impairment of meiotic
heterochromatin, testis spread preparations and cryosections were
analyzed with the .alpha.-methH3-K9 antibodies. In wt preparations,
the .alpha.-methH3-K9 antibodies decorate heterochromatic foci in
spermatogonia (B-Sg) and in pre-leptotene spermatocytes (preL-Sc)
(FIG. 13B, left images, top panel). In early meiotic prophase
(Zyg-Sc) and early pachytene, the .alpha.-methH3-K9 staining was
not exclusive for heterochromatin but also extended into
euchromatin. From mid-pachytene through diplotene and in
diakinesis, the .alpha.-methH3-K9 staining was restricted to
heterochromatic clusters which condense into one block of
heterochromatin in elongating spermatids (FIG. 13B, top panels).
MethH3-K9 signals in elongated spermatids and mature spermatozoa,
in which histones are replaced by protamines, were not detect. The
authenticity of this staining pattern had been confirmed in
co-localisation analyses with antibodies that recognize the
synaptonemal complex (Offenberg et al., 1991; Lammers et al.,
1995), HP1.beta. (Motzkus et al., 1999) and phosH3 (Cobb et al.,
1999). Thus, in analogy to the somatic stainings shown above for
PMEFs, these results indicate that methylation of H3-K9 is also a
specific marker for meiotic heterochromatin in differentiating male
germ cells.
[0190] c) Impaired H3-K9 Methylation and Aneuploidies in Suv39h dn
Spermatogonia
[0191] In preparations from Suv39h dn testis spreads, H3-K9
methylation was absent in spermatogonia and pre-leptotene
spermatocytes (FIG. 13B, left images, bottom panel). Further, the
pronounced euchromatic staining that characterizes early
spermatocytes (Zyg-Sc) at the onset of meiotic prophase was not
observed. The impairment of H3-K9 methylation was accompanied by a
dispersed distribution of phosH3 in .apprxeq.60% of Suv39h dn
spermatogonia. By contrast, HP1.beta. was largely undetectable in
both wt and Suv39h dn spermatogonia.
[0192] Surprisingly, from mid-pachytene onwards, wild-type staining
for methH3-K9 at pericentric heterochromatin was observed (FIG.
13B, bottom panel). HP1.beta. localisation and phosH3 signals at
autosomes ocurred normally in Suv39h dn late spermatocytes. Thus,
these results demonstrate that the Suv39h HMTases selectively
regulate H3-K9 methylation in spermatogonia and at the very early
stages of meiotic prophase. Similar to the analysis with PMEFs (see
above), an .apprxeq.5-fold increased rate for complete chromosome
mis-segregation in Suv39h dn spermatogonia that results in the
occurence of tetraploid spermatocytes ws observed (see FIG. 14C,
below). In summary, these data define an early and stage-specific
meiotic role for the Suv39h HMTases, and further suggest the
existence of a novel H3-K9 HMTase(s) which can methylate
heterochromatin during meiotic prophase, diakinesis and in
spermatids.
[0193] FIG. 13 shows the spermatogenic failure and H3-K9
methylation in germ cells of Suv39h dn mice as follows: (A) Overall
size and histology of wild-type and Suv39h dn testes at .apprxeq.5
months of age. The Suv39h dn testis section reveals many
seminiferous tubules that lack spermatocytes (Sc) and spermatids
(St). In particular, although a few seminiferous tubules (1)
contain zygotene spermatocytes (Zyg-Sc), more advanced
differentiation stages (2) display apoptotic spermatocytes (arrows)
at pachytene. At even later differentiation stages (3), pachytene
spermatocytes are almost completely absent. Some tubules (4) harbor
only Sertoli cells (SeC). Abbreviations: Intermediate (In-Sg) and
B-type spermatogonia (B-Sg); pre-leptotene (PreL-Sc), zygotene
(Zyg-Sc), mid-pachytene (mPach-Sc), late-pachytene (lPach-Sc),
diplotene (Diplo-Sc) and diakinesis/M-I (M-I-Sc) spermatocytes;
round (rSt), elongating (elSt) and elongated (eSt) spermatids;
Sertoli cells (SeC).
