From the Department of Cell Biology, Institute for Virus Research,
Kyoto University, Shogoin Kawara-cho, Kyoto 606-8507, the
Laboratory of Applied Molecular Biology, Department of
Applied Biochemistry, Osaka Prefecture University, 1-1 Gakuen-cho,
Sakai, Osaka 599-8501, and the § Department of
Microbiology and Immunology, Nippon Medical School, Sendagi,
Bunkyo-ku, Tokyo 113-8602, Japan
Received for publication, March 2, 2001, and in revised form, April 12, 2001
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ABSTRACT |
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The covalent modification of histone tails has
regulatory roles in various nuclear processes, such as control of
transcription and mitotic chromosome condensation. Among the different
groups of enzymes known to catalyze the covalent modification, the most recent additions are the histone methyltransferases (HMTases), whose
functions are now being characterized. Here we show that a SET
domain-containing protein, G9a, is a novel mammalian lysine-preferring HMTase. Like Suv39 h1, the first identified lysine-preferring mammalian
HMTase, G9a transfers methyl groups to the lysine residues of histone
H3, but with a 10-20-fold higher activity. It was reported that
lysines 4, 9, and 27 in H3 are methylated in mammalian cells. G9a was
able to add methyl groups to lysine 27 as well as 9 in H3, compared
with Suv39 h1, which was only able to methylate lysine 9. Our data
clearly demonstrated that G9a has an enzymatic nature distinct from
Suv39 h1 and its homologue h2. Finally, fluorescent protein-labeled G9a was shown to be localized in the nucleus but not in
the repressive chromatin domains of centromeric loci, in which Suv39 h1
family proteins were localized. This finding indicates that G9a
may contribute to the organization of the higher order chromatin
structure of non-centromeric loci.
In eukaryotes, organization of higher order chromatin structure is
thought to be essential for both epigenetic gene control and proper
chromosome condensation in mitosis. Targeted covalent modification of
the amino-terminal tails of the core histones in nucleosomes has
emerged as one of the important mechanisms in this process.
Posttranslational acetylation and phosphorylation of histone tails are
intensively studied among these modifications. Transcriptionally active
euchromatic regions of the chromosome are often associated with
hyperacetylated histones, and silent heterochromatic regions are often
associated with hypoacetylated forms (1). Steady-state levels of
histone acetylation in vivo are maintained by the opposing
activities of histone acetyl transferase and histone deacetylase
(2, 3). Besides acetylation, the histone H3 amino terminus is
also phosphorylated in association with different cellular
processes. Phosphorylation of serine 10 in histone H3 correlated
closely with chromosomal condensation and was shown to be required for
proper chromosome condensation and segregation (4, 5). The
phosphorylation of serine 10 also correlated with transcriptional
activation of immediate-early genes in a rapid and transient manner
upon mitogen stimulation (6, 7). Recently, several kinases have been
identified as the enzymes that contribute to the mitogen-stimulated
histone H3 phosphorylation (8, 9).
In addition to acetylation and phosphorylation, it has also been
reported that core histones, especially H3 and H4, are methylated (10,
11). In mammalian HeLa cells, lysines 4, 9, and 27 in H3 and lysine 20 in H4 were shown to be methylated (12). Although the biological
significance of histone methylation remains unclear, several histone
methyltransferases (HMTases)1
have recently been identified (12-14). Among these enzymes, Suv39 h1
and its homologue, h2, are the first known mammalian lysine-preferring HMTases and SET domain-containing proteins. Both enzymes preferentially methylate H3 in a mixture of histones in vitro, and their
catalytic activities are dependent on the SET domain. By using a
peptide encoding the first 20 residues of the H3 amino terminus, it was shown that the Suv39 h protein could transfer methyl groups to lysine
9. The enzymes that catalyze methylation of the other residues, lysines
4 and 27 in H3 and lysine 20 in H4, remain to be identified.
The SET domains in Suv39 h1 and h2 are evolutionarily conserved from
yeast to mammals and are found in many nuclear proteins called
"chromatin modulators" (13). Therefore, SET domain-containing proteins have diverse functions. For example, some members of the
Drosophila polycomb and trithorax group (Pc-G and
trx-G) protein families, which have been shown to act as repressors and
activators, respectively, of the homeotic selector genes, contain the
SET domain in their carboxyl termini (14). Among several SET
domain-containing protein families, Suv39 h family proteins are
characterized as a group possessing a chromo-domain, which is also
considered as a chromatin regulator motif. Members of this family were
identified as CLR4 in yeast (15), Su(VAR)3-9 in fruit fly (16), Suv39 h1 and h2 in mouse, and SUV39H1 in human (17, 18). The Suv39 h family
proteins have been considered to contribute to the organization of
repressive chromatin regions such as the centromere (13).
