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• W2092794927 abstract "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. 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. histone methyltransferase human kilobase polymerase chain reaction enhanced green fluorescent protein Discosoma striata red heterochromatic protein 1 mouse glutathione S-transferase centromere protein telomeric repeat binding factor ankyrin 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 (1Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2419) Google Scholar). Steady-state levels of histone acetylation in vivo are maintained by the opposing activities of histone acetyl transferase and histone deacetylase (2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6679) Google Scholar, 3Grunstein M. Cell. 1998; 93: 325-328Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). 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 (4Wei Y. Mizzen C.A. Cook R.G. Gorovsky M.A. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7480-7484Crossref PubMed Scopus (357) Google Scholar, 5Wei Y., Yu, L. Bowen J. Gorovsky M.A. Allis C.D. Cell. 1999; 97: 99-109Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar). The phosphorylation of serine 10 also correlated with transcriptional activation of immediate-early genes in a rapid and transient manner upon mitogen stimulation (6Mahadevan L.C. Willis A.C. Barratt M.J. Cell. 1991; 65: 775-783Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 7Thomson S. Mahadevan L.C. Clayton A.L. Semin. Cell Dev. Biol. 1999; 10: 205-214Crossref PubMed Scopus (189) Google Scholar). Recently, several kinases have been identified as the enzymes that contribute to the mitogen-stimulated histone H3 phosphorylation (8Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (428) Google Scholar, 9Thomson S. Clayton A.L. Hazzalin C.A. Rose S. Barratt M.J. Mahadevan L.C. EMBO J. 1999; 18: 4779-4793Crossref PubMed Scopus (404) Google Scholar). In addition to acetylation and phosphorylation, it has also been reported that core histones, especially H3 and H4, are methylated (10von Holt C. Brandt W.F. Greyling H.J. Lindsey G.G. Retief J.D. Rodrigues J.D. Schwager S. Sewell B.T. Methods Enzymol. 1989; 170: 431-523Crossref PubMed Scopus (121) Google Scholar,11Duerre J.A. DiMaria P. Kim S. Paik W.K. Crit. Rev. Oncog. 1991; 2: 97-108PubMed Google Scholar). In mammalian HeLa cells, lysines 4, 9, and 27 in H3 and lysine 20 in H4 were shown to be methylated (12Strahl B.D. Ohba R. Cook R.G. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14967-14972Crossref PubMed Scopus (414) Google Scholar). Although the biological significance of histone methylation remains unclear, several histone methyltransferases (HMTases)1have recently been identified (12Strahl B.D. Ohba R. Cook R.G. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14967-14972Crossref PubMed Scopus (414) Google Scholar, 13Jenuwein T. Laible G. Dorn R. Reuter G. Cell. Mol. Life Sci. 1998; 54: 80-93Crossref PubMed Scopus (304) Google Scholar, 14Kennison J.A. Annu. Rev. Genet. 1995; 29: 289-303Crossref PubMed Scopus (354) Google Scholar). 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” (13Jenuwein T. Laible G. Dorn R. Reuter G. Cell. Mol. Life Sci. 1998; 54: 80-93Crossref PubMed Scopus (304) Google Scholar). Therefore, SET domain-containing proteins have diverse functions. For example, some members of theDrosophila 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 (14Kennison J.A. Annu. Rev. Genet. 1995; 29: 289-303Crossref PubMed Scopus (354) Google Scholar). 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 (15Allshire R.C. Nimmo E.R. Ekwall K. Javerzat J.P. Cranston G. Genes Dev. 1995; 9: 218-233Crossref PubMed Scopus (385) Google Scholar), Su(VAR)3–9 in fruit fly (16Tschiersch B. Hofmann A. Krauss V. Dorn R. Korge G. Reuter G. EMBO J. 1994; 13: 3822-3831Crossref PubMed Scopus (475) Google Scholar), Suv39 h1 and h2 in mouse, and SUV39H1 in human (17Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2199) Google Scholar, 18O'Carroll D. Scherthan H. Peters A.H. Opravil S. Haynes A.R. Laible G. Rea S. Schmid M. Lebersorger A. Jerratsch M. Sattler L. Mattei M.G. Denny P. Brown S.D. Schweizer D. Jenuwein T. Mol. Cell. Biol. 2000; 20: 9423-9433Crossref PubMed Scopus (250) Google Scholar). The Suv39 h family proteins have been considered to contribute to the organization of repressive chromatin regions such as the centromere (13Jenuwein T. Laible G. Dorn R. Reuter G. Cell. Mol. Life Sci. 1998; 54: 80-93Crossref PubMed Scopus (304) Google Scholar). 