[0194] (B) Double-labelling immunofluorescence of wt (top panel)
and Suv39h dn (bottom panel) germ cells with .alpha.-methH3-K9
(pink) and .alpha.-Scp3 (green) antibodies. DNA was counterstained
with DAPI (blue). In Suv39h dn germ cells, H3-K9 methylation is
absent in proliferating spermatogonia (B-Sg) and in pre-leptotene
spermatocytes (PreL-Sc), and is highly reduced in zygotene
spermatocytes (Zyg-Sc) where only residual signals are detected at
pericentric heterochromatin (arrowheads). At later stages, H3-K9
methylation appears in a wild-type staining (compare top and bottom
panels), although Suv39h dn sex chromosomes (arrows) remain more
intensely labeled at diplotene and diakinesis. The double arrow
indicates the pseudo-autosomal region (PAR).
EXAMPLE 13
[0195] a) Non-Homologous Interactions and Delayed Synapsis in
Suv39h dn Spermatocytes
[0196] The absence of pericentric H3-K9 methylation in
spermatogonia and early spermatocytes is suggestive for a role of
the Suv39h HMTases in defining a higher-order structure that may be
required for the initial alignments and clustering of meiotic
chromosomes. Therefore chromosome synapsis was analyzed by
immunofluorescence of pachytene spreads with antibodies that are
specific for the axial/lateral and central elements of the
synaptonemal complex (SC) (FIGS. 14A,B). Intriguingly, in
.apprxeq.15% (n=90) of Suv39h dn spermatocytes, non-homologous
interactions between autosomes were observed (FIG. 14J).
Non-homologous interactions were even more frequent (.apprxeq.35%)
between sex chromosomes and autosomes (X/Y-A). Interestingly, these
illegitimate associations occurred predominantly between the
acrocentric ends (cen-cen) of non-homologous chromosomes, to a
lesser extent between centromeres and telomeres (cen-tel) and only
very rarely between telomeres (tel-tel) (FIG. 14J). In addition,
Suv39h dn spermatocytes contained unsynapsed sex chromosomes (see
below) and autosomal bivalents that were delayed in synapsis.
Delayed synapsis of autosomes (A-del) almost invariably was
correlated with engagement in non-homologous associations (FIG.
14A), suggesting that both processes may be functionally
related.
[0197] The illegitimate associations were further confirmed by
transmission electron microscopy (FIGS. 14D-G). These
ultrastructural analyses revealed the presence of physical
connections and bridge-like structures between the ends of
non-homologous chromosomes (double arrow in FIGS. 14D,C,F). The
incidence of partner exchange (FIG. 14G) and non-homologous
alignments were also observed. None of these aberrant chromosomal
interactions were detected in EM preparations from wt
spermatocytes.
[0198] b) Bivalent Mis-Segregation at Meiosis I in Suv39h dn
Spermatocytes
[0199] To detemine whether the absence of methH3-K9 in early
prophase may affect chromosome dynamics and segregation during the
meiotic divisions, testis spread preparations were next analyzed
for diakinesis/metaphase I (M-I) and metaphase II (M-II) cells. At
diakinesis/M-I, most Suv39h dn spermatocytes revealed bivalents
with wt-like morphology, indicating that chromosome condensation
and chiasmata formation was unperturbed (but see FIGS. 15B-DD,
below). However, at M-II, .apprxeq.14% of secondary spermatocytes
were tetraploid, indicating segregation failure of all bivalents
during the first meiotic division (FIGS. 14I and 14K). Therefore,
the Suv39h-induced defects at pericentric heterochromatin persist
throughout the first meiotic division and do not appear to be
`rescued` by the additional H3-K9 methylation that occurs during
mid- to late meiotic prophase (see FIG. 13B).
[0200] FIG. 14 shows the illegitimate associations and delayed
synapsis of Suv39h dn meiotic chromosomes as follows: (A-C)
Double-labelling immunofluorescence of Suv39h dn pachytene
spermatocytes with antibodies that are specific for the
axial/lateral elements .alpha.-Scp3 (in green) and central elements
.alpha.-Scp1 (in red) of the synaptonemal complex (SC). This
co-labelling reveals unsynapsed chromosomes in a green-like
staining and synapsed chromosomes in an orange-red colour. DNA was
counterstained with DAPI (blue) which highlights pericentric
heterochromatin in a more intense blue contrast. (A) Two
mid-pachytene spermatocytes (mPach-Sc) showing multiple
illegitimate associations (arrowheads) between non-homologous
autosomes (A) and between autosomes and sex chromomes (X, Y).