In this study, a SET domain-containing protein, G9a, was identified as
a novel mammalian lysine-preferring HMTase. Compared with Suv39 h1, G9a
possesses an apparently unique molecular nature. It is much more
efficient at transferring methyl groups to histone H3 in
vitro. In addition, not only lysine 9, but also lysine 27 in H3,
is a target for methylation by G9a. Finally, the nuclear localization of ectopically expressed G9a differs significantly from
that of Suv39 h1, suggesting that G9a may not be involved in the
organization of repressive chromatin domains but may contribute to the
regulation of chromatin structure in other loci.
Plasmids--
Full-length human G9a (hG9a) cDNA (19) was
obtained by ligation of a 1.2-kb BamHI/ApaI
cDNA fragment encoding amino acids 1-399 to a
BamHI/ApaI-digested partial hG9a cDNA
(encoding amino acids 350 to termination), which was previously cloned
into pBluescript (Stratagene). The former was PCR-amplified from a
Jurkat leukemia cDNA library using specific primers
5'-GCATGAGTGATGATGTCCACTCAC and 5'-TCACGGACAGGTACAACTGCC. For protein
expression in mammalian cells, the resulting 3.0-kb
HindIII/NotI-digested cDNA fragment containing the entire hG9a open reading frame was inserted into the
respective site of the pEGFP-C1 vector (CLONTECH)
to generate an Aequorea Victoria green fluorescent protein
(EGFP) (20) fusion protein. Full-length mouse Suv39 h1 cDNA was
PCR-amplified from a mouse B cell leukemia cDNA library using
specific primers 5'-ATAGAATTCGATGGCGGAAAATTTAAAAG and
5'-ATAGAATTCCTAGAAGAGGTATTTTCGGCA. The obtained cDNA fragment was
inserted into the EcoRI sites of pEGFP-C1 and pDsRed1-C1
vectors (CLONTECH) in an in-frame fashion.
Expression plasmids for EGFP and Discosoma striata red
(DsRed) (21)-human heterochromatic protein 1
For protein expression in Escherichia coli, mouse G9a (mG9a)
cDNA was isolated from a uni-ZAP murine testis cDNA library
(Stratagene) using the 1.2-kb hG9a cDNA described above as a probe.
A 1.4-kb EcoRI/XhoI cDNA fragment encoding
amino acids 621-1000 of mG9a was subcloned into the bacterial
expression vector pGEX-4T-3 (Amersham Pharmacia Biotech) to
obtain an amino-terminal glutathione S-transferase (GST)-mG9a fusion molecule. A 1.0-kb cDNA encoding amino acids 82-412 of Suv39 h1 was PCR-amplified and inserted into the
EcoRI site of pGEX4T-3. Mouse histone H3 cDNA (H3.1,
GenBankTM accession number 193861) was PCR-amplified from a
mouse embryonic stem cell line, TT2 cDNA library using specific
primers 5'-TAAGAATTCGGCTCGTACTAAGCAGACCGC and
5'-TATACTCGAGTTAAGCCCTCTCCCCGCGGAA. The obtained PCR product was
digested with EcoRI/XhoI and subcloned into the
respective sites of pZErO 2-1 (Invitrogen). A 180-base pair
EcoRI/SalI fragment of the histone H3 cDNA
encoding amino acids 1-57 was introduced into pGEX-4T-3. All
PCR-amplified cDNA products were confirmed by sequencing. Point
mutations within the SET domain of mG9a and Suv39 h1 and the amino
terminus of histone H3 were engineered by the standard double
PCR mutagenesis method. The amino terminus (residues 1-52) of human
centromere protein-A (CENP-A) and a full-length HP1HS Bacterial Protein Expression--
Recombinant proteins were
produced in E. coli BL21 codon-plus RIL strain
(Stratagene) and purified with glutathione-Sepharose beads (Amersham
Pharmacia Biotech) by the recently reported method (17). Concentrations
of recombinant proteins used were determined by Coomassie Brilliant
Blue R-250 staining in SDS-polyacrylamide gel electrophoresis gels.
In Vitro HMTase Assays--
All native histones (a mixture of
H1, 2A, 2B, 3, and 4; and single H1, H2A, and H3) were purchased from
Roche Molecular Biochemicals. Methyltransferase assays were performed
as reported (17). Briefly, 50 µl of reaction mixture containing
substrates, enzymes, and 250 nCi of
S-adenosyl-[methyl-14C]-L-methionine
in methylase activity buffer (50 mM Tris, pH 8.5, 20 mM KCl, 10 mM MgCl2, 10 mM Cell Culture--
293 human embryonic kidney cells were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 10% fetal calf serum at 37 °C in a 5% CO2
atmosphere. To generate 293 cells stably expressing both EGFP- and
DsRed-fusion molecules, individual protein expression vectors were
sequentially introduced into 293 cells in concert with different
drug-resistant gene-expressing vectors such as the PGK
promoter-driven puromycin- or hygromysin-resistant gene expression
vectors. Transfections were performed by using a lipofection reagent,
LipofectAMINE (Life Technologies, Inc.).