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. Full-length human G9a (hG9a) cDNA (19Milner C.M. Campbell R.D. Biochem. J. 1993; 290: 811-818Crossref PubMed Scopus (66) Google Scholar) was obtained by ligation of a 1.2-kb BamHI/ApaI cDNA fragment encoding amino acids 1–399 to aBamHI/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-kbHindIII/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) (20Yang T.T. Cheng L. Kain S.R. Nucleic Acids Res. 1996; 24: 4592-4593Crossref PubMed Scopus (352) Google Scholar) 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) (21Matz M.V. Fradkov A.F. Labas Y.A. Savitsky A.P. Zaraisky A.G. Markelov M.L. Lukyanov S.A. Nature Biotechnol. 1999; 17: 969-973Crossref PubMed Scopus (1540) Google Scholar)-human heterochromatic protein 1 α-isoform (HP1HSα) (22Sugimoto K. Yamada T. Muro Y. Himeno M. J. Biochem. (Tokyo). 1996; 120: 153-159Crossref PubMed Scopus (41) Google Scholar) fusion molecules were constructed by similar procedures. 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 theEcoRI 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 pairEcoRI/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 HP1HSα cDNA were cloned into pGEX vectors as previously described in Refs.23Muro Y. Azuma N. Onouchi H. Kunimatsu M. Tomita Y. Sasaki M. Sugimoto K. Clin. Exp. Immunol. 2000; 120: 218-223Crossref PubMed Scopus (29) Google Scholar and 22Sugimoto K. Yamada T. Muro Y. Himeno M. J. Biochem. (Tokyo). 1996; 120: 153-159Crossref PubMed Scopus (41) Google Scholar, respectively. cDNAs encoding mouse telomeric repeat binding factor 1 (TRF1) and TRF2 were isolated from a λgt11 mouse thymus cDNA library and subcloned into the pGEX-4T vectors. 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 (17Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2199) Google Scholar). Concentrations of recombinant proteins used were determined by Coomassie Brilliant Blue R-250 staining in SDS-polyacrylamide gel electrophoresis gels. All native histones (a mixture of H1, 2A, 2B, 3, and 4; and single H1, H2A, and H3) were purchased fromRoche Molecular Biochemicals. Methyltransferase assays were performed as reported (17Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2199) Google Scholar). Briefly, 50 μl of reaction mixture containing substrates, enzymes, and 250 nCi ofS-adenosyl-[methyl-14C]-l-methionine in methylase activity buffer (50 mm Tris, pH 8.5, 20 mm KCl, 10 mm MgCl2, 10 mm β-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). 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% CO2atmosphere. 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.). 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-ΔSET (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. 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. 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. (17Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2199) Google Scholar) 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 (19Milner C.M. Campbell R.D. Biochem. J. 1993; 290: 811-818Crossref PubMed Scopus (66) Google Scholar). 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)φφNHSC motif (where φ 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 (17Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2199) Google Scholar). HG9a possesses the yeast CLR4-type catalytic core motif in the SET domain (900R φφNHLC906), with arginine in the first position of the core motif. 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) andS-adenosyl-[methyl-14C]-l-methionine as a methyl donor. Reaction products were separated by SDS-polyacrylamide gel electrophoresis, andmethyl-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.Figure 4G9a 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. Band 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-ΔSET-(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.View Large Image Figure ViewerDownload (PPT) 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) (23Muro Y. Azuma N. Onouchi H. Kunimatsu M. Tomita Y. Sasaki M. Sugimoto K. Clin. Exp. Immunol. 2000; 120: 218-223Crossref PubMed Scopus (29) Google Scholar) and full-length constructs of human CENP-B (24Sugimoto K. Muro Y. Himeno M. J. Biochem. (Tokyo). 1992; 111: 478-483Crossref PubMed Scopus (34) Google Scholar), human CENP-C (25Sugimoto K. Yata H. Muro Y. Himeno M. J. Biochem. (Tokyo). 1994; 116: 877-881Crossref PubMed Scopus (80) Google Scholar), human HP1HSα (22Sugimoto K. Yamada T. Muro Y. Himeno M. J. Biochem. (Tokyo). 1996; 120: 153-159Crossref PubMed Scopus (41) Google Scholar), mouse TRF1 (26Smith S. de Lange T. Trends Genet. 1997; 13: 21-26Abstract Full Text PDF PubMed Scopus (108) Google Scholar), mouse TRF2 (27van Steensel B. Smogorzewska A. de Lange T. Cell. 1998; 92: 401-413Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar), and human p53 (28Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar), 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. Because methylation of H3 occurs dominantly at lysines 4, 9, and 27 in HeLa cells (12Strahl B.D. Ohba R. Cook R.G. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14967-14972Crossref PubMed Scopus (414) Google Scholar), and Suv39 h1 and h2 proteins were shown to methylate only