Several autosomes are also delayed in synapsis (Adel). (B) Late
pachytene (lPach-Sc) spermatocyte containing two autosomes which
are engaged in non-homologous interaction through their pericentric
regions (arrowhead). In addition, the sex chromosomes failed to
pair. (C) Tetraploid spermatocyte resulting from complete
mis-segregation of all chromosomes in the preceding mitotic
division of a Suv39h dn spermatogonium.
[0201] (D-G) Transmission electron microscopy of Suv39h dn
pachytene chromosomes, confirming that non-homologous chromosome
associations mainly occur through pericentric heterochromatin which
is visulised by the more granular silver staining (arrowhead and
double arrows). The chromosomes displayed in panel G show multiple
engagements of partner exchange.
[0202] (H, I) Giemsa-stained metaphase II chromosomes of wt and
Suv39h dn secondary spermatocytes illustrating complete
mis-segregation in the preceeding meiosis I division of Suv39h dn
cells.
[0203] (J) Histogram for the frequency of non-homologous chromosome
associations and delayed synapsis in wt (n=80) and Suv39h dn (n=90)
pachytene spermatocytes. (K) Histogram for the frequency of meiosis
I mis-segregation of chromosome bivalents in wt (n=40) and Suv39h
dn (n=30) secondary spermatocytes.
EXAMPLE 14
Suv39h Deficiency Interferes with Sex Chromosome Segregation
[0204] Spermatogenesis in male mammals is specialised by the
presence of the heteromorphic sex chromosomes which form a unique
chromatin region known as the sex vesicle or XY body (Solari,
1974). Moreover, the Y chromosome is the most heterochromatic
chromosome in the mouse (Pardue and Gall, 1970). Homolog pairing
and cross-over between sex chromosomes is dependent upon the
presence of a small, pseudo-autosomal region called PAR (Burgoyne,
1982). The absence of Suv39h function interferes with the chromatin
organization and segregation of the sex chromosomes in several
ways.
[0205] First, although methH3-K9 signals at the XY body (arrows in
FIG. 13B) were detected at comparable levels in wt and mutant
pachytene spermatocytes, Suv39h dn sex chromosomes remain more
heavily methylated in diplotene and diakinesis (see FIG. 13B,
bottom panels). Correspondingly, prolonged HP1.beta. binding to the
XY body during diplotene was observed. Second, at diakinesis/M-I,
the proximal region of the long arm of the Y chromosome appears
hypo-condensed in 10% of Suv39h dn cells (FIGS. 15B, E). Moreover,
the mutant Y chromosomes display premature separation of their arms
or even complete separation of the two sister chromatids (FIGS.
15D, E). Third, H3-K9 methylation is present at the PAR (double
arrows in FIG. 13B) in both wt and Suv39h dn sex chromosomes, and
the PAR is also decorated with HP1.beta.. Despite these similar
staining patterns, the sex chromosomes failed to synapse in
.apprxeq.15% of Suv39h dn pachytene spermatocytes (FIGS. 14A, B).
At diakinesis/M-I (FIGS. 15B, C), the presence of XY univalents was
4-fold increased as compared to wt cells (FIG. 15F). Together,
these data indicate a role for the Suv39h HMTases in co-regulating
the specialised chromatin structure of the sex chromosomes, in
particular of the highly heterochromatic Y chromosome.
[0206] FIG. 15 shows the aberrant function of the Y chromosome
during meiosis of Suv39h dn spermatocytes as follows:
Giemsa-stained diakinesis/metaphase-I chromosomes of wt (A) and
Suv39h dn (B-D) primary spermatocytes illustrating univalency (B,
C), impaired condensation (B, C) and premature sisterchromatid
separation of the Y chromosome (C, D). (E) Histogram for the
frequency of diakinesis/M-I cells with abnormal condensation or
premature sisterchromatid separation of the Y chromosome (wt:
n=190; Suv39h dn: n=170). (F) Histogram for the frequency of XY
univalency at pachytene (wt: n=80; Suv39h dn: n=80) or
diakinesis/M-I (wt: n=190; Suv39h dn: n=170).
EXAMPLE 15
Screening for Moduators of Suv39h1 MTase Activity.
[0207] All steps are automated and the position of the different
test compounds are registered on computer for later reference.