Immunoprecipitation of EGFP-tagged Proteins--
293 cells were
cultured in 6-cm diameter dishes under the condition described above.
Cells about 50% confluent conditions were transfected with
EGFP-hG9a or its SET domain deletion mutant, EGFP- Fluorescent Microscope Analysis--
Aliquots of cells were
grown on glass coverslips in 35-mm dishes for 48 h, fixed with 4%
paraformaldehyde/phosphate-buffered saline for 20 min, and treated with
0.1% Triton X-100 for 5 min. After staining with
4',6-diamidino-2-phenylindole (1 µg/ml) for 5 min, cells were
observed under a fluorescent microscope (Eclipse E600, Nikon) equipped
with a PlanApo 60 x (NA 1.40) and a MicroMAX 1300Y cooled
CCD camera (Princeton Instruments). Images were acquired by MetaMorph
software (Universal Imaging Corp.) by collecting a Z-series of 0.5-µm
optical sections.
G9a Contains a SET Domain and Adjacent Cysteine-rich Regions,
Essential for HMTase Activity--
Among SET domain-containing
proteins, HMTase activity has so far been observed only in Suv39 h
family proteins including yeast CLR4 and mammalian Suv39 h1 and h2.
Because only Suv39 h family proteins possess two cysteine-rich regions
adjacent to the SET domain, Rea et al. (17) suggested that
to exert HMTase activity, the SET domain requires combination with
these adjacent cysteine-rich regions. Taking this viewpoint, we focused
on G9a, a ubiquitously expressed SET domain-containing protein that was
originally characterized as a molecule encoded by a gene mapped in the
class III region of the human major histocompatibility complex locus
(19). G9a resembles CLR4 and Suv39 h, because it contains a SET domain
flanked by two cysteine-rich regions in its carboxyl terminus, but
other regions are quite different (illustrated in Fig.
1A).
Amino acid sequence alignment of the two cysteine-rich regions flanking
the SET domain from hG9a and mouse Suv39 h1 revealed that they are
highly related, and the positions of their cysteine residues are highly
conserved (Fig. 1B, top panel). Within the SET
domain, the H(R) G9a Possesses Higher HMTase Activity--
To investigate whether
mG9a carboxyl terminus has HMTase activity, we performed an in
vitro methylation assay using native histones as substrates.
Recombinant GST-mG9a (residues 621-1000) and GST-Suv39 h1 (residues
82-412) were incubated with native histones (a mixture of H1, 2A, 2B,
3, and 4) and
S-adenosyl-[methyl-14C]-L-methionine
as a methyl donor. Reaction products were separated by
SDS-polyacrylamide gel electrophoresis, and
methyl-14C-transferred proteins were visualized
by an imaging analyzer. We observed that GST-mG9a, as well as GST-Suv39
h1, showed HMTase activity and preferentially methylated H3.
Furthermore, GST-mG9a exhibited higher HMTase activity, and 1 µg of
GST-mG9a was capable of transferring methyl groups to histone H3 more
efficiently than was 10 µg of GST-Suv39 h1 (Fig.
2). Serial dilution analysis revealed that GST-mG9a possessed about 20-fold higher HMTase activity than Suv39
h1 (data not shown). However, addition of excess amounts of the
GST-mG9a amino terminus molecule (residues 138-698) did not result in
any detectable HMTase activities, indicating that G9a catalyzes histone
H3 methylation in a carboxyl-terminal SET domain-dependent
manner. To confirm that the HMTase activity is an intrinsic property of
G9a, we repeated the in vitro HMTase assay using hG9a that
was ectopically expressed in mammalian cells. EGFP fused with
full-length hG9a (residues 1-1001) was expressed in the human
embryonic kidney cell line 293, harvested by using anti-EGFP specific
antibodies, and introduced into the HMTase assay. As shown in Fig. 4,
D and E, EGFP-hG9a fusion molecules also exerted
HMTase activity and preferentially methylated H3 in the mixture of
native histones.
We also performed the HMTase assay for G9a by using single native
histone as a substrate. It was shown that not only H3 but also H1 can
be efficiently methylated by Suv39 h1 if H1 was used alone. When
purified native H1, H2A, and H3 were applied to the assay as single
substrates, G9a also significantly methylated not only native H3 but
also H1. Slightly methylated proteins were detected in the lane of H2A
(Fig. 3, lane 2), possibly due
to incomplete elimination of H1 and H3 during the purification
processes. The physiological significance of the H1 methylation is
currently unknown but poses an interesting issue for future
studies.