Compounds being tested for modulating activity are aliquoted into
384 well plates in duplicate. 20-200 nmol of recombinant GST tagged
SUV39H1 in MAB buffer, is then added to the reaction. 20 nmol of
branched peptide ([TARKST].sub.4-K.sub.2-K-cys) which has been
labelled with europium is then added, followed by 100 nmol of
S-adenosyl methionine. This reaction is left at room temperature
for 40 mins, then transferred onto a second plate to which the
.alpha.-methH3-K9 antibody has been coated. This reaction is then
left at room temperature for 40 mins to allow the antibody to bind
methylated substrate. Following capture of methylated substrate,
unbound non-methylated substrate is washed off in 50 mM tris pH
8.5. The europium label is then cleaved from the peptide in 50
.mu.l pH 4.5 enhancement solution for 25 mins. The chelated
europium molecules are then excited at 360 nm and the level of
emitted fluorescence at 620 nm is then calculated using
time-resolved fluorescence in a PolarStar plate reader. The results
are then automatically graphed.
[0208] The level of fluorescence is directly related to the level
of MTase activity. The effect of the different compounds on the
MTase activity can be clearly seen on the graph when compared to
control reactions with no componds added or with no enzyme
added.
[0209] FIG. 17 illustrates the principle of the screening method as
follows:
[0210] a) A Suv39h1-like MTase is incubated with S-Adenosyl
Methionine (SAM) and a chromogenically labelled unmodified peptide
substrate (e.g. branched peptide [TARKST]4-K2-K-cys). Following
methylation of this substrate the substrate becomes an epitope for
a Lys9-methyl specific antibody which has been immobilised on a
microtiter plate. The level of bound peptide can then be quantified
by the level of fluorescence of from the chromogenic label.
[0211] b) In the presence of a modulator (e.g. an inhibitor, I) the
transfer of methyl groups by the MTase will be affected
(decreased), this in turn will affect the amount of substrate
captured by the immobilised antibody, which is quantified by the
level of fluorescence. A compound with inhibitory effects will
result in a decrease in fluorescent signal, whereas a compound with
enhancing effects will result in an increase in fluorescent
signal.
1TABLE I Viability of Suv39h double null mice. cross N1H2 .times.
H1H2.sup.a N1H2 .times. N1H2 N1H2 .times. H1N2 dn mice expected 1:8
1:4 1:4 total total # mice 81 89 27 197 born # dn mice 11 27 8 46
expected.sup.b # dn mice 4 8 3 15 observed % dn mice 36.4 29.6 37.5
32.6 viable .sup.ai.e.: N1H2 .times. H1H2: .male..male. Suv39h1-/-,
Suv39h2+/- .times. .female..female. Suv39hl+/-, Suv39h2+/-
.sup.bBased on number of mice born with other Suv39h1 and Suv39h2
allelic combinations which show no reduced prenatal viability.
[0212]
2TABLE II Incidence of B-cell lymphomas in mice with reduced Suv39h
gene dosage Suv39h # of mice total # % of mice Genotype gene dosage
with tumor of mice with tumor W1W2 3 0 57 0 W1H2, W1N2, 0-2 1 22
4.6 H1N2 H1W2, N1W2 2-3 8 26 30.8 H1H2, N1H2* 1-2 20 72 27.8 N1N2 0
2 6 33.3 *i.e.: N1H2: Suv39h1-/-, Suv39h2+/-
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immune defects, and thymic lymphoma. Genes Dev, 10, 2411-2422.
[0299] Yoshida, K., Kandoh, G., Matsuda, Y., Habu, T., Nishimune,
Y., and Morita, T. (1998). The mouse RecA-like gene Dmc1 is
required for homologous chromosome synapsis during meiosis. Mol
Cell, 1, 707-718.
[0300] Yuan, L., Liu, J. G., Zhao, J., Brundell, E., Daneholt, B.,
and Hoog, C. (2000). The murine Scp3 gene is required for
synaptonemal complex assembly, chromosome synapsis, and male
fertility. Mol. Cell, 5, 73-83.
[0301] Zheng, Q., Simel, E. J., Klein, P. E., Royer, M. T. and
Houtz, R. L. (1998) Expression, purification, and characterization
of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase
large subunit N-epsilon-methyltransferase. Protein Expr Purif, 14,
104-12.
Sequence CWU 1
1
19 1 1452 DNA Mus musculus 3'UTR (1)..(18) CDS (19)..(1452) 1
gaatgaaagc tccgcaag atg gcg acg gcc agg gcc aag gca cgg ggc agt 51
Met Ala Thr Ala Arg Ala Lys Ala Arg Gly Ser 1 5 10 gag gca gga gcg
cgg tgt cac cgg gct cca ggt ccg ccc ccg agg ccc 99 Glu Ala Gly Ala
Arg Cys His Arg Ala Pro Gly Pro Pro Pro Arg Pro 15 20 25 aag gcc
agg cga acg gcg aga cgc cgc cgc gcg gag acc ctg acg gcg 147 Lys Ala
Arg Arg Thr Ala Arg Arg Arg Arg Ala Glu Thr Leu Thr Ala 30 35 40
cga cgc tcg cgg ccg tct gcg ggc gag agg cgc gcc ggc tcc cag cga 195
Arg Arg Ser Arg Pro Ser Ala Gly Glu Arg Arg Ala Gly Ser Gln Arg 45
50 55 gcg tgg tcc gga gct ccg cgg gcc gcg gtc ttt ggc gac gag tgt
gca 243 Ala Trp Ser Gly Ala Pro Arg Ala Ala Val Phe Gly Asp Glu Cys
Ala 60 65 70 75 cga ggt gcc tta ttc aag gcc tgg tgt gtg cct tgc cta
gtt tca ctt 291 Arg Gly Ala Leu Phe Lys Ala Trp Cys Val Pro Cys Leu
Val Ser Leu 80 85 90 gat act ctc cag gaa tta tgt aga aaa gaa aag
ctc aca tgt aaa tcg 339 Asp Thr Leu Gln Glu Leu Cys Arg Lys Glu Lys
Leu Thr Cys Lys Ser 95 100 105 att gga atc acc aaa agg aat cta aac
aat tat gag gtg gag tac ttg 387 Ile Gly Ile Thr Lys Arg Asn Leu Asn
Asn Tyr Glu Val Glu Tyr Leu 110 115 120 tgt gac tac aag gta gca aag
ggt gtg gaa tat tat ctt gta aaa tgg 435 Cys Asp Tyr Lys Val Ala Lys
Gly Val Glu Tyr Tyr Leu Val Lys Trp 125 130 135 aaa gga tgg cca gat
tct aca aac acc tgg gag ccc ttg aga aac ctc 483 Lys Gly Trp Pro Asp
Ser Thr Asn Thr Trp Glu Pro Leu Arg Asn Leu 140 145 150 155 agg tgt
cca cag ctc ctg cgg cag ttc tct gat gac aag aag act tac 531 Arg Cys
Pro Gln Leu Leu Arg Gln Phe Ser Asp Asp Lys Lys Thr Tyr 160 165 170
tta gct cag gaa agg aaa tgc aag gct gtc aat tca aaa tcc ttg caa 579
Leu Ala Gln Glu Arg Lys Cys Lys Ala Val Asn Ser Lys Ser Leu Gln 175
180 185 cct gca att gct gag tat att gta cag aaa gct aag caa aga ata
gct 627 Pro Ala Ile Ala Glu Tyr Ile Val Gln Lys Ala Lys Gln Arg Ile
Ala 190 195 200 ctg cag aga tgg caa gat tac ctc aac aga aga aag aac
cat aag ggg 675 Leu Gln Arg Trp Gln Asp Tyr Leu Asn Arg Arg Lys Asn
His Lys Gly 205 210 215 atg ata ttt gtt gaa aac act gtt gac ttg gag
ggc cca cct tta gac 723 Met Ile Phe Val Glu Asn Thr Val Asp Leu Glu
Gly Pro Pro Leu Asp 220 225 230 235 ttc tac tac att aac gag tac agg
cca gct ccc ggg atc agc ata aac 771 Phe Tyr Tyr Ile Asn Glu Tyr Arg
Pro Ala Pro Gly Ile Ser Ile Asn 240 245 250 agt gaa gcc acc ttt gga
tgt tca tgt aca gac tgc ttc ttt gac aag 819 Ser Glu Ala Thr Phe Gly
Cys Ser Cys Thr Asp Cys Phe Phe Asp Lys 255 260 265 tgt tgt cct gct