We further investigated the substrate specificity of G9a as a
methyltransferase using several other nuclear proteins, which were
shown to be covalently modified by other enzymes such as kinases or are
possible substrate candidates of these enzymes. We introduced several
recombinant GST-fused nuclear proteins, which were human CENP-A amino
terminus (residues 1-52) (23) and full-length constructs of
human CENP-B (24), human CENP-C (25), human HP1HS G9a Transferred Methyl Groups to Lysines 9 and 27 of Histone
H3--
Because methylation of H3 occurs dominantly at lysines 4, 9, and 27 in HeLa cells (12), and Suv39 h1 and h2 proteins were shown to
methylate only lysine 9 of H3 (18), the enzymes involved in the
reactions at lysines 4 and 27 in H3 remained to be identified. We next
examined the residues of H3 targeted by G9a, focusing on these lysines.
Because recombinant H3N was found to be a more suitable substrate than
the native histone H3, we introduced several lysine-to-arginine
replacements into H3N, as summarized in Fig. 4A. When we
used NT as a substrate in which all three lysine residues were
replaced with arginine, no methyl groups were introduced by GST-mG9a
(Figs. 4B and 6A), suggesting that the G9a target site(s) in H3 is also lysine. When single residue-replaced mutants 4R,
9R, and 27R were used for the assay, Suv39 h1 only methylated mutants in which lysine 4 or 27 was replaced with arginine (4R or 27R)
but not 9R (lysine 9 to arginine replacement), consistent with previous
results (4). However, all the single lysine-replaced mutants including
9R were clearly methylated by GST-mG9a, although the methylation
efficiency of 9R was only 20% of N3H (Fig. 6A). This result
suggested that G9a could methylate two or more lysine sites. Further
methylation assays using double lysine to arginine-replaced H3N mutants
demonstrated this clearly. Not only N9 (lysines 4 and 27 were changed
to arginine), but also N27 (lysines 4 and 9 were changed to arginine)
was substantially methylated by GST-mG9a (Figs. 4B and
5A). Methylation efficiencies of N9 and N27 by GST-mG9a were
about 50 and 20% of H3N, respectively. In contrast, N4 (lysines 9 and
27 were replaced with arginine) was no longer targeted by GST-mG9a. GST-Suv39 h1 again showed its target specificity for lysine 9 and only methylated N9 among the double lysine-replaced mutants (Figs.
4C and 6B). Therefore, we concluded that G9a is a
lysine-preferring H3 HMTase and speculated that G9a may be the responsible methyltransferase of lysines 9 and 27 in histone H3 in vivo.
To address whether these enzymatic properties of G9a are intrinsic,
transiently expressed EGFP-fused hG9a in 293 cells was again introduced
into the above in vitro HMTase assays. As shown in Fig.
4E, EGFP-hG9a expressed in mammalian cells clearly
methylated both N9 and N27. Furthermore, relative methylation
efficiencies among the substrates used were comparable between
EGFP-hG9a produced in mammalian cells and GST-hG9a produced in E. coli. (values shown at the bottom of Figs. 4E and
6A).
Lysine 27 Could Not Be Methylated by Hyperactive Suv39 h1
Mutant--
Because the first position of the catalytic core motif in
the mG9a SET domain (899R Ectopically Introduced EGFP-G9a Localizes in Nuclear Loci Different
from Suv39 h1--
The above experiments clearly demonstrated that G9a
possesses a distinct enzymatic nature in vitro compared with
the first identified lysine-preferring HMTase, Suv39 h1. To estimate
the in vivo function of G9a, we examined the nuclear
localization of ectopically introduced G9a in mammalian cells.
Expression constructs of EGFP-hG9a and DsRed fused with
HP1HS In this report, we demonstrated that G9a is a lysine-preferring H3
HMTase that shows stronger HMTase activity than Suv39 h1. Mutagenesis
experiments demonstrated that the hyperenzymatic activity of G9a is
also dependent on the first residue of the core motif in the SET
domain, arginine 899 in mouse, as shown by the change of histidine 320 to arginine at this position in Suv39 h1 (17).