gaa gct gga gtt gtg ttg gct tat aat aag aag caa 867 Cys Cys Pro Ala
Glu Ala Gly Val Val Leu Ala Tyr Asn Lys Lys Gln 270 275 280 caa att
aaa atc caa cca ggc act ccc atc tac gaa tgc aac tca agg 915 Gln Ile
Lys Ile Gln Pro Gly Thr Pro Ile Tyr Glu Cys Asn Ser Arg 285 290 295
tgt cga tgt gga cct gaa tgt ccc aat agg att gta caa aaa ggc aca 963
Cys Arg Cys Gly Pro Glu Cys Pro Asn Arg Ile Val Gln Lys Gly Thr 300
305 310 315 caa tat tca ctg tgc atc ttt aaa act agc aat ggc tgt ggt
tgg ggt 1011 Gln Tyr Ser Leu Cys Ile Phe Lys Thr Ser Asn Gly Cys
Gly Trp Gly 320 325 330 gta aaa acc ctt gtg aag att aaa aga atg agt
ttt gtc atg gaa tat 1059 Val Lys Thr Leu Val Lys Ile Lys Arg Met
Ser Phe Val Met Glu Tyr 335 340 345 gtt gga gag gtg atc aca agt gaa
gag gcc gag aga cgg gga cag ttc 1107 Val Gly Glu Val Ile Thr Ser
Glu Glu Ala Glu Arg Arg Gly Gln Phe 350 355 360 tat gac aac aaa ggg
atc acc tac ctc ttt gac ctg gac tac gag tct 1155 Tyr Asp Asn Lys
Gly Ile Thr Tyr Leu Phe Asp Leu Asp Tyr Glu Ser 365 370 375 gat gag
ttc aca gtg gat gca gct cga tat gga aac gta tcc cat ttt 1203 Asp
Glu Phe Thr Val Asp Ala Ala Arg Tyr Gly Asn Val Ser His Phe 380 385
390 395 gtg aat cat agt tgt gac cca aat ctt cag gtg ttt agt gtt ttc
atc 1251 Val Asn His Ser Cys Asp Pro Asn Leu Gln Val Phe Ser Val
Phe Ile 400 405 410 gat aac ctt gat act cgg ctg ccc agg ata gca ttg
ttc tct aca aga 1299 Asp Asn Leu Asp Thr Arg Leu Pro Arg Ile Ala
Leu Phe Ser Thr Arg 415 420 425 acc ata aac gct gga gaa gag ctg act
ttt gac tat caa atg aaa ggt 1347 Thr Ile Asn Ala Gly Glu Glu Leu
Thr Phe Asp Tyr Gln Met Lys Gly 430 435 440 tct gga gaa gca tct tca
gac tcc att gac cac agc cct gcc aaa aaa 1395 Ser Gly Glu Ala Ser
Ser Asp Ser Ile Asp His Ser Pro Ala Lys Lys 445 450 455 agg gtc aga
acc caa tgt aaa tgt gga gcc gag act tgc aga ggt tac 1443 Arg Val
Arg Thr Gln Cys Lys Cys Gly Ala Glu Thr Cys Arg Gly Tyr 460 465 470
475 ctc aac tga 1452 Leu Asn 2 477 PRT Mus musculus 2 Met Ala Thr
Ala Arg Ala Lys Ala Arg Gly Ser Glu Ala Gly Ala Arg 1 5 10 15 Cys
His Arg Ala Pro Gly Pro Pro Pro Arg Pro Lys Ala Arg Arg Thr 20 25
30 Ala Arg Arg Arg Arg Ala Glu Thr Leu Thr Ala Arg Arg Ser Arg Pro
35 40 45 Ser Ala Gly Glu Arg Arg Ala Gly Ser Gln Arg Ala Trp Ser
Gly Ala 50 55 60 Pro Arg Ala Ala Val Phe Gly Asp Glu Cys Ala Arg
Gly Ala Leu Phe 65 70 75 80 Lys Ala Trp Cys Val Pro Cys Leu Val Ser
Leu Asp Thr Leu Gln Glu 85 90 95 Leu Cys Arg Lys Glu Lys Leu Thr
Cys Lys Ser Ile Gly Ile Thr Lys 100 105 110 Arg Asn Leu Asn Asn Tyr