More importantly, G9a was found to be able to transfer methyl groups to
not only lysine 9 but also lysine 27 in H3 in vitro, suggesting that G9a may be the responsible enzyme for the lysine 27 methylation described in HeLa cells. Furthermore, studies with the
mutant SuvH320R suggested that the target lysine specificity of G9a is
probably independent of its hyperenzymatic activity. From the in
vitro HMTase assays using various H3N mutants, the target lysine
residue preference of G9a was examined. GST-mG9a transferred methyl
groups to lysine 27 even in the H3N mutant N27 (lysines 4 and 9 were
replaced with arginine), the most preferred target site of G9a was
lysine 9, and N9 (lysines 4 and 27 were replaced) gave the highest
methyl-14C counts among the double
lysine-replaced mutants (Fig. 6A). It is also worth noting
that the total incorporation rate of methyl-14C
from the double lysine-replaced H3N substrates never reached the levels
of the wild-type H3N for G9a and also Suv39 h1 (Fig. 6, A
and B), which suggests that methylation of lysine residues, at least at positions 9 and 27, may synergistically affect
each other.
In mammals, three lysine-preferring HMTases including G9a and some
arginine methyltransferases that can methylate histones were
identified. However, we still have very limited information about the
physiological significance of histone methylation. A nuclear receptor
coactivator-interacting protein, CARM1, which contains an
arginine-specific methyltransferase motif and shows HMTase activity
in vitro, provided suggestive evidence that histone methylation may contribute to transcriptional activation, although the
significance of arginine methylation in H3 has not been defined (32).
In addition, lysine-selective HMTase activities were detected in
transcriptionally active Tetrahymena macronuclei but not in transcriptionally inert micronuclei (12). On the other hand, a possible
correlation between methylation of lysine 9 in H3 and transcriptional
silencing has been also proposed. If all of these findings are
physiologically relevant, histone methylation may both positively and
negatively regulate transcription. How might histone methylation
regulate transcription? One possible mechanism was proposed by the
studies of Suv39 h1 function, in which methylation of lysine 9 inhibited phosphorylation of serine 10 in the H3 amino terminus
peptides and vice versa (17). The phosphorylated serine 10 was shown to
accelerate histone acetyl transferase acetylation of lysine 14 in H3
(33). Therefore, it is proposed that HMTases may function through
regulation of other histone-modifying activities, such as kinase and
histone acetyl transferase, to positively or negatively regulate
transcription (33, 34). Interestingly, lysine 27 of H3 is embedded
within a similar sequence context as lysine 9 (RKS),
and their adjoining serines 10 and 28 were both capable of being
phosphorylated in mitotic chromosomal condensation (35). In this
context, G9a may also suppress phosphorylation of serine 28 by
methylation of lysine 27 and control serine 28 phosphorylation-mediated
function. An essential function of HMTase may be simply to mark
specific histone residues with methyl groups, as previously proposed
(2). The specific methyl marking may recruit various regulatory
molecule(s) involved in transcription or higher order chromatin
organization.2 If so, H3
methylation by G9a and Suv39 h1 may result in recruitment of distinct
molecule(s) to the nucleosome.
Among the native histone molecules we used as a single substrate, H1
was also found to be a good in vitro substrate for G9a. A
similar finding has also been reported for Suv39 h1 (17). Although H1
methylation is currently not well understood in mammalian cells, some
H1 functions may be regulated by methylation.
Other than H3N, none of the other analyzed GST-fused nuclear
proteins, such as CENP-A, CENP-B, CENP-C, HP1HS Finally, we found that EGFP-hG9a had a quite different localization
pattern in interphase nuclei than that of Suv39 h1. Endogenous Suv39 h1
and its human counterpart SUV39H1 were enriched at heterochromatic foci
in interphase nuclei in mammalian cells (39) and associated with HP1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-isoform
(HP1HS
) (22) fusion molecules were constructed by
similar procedures.
cDNA were cloned into pGEX vectors as previously described in Refs.
23 and 22, respectively. cDNAs encoding mouse telomeric repeat
binding factor 1 (TRF1) and TRF2 were isolated from a
gt11 mouse
thymus cDNA library and subcloned into the pGEX-4T vectors.
-mercaptoethanol, 250 mM sucrose) was
incubated for 60 min at 37 °C. The reaction products were separated
by 15% SDS-polyacrylamide gel electrophoresis and visualized by
Coomassie Brilliant Blue R-250 staining. Gels were dried, and detection
of methyl-14C was performed using a BAS-5000
imaging analyzer (Fuji Film).
SET (see Fig. 5)
by the method described above. After 3 days of culture, cells were
harvested and lysed in phosphate-buffered saline containing 0.5%
Nonidet P-40 and a mixture of protease inhibitors (Roche Molecular
Biochemicals). After sonication and centrifugation (16,000 × g, 15 min), supernatants were incubated with anti-EGFP
specific antibodies (CLONTECH). The immune
complexes were collected with protein-A-Sepharose beads (Amersham
Pharmacia Biotech) and used in the Western blotting and in
vitro HMTase assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence comparison of human G9a and Suv39
h1. A, schematic relationship between human G9a and
Suv39 h1. The SET domain is shown as a black box.