Glu Val Glu Tyr Leu Cys Asp Tyr Lys Val 115 120 125 Ala Lys Gly Val
Glu Tyr Tyr Leu Val Lys Trp Lys Gly Trp Pro Asp 130 135 140 Ser Thr
Asn Thr Trp Glu Pro Leu Arg Asn Leu Arg Cys Pro Gln Leu 145 150 155
160 Leu Arg Gln Phe Ser Asp Asp Lys Lys Thr Tyr Leu Ala Gln Glu Arg
165 170 175 Lys Cys Lys Ala Val Asn Ser Lys Ser Leu Gln Pro Ala Ile
Ala Glu 180 185 190 Tyr Ile Val Gln Lys Ala Lys Gln Arg Ile Ala Leu
Gln Arg Trp Gln 195 200 205 Asp Tyr Leu Asn Arg Arg Lys Asn His Lys
Gly Met Ile Phe Val Glu 210 215 220 Asn Thr Val Asp Leu Glu Gly Pro
Pro Leu Asp Phe Tyr Tyr Ile Asn 225 230 235 240 Glu Tyr Arg Pro Ala
Pro Gly Ile Ser Ile Asn Ser Glu Ala Thr Phe 245 250 255 Gly Cys Ser
Cys Thr Asp Cys Phe Phe Asp Lys Cys Cys Pro Ala Glu 260 265 270 Ala
Gly Val Val Leu Ala Tyr Asn Lys Lys Gln Gln Ile Lys Ile Gln 275 280
285 Pro Gly Thr Pro Ile Tyr Glu Cys Asn Ser Arg Cys Arg Cys Gly Pro
290 295 300 Glu Cys Pro Asn Arg Ile Val Gln Lys Gly Thr Gln Tyr Ser
Leu Cys 305 310 315 320 Ile Phe Lys Thr Ser Asn Gly Cys Gly Trp Gly
Val Lys Thr Leu Val 325 330 335 Lys Ile Lys Arg Met Ser Phe Val Met
Glu Tyr Val Gly Glu Val Ile 340 345 350 Thr Ser Glu Glu Ala Glu Arg
Arg Gly Gln Phe Tyr Asp Asn Lys Gly 355 360 365 Ile Thr Tyr Leu Phe
Asp Leu Asp Tyr Glu Ser Asp Glu Phe Thr Val 370 375 380 Asp Ala Ala
Arg Tyr Gly Asn Val Ser His Phe Val Asn His Ser Cys 385 390 395 400
Asp Pro Asn Leu Gln Val Phe Ser Val Phe Ile Asp Asn Leu Asp Thr 405
410 415 Arg Leu Pro Arg Ile Ala Leu Phe Ser Thr Arg Thr Ile Asn Ala
Gly 420 425 430 Glu Glu Leu Thr Phe Asp Tyr Gln Met Lys Gly Ser Gly
Glu Ala Ser 435 440 445 Ser Asp Ser Ile Asp His Ser Pro Ala Lys Lys
Arg Val Arg Thr Gln 450 455 460 Cys Lys Cys Gly Ala Glu Thr Cys Arg
Gly Tyr Leu Asn 465 470 475 3 543 DNA Homo sapiens misc_feature
(1)..(543) EST Acc. No.173625 3 ggccatgtgg ttgancccct ggntttaccn
nnccntggnn ggnnttgann ccccttagat 60 tatagtccag aatcattgtt
gtcatataac tgccctcatc tttcagcttc gtcacttgtg 120 attacctttc
caacttattc catgacaaaa cttattcttt taatcttcac atgggttttt 180
acaccccagc catggtcatt gatactgtga aagatgcaaa gtgaattact gtgtgccttt
240 ttgtacaatc ctattggtac agtgaggtcc acattgacag attgagatgc
atttatagat 300 gggagtaaca ggtgggattt taatttgttg gtttttacta
taagccaaaa gaattccagc 360 ttcaccaaga caacattttt catagaagca
atctgtgcat gaacaacaaa aggtagcttc 420 atttactaag ctgattccag
gagctggttt gtattcatca atatagcaga agtctgaagg 480 tgggccttct
aagtgaaccc tattntcaac aaatatcact cctttattat tctgtcttct 540 gcg 543
4 579 DNA Homo sapiens misc_feature (1)..(579) EST Acc. No.