Hatched bars represent cysteine-rich regions. The putative
HMTase region is indicated by scattered dots between the two
molecules. G9a protein contains a polyglutamic acid stretch
(E), another cysteine-rich region, and six contiguous
ankyrin repeats (ANK) upstream of the putative HMTase
region, whereas the Suv39 h1 protein contains a chromo-domain (shown as
dense dots). B, amino acid alignment of the
putative HMTase core regions. Top, comparison of the two
cysteine-rich regions upstream and downstream of the SET domain.
Cysteine residues are shaded in gray. Conserved and similar
residues (+) are shown between these two sequences.
Bottom, comparison of the carboxyl-terminal amino acid
sequence of the SET domains containing a possible catalytic core motif
(boxed). The amino acid at the hyperactive position is
indicated by gray shading.
NHSC motif (where
indicates a hydrophobic residue) was previously shown to be a possible catalytic core motif
responsible for the enzymatic activity (shown boxed in Fig. 1B, bottom panel), because amino acid replacement
of histidine 324 and cysteine 326 in Suv39 h1 abolished enzymatic
activity. Replacement of histidine 320 of Suv39 h1 with arginine
(SuvH320R) results in a 20-fold increase in enzymatic activities. Yeast
CLR4, which possesses arginine at this position of the putative
catalytic core motif, exhibits hyperenzymatic activities similar to the SuvH320R mutant, supporting the importance of this position in determination of the strength of activity (17). HG9a possesses the
yeast CLR4-type catalytic core motif in the SET domain (900R
NHLC906), with arginine in the first position of the core motif.
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Fig. 2.
G9a possess strong in vitro
HMTase activity in comparison to Suv39 h1. Recombinant GST
fusion proteins containing mG9a amino terminus (residues 138-698),
Suv39 h1-(82-412), and mG9a carboxyl terminus (residues
621-1000) were introduced into in vitro HMTase assays with
20 µg of free core histones (left four lanes) and 5 µg
of sole histone H3 (right four lanes) as substrates and
S-adenosyl-[methyl-14C]-L-methionine
as a methyl donor. Coomassie Brilliant Blue R-250 staining (top
panel) shows enzymes (arrows) and purified histones.
Autoradiography (bottom panels) indicates specific
methylation of H3 and H1 by GST-mG9a. All the presented data of
methyltransferase assays are representative of multiple independent
studies. E, enzyme.
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Fig. 3.
Native histones H1 and H3 and recombinant H3
amino terminus (residues 1-57) are suitable
substrates for in vitro methylation by G9a. In
vitro methylation assays were carried out using 10 µg each of
purified histones H1, H2A, and H3 and several recombinant GST fusion
proteins, such as 10 µg of CENP-A amino terminus (residues 1-52), 10 µg of histone H3 amino terminus (residues 1-57) (termed H3N, see
Fig. 4A), 15 µg of human HP1HS , 5 µg of
mouse TRF1, and 5 µg of human p53, as substrates. The catalytic
activity of G9a is highly specific to native H1, H3, and recombinant
H3N. E, enzyme; S, substrate.
(22),
mouse TRF1 (26), mouse TRF2 (27), and human p53 (28), into the in
vitro methyltransferase assays. Among these substrates, only the
GST-fused histone H3 amino-terminal (residues 1-57) molecule (H3N, see
Fig. 4A) was actively
methylated by GST-mG9a, whereas methylation of the other GST fusion
molecules was almost undetectable (Fig. 3 and data not shown). In
addition, H3N was a more efficient substrate of G9a than was the native
purified histone H3, presumably due to the pre-existence of methyl
groups on the targeted residue(s) of native H3.
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Fig. 4.
G9a can transfer methyl groups to lysines 9 and 27 of histone H3. A, recombinant GST fusion histone
H3 amino terminus (residues 1-57), H3N, and the mutants used as
substrates for in vitro methylation assays. The amino acid
sequence of the mouse H3 amino terminus is shown on the top. The H3 amino terminus region
(residues 1-57) fused to GST is shaded. All the lysine
(K) to arginine (R) replacements at position 4, 9, and/or 27 in each of the mutants are boxed. B
and C, in vitro HMTase assays using 20 µg of
H3N mutants as substrates. GST-mG9a-(621-1000) (B) and
GST-Suv39 h1-(82-412) (C) were used for the enzyme assay.
In cases employing the single lysine mutants 4R, 9R, and 27R as
substrates, both enzymes could methylate 4R and 27R dominantly as
substrates, indicating that lysine 9 is the dominant methylation
residue. Apparent HMTase activity against 9R was also detected by
GST-mG9a but not Suv39 h1, suggesting the existence of an additional
target site for G9a. Among the double lysine mutants N4, N9, and N27,
N9 was found to be a suitable substrate (S) for both enzymes
(E); however, N27 could also serve as the substrate for G9a
methylation, indicating the ability of GST-mG9a to methylate lysine 27. D and E, methylation of lysine 27 was an
intrinsic property of G9a. Transiently expressed EGFP-hG9a-(1-1000)
and its SET domain deletion mutant, EGFP- SET-(1-755) in 293 cells
were harvested with anti-EGFP antibodies. Western blot analysis
confirmed that nearly the same amounts of fusion molecules were
precipitated (arrows in D). The
immunoprecipitated molecules were then introduced into in
vitro HMT assays using native histones or H3N mutants as
substrates, and the relative methylation efficiencies (%) were
determined (E). The relative methylation efficiency (%) was
calculated as (14C counts of mutant H3N
14C counts of GST)/(14C counts of H3N
14C counts of GST) × 100. IP,
immunoprecipitation.
NHLC905) is arginine, which
also exists in the core motif of the active HMTase CLR4
(406R
NHSC412), we speculated that arginine 899 might
be responsible for the strong HMTase activity of G9a. A previous
observation that a single amino acid replacement of histidine 320 in
the stringent HMTase Suv39 h1 (carrying 320H
NHSC326)
with arginine produced a hyperactive enzyme that possessed more than 20 times higher activity than the original (17) supported this
possibility. In addition to the higher enzymatic activity, we found
that G9a could catalyze the methylation of lysines 9 and 27 in H3N
in vitro, whereas Suv39 h1 was limited to lysine 9. Therefore, we also investigated the possibility that the first amino
acid residue of the catalytic core motif of G9a, called the
"hyperactive position" (shown shaded in gray in Fig.
1B, bottom panel), might also contribute to the target specificity of G9a, especially with regard to lysine 27 of H3.
To address these issues, we constructed two recombinant enzymes, in
which the amino acid at the hyperactive position was substituted
by site-directed mutagenesis. G9aR899H protein was produced by amino
acid substitution of arginine 899 in mG9a with histidine, and SuvH320R
was produced by replacement of histidine 320 in Suv39 h1 with arginine.
As expected, the catalytic activity of G9aR899H was dramatically
reduced to an almost undetectable level even though 10 times more
protein was used (Fig. 5A,
right panel). Because of this poor enzymatic activity of
G9aR899H, lysine selectivity could not be determined. In contrast to
this result, SuvH320R gained hyperactivity by this substitution, as
described previously, and quite efficiently methylated H3N and N9 but
remained unable to transfer methyl groups to N27 (Fig. 5B,
right panel and summarized in Fig.
6C). Therefore, these data
indicate that the hyperactive position may not influence the target
selectivity of lysine residues in H3, at least in the case of Suv39
h1.
View larger version (59K):
[in a new window]
Fig. 5.
Histidine 320 of Suv39 h1 is responsible for
hyper-HMTase activity but not for lysine selectivity in H3. To
determine whether the amino acid residue at the hyperactive position
also contributes to lysine selectivity, amino acid-substituted mutants
of GST-mG9a and GST-Suv39 h1 were introduced in in vitro
HMTase assays. A, HMTase activities of G9aR899H (arginine
899 replaced with histidine) were measured (right panel).
Significant reduction of the HMTase activity of G9aR899H was observed,
making estimation of substrate specificity difficult (right
panel). B, recombinant GST-Suv39 h1-(82-412) and its
hyperactive mutant SuvH320R (replaced histidine 320 with arginine) were
assayed as shown in A. Although it gained in
hyperactivity as previously reported (17), SuvH320R still methylated
only lysine 9 (right panel), indicating that the amino acid
residue at the hyperactive position may not contribute to lysine
selectivity. E, enzyme; S, substrate.
View larger version (32K):
[in a new window]
Fig. 6.
The different lysine selectivity between
GST-G9a and GST-Suv39 h1. In vitro HMTase assays using
GST-mG9a (A), GST-Suv39 h1(B), and its active
form, H320R (C), in the conditions described in the legend
to Fig. 4 are summarized. Relative methylation efficiencies were
calculated as described in the legend to Fig. 4E. Results
from three independent experiments are shown, and error bars indicate
the S.D. The different methylation profiles strongly suggest that only
GST-mG9a can transfer methyl groups to lysine 27 in H3.
were cointroduced into 293 cells, and the stably
expressed fluorescent tagged proteins were simultaneously observed.
HP1HS
has been shown to be localized to the centromeric
heterochromatin region in human cells (29). We observed that
DsRed-HP1HS
was also localized in the centromeric
regions in metaphase chromosomes (data not shown), similar to previous
reports of EGFP-HP1HS
(30). As shown in Fig.