AQ494637 4 gcttctcata catgatacgt gttcngctct gnngtntnng tttangaata
cntaaaanaa 60 aaggnagggg ngncntttga ttcgtgtgat tccatagatg
cactcatatg gaactgtatt 120 tcattntgtg aatcatagta gtgacccaaa
tcttcatatg ttctatgntn tcactgataa 180 cttgacactg gccttcccta
tatagctctg tgttccatga gaactataaa tgctggagaa 240 gagttgattt
ttgacaatca aacaaaaagt tctggggata tatcttcaga gtttattgac 300
cacagctcag ccaaaaagag ggtcagaact gtatgtaaat gtggagctgt gacttgcaga
360 ggttgcctca aatgaatttt caggaaatag aaatgatgat aattggtagt
tgtttctttt 420 ttctaatgtt atcattctaa aaataagtat ttggaactct
cttttcatat tatcaagatt 480 attactatgt taaattgaca tncatggttc
aaggcattta ccanatgcat tactgatgcc 540 tcttgagaga gggccactgt
gttgcataga ctgatctga 579 5 565 DNA Homo sapiens misc_feature
(1)..(565) EST Acc. No. AQ691972 5 agaggatgag catggatcnt cgctatagca
aaccacanat anaatcccac ctgttactcc 60 catctataaa tgcatctcaa
tctgtcaatg tggaccttac tgtaccaata ggattgtaca 120 aaaaggcaca
cagtaattca ctttgcatct ttcacagtat caatgaccat ggctggggtg 180
taaaaaccca tgtgaagatt aaaagaataa gttttgtcat ggaataagtt ggaaaggtaa
240 tcacaagtga cgaagctgaa agatgagggc agttatatga caacaaatga
tctggactat 300 gaatctgatg aattcacaga ggatgcagct caatatggaa
ctgtatttca ttntgtgaat 360 cataagtagt gacccaaact tcatatgttc
aatgttntca ttgataactt gacactggcc 420 tttccttaat agctctgtgt
tccatgagaa ctataaatgc tggagaagaa gtgatttttg 480 acatcaacaa
aagttctggg attatcttca aagttattgc cacagttacc aaaagaaggc 540
aaactgttgt aatgtgagct gtact 565 6 535 DNA Homo sapiens misc_feature
(1)..(535) EST Acc. No. AQ554070 6 tcagactcat agtccagatc aaagagattc
tgtgattccc ttgttgtcat agaactgtcc 60 tcgtctttca gcttcttcac
ttgtgattac ctaaacagaa aaaactgtaa gtatattacg 120 tagctactga
accaaagaag cattcatcta cctatctact aatatgcgaa tacctacaaa 180
tatttaaaaa gtaagaaatt caggtgtcat caaagcaaac attcacacaa actaagactc
240 agatgcaaag aggtgggaaa atgaggggaa gaaaaatgat aatgcaaaag
actgatgacc 300 tttttttttt aaacagggtc tcactctgtc actcaggcta
gaatgcggtg gtgccatcat 360 gactccctgt atcctttaac tcctgggatc
aagcgatctt cctgcctcag cctcctgact 420 agctggatca caggtgcata
ccgccatgcc cagctaatga tttagttttt atagagatgt 480 ggggtctcac
tatgttgccc acactggtct ggaactcctg ggctcaagtg agcct 535 7 20 PRT Homo
sapiens 7 Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys
Ala Pro 1 5 10 15 Arg Lys Gln Leu 20 8 20 PRT Homo sapiens 8 Met
Gly Pro Arg Arg Arg Ser Arg Lys Pro Glu Ala Pro Arg Arg Arg Ser Pro
1 5 10 15 Ser Pro 20 9 22 PRT Rattus rattus 9 Met Glu Thr Ser Ser
Arg Gly Gly Lys Lys Lys Ser Thr Lys Thr Ser Arg Ser 1 5 10 15 Ala
Lys Ala Gly 20 22 10 25 DNA Artificial Sequence Description of
Artificial Sequence Primer 10 atgggggcag ggttttcggg tagac 25 11 26
DNA Artificial Sequence Description of Artificial Sequence Primer
11 aaatggtatt tgcaggccac ttcttg 26 12 26 DNA Artificial Sequence
Description of Artificial Sequence Primer 12 aaatggtatt tgcaggccac
ttcttg 26 13 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 13 ggatgggatg gtggaatggt ttttat 26 14 26 DNA
Artificial Sequence Description of Artificial Sequence Primer 14
aaatggtatt tgcaggccac ttcttg 26 15 26 DNA Artificial Sequence
Description of Artificial Sequence Primer 15 aaatggtatt tgcaggccac
ttcttg 26 16 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 16 gactgcctag tctggcactg aact 24 17 25 DNA
Artificial Sequence Description of Artificial Sequence Primer 17
gatcactgcg tacatataca ctgat 25 18 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 tagacttcta ctacattaac
g 21 19 20 DNA Artificial Sequence Description of Artificial
Sequence Primer 19 gatgtcagtg gctatgaatg 20
* * * * *