7, left panels, G9a
(green) was strictly localized in nuclei and visualized as
discrete clear speckles, whereas HP1HS
(red)
appeared as heterogeneous dots in the interphase nuclei. When the two
images were merged, almost no overlapping of foci was seen except in a
few cases (shown as yellow dots in upper nuclei in Fig.
7D). We also established 293 cells expressing both EGFP-Suv39 h1 and DsRed-HP1HS
(Fig. 7, right
panels). In the interphase nuclei, Suv39 h1 and HP1HS
signals were observed as completely overlapped
dots (Fig. 7L), consistent with a similar report (31). When
EGFP-G9a and DsRed-Suv39 h1 were cointroduced, again these two signals
were found to be mainly independent and non-overlapping (Fig. 7,
middle panels). These data indicated that most of the G9a
molecules are outside of the heterochromatin domains around centromeric
loci, in which both Suv39 h1 and HP1 proteins have been shown to be
localized.
View larger version (45K):
[in a new window]
Fig. 7.
Different nuclear localization of
EGFP-fused G9a from Suv39 h1 and HP1. Fluorescent analysis of
interphase nuclei in 293 cells stably expressing EGFP-fused full-length
human G9a (EGFP-hG9a) and DsRed-fused human HP1HS
(DsRed-HP1HS
) (A-D), EGFP-hG9a and
DsRed-Suv39 h1 (E-H), and EGFP-Suv39 h1 and
DsRed-HP1HS
(I-L). Nuclei were stained with
4',6-diamidino-2-phenylindole (DAPI) (A,
E, and I). The images of GFP fusion proteins
(B, F, and J) and DsRed fusion
proteins (C, G, and K) were seen as
green and red signals, respectively. The
green and red images were overlaid in
D, H, and L.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, TRF1,
TRF2, and p53, were methylated by G9a under our conditions. However, another histone modification activity, acetylation, is not
restricted to histones. For example, histone acetyl transferase activity was shown to catalyze p53 acetylation, and this modification is regarded to be physiologically relevant (28). Considering the fact
that multiple SET domain-containing proteins, including functionally
uncharacterized ones, exist within a single organism, it should not be
surprising that SET domain-mediated methylation is not restricted to
histones. Furthermore, we could not eliminate the possibility that some
of the nuclear proteins we analyzed might be methylated by a different
type of methyltransferase that regulates their functions in
vivo.
(40), indicating their likely contribution to the structural
organization of the centromere. EGFP-hG9a colocalized with neither
DsRed-Suv39 h1 nor HP1HS
, whereas EGFP-Suv39 h1 and
DsRed-HP1HS
were strictly colocalized. Amino acid
sequences between G9a and Suv39 h1 were quite different except for the
conserved regions essential for HMTase activity (Fig. 1A).
Suv39 h1 contains a 60-amino acid chromo-domain, and G9a contains six
contiguous 33-amino acid ankyrin (ANK) repeats upstream of their
respective SET domains. Whereas the chromo-domain directs euchromatic
or heterochromatic localizations (41), ANK repeats have been identified
in many proteins with various subcellular localizations, such as Notch protein in the inner face of the plasma membrane (42), NF-
B in
cytoplasm (43), and SW14 in nucleus (44). ANK repeats are considered as
a motif that serves to interact with other proteins; therefore G9a may
be recruited to specific chromosomal loci via interaction between the
ANK repeats of G9a and additional regulatory proteins. Although our
data should be confirmed by specific antibodies against endogenous
molecules, the current findings suggest the possibility that
HMTase-mediated higher order chromatin structure may not be restricted
to centromeric loci but may also occur in other chromosomal
loci. Further characterization of the loci, in which G9a protein
accumulation was observed, will facilitate the elucidation of the
in vivo function of G9a.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. H. Kato and Ruth T. Yu for critical reading of this manuscript and A. Kaida for providing the p53 plasmid.
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FOOTNOTES |
---|
* This work was supported by grants from the Ministry of Education, Science, Culture, and Sports of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-75-751-3990; Fax: 81-75-751-3991; E-mail: yshinkai@virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, April 20, 2001, DOI 10.1074/jbc.M101914200
2 In accordance with this idea, several groups showed most recently that the chromo-domain of HP1 could bind specifically to methylated lysine 9 of H3 (36-38).
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ABBREVIATIONS |
---|
The abbreviations used are: HMTase, histone methyltransferase; h, human; kb, kilobase; PCR, polymerase chain reaction; EGFP, enhanced green fluorescent protein; DsRed, Discosoma striata red; HP1, heterochromatic protein 1; m, mouse; GST, glutathione S-transferase; CENP, centromere protein; TRF, telomeric repeat binding factor; ANK, ankyrin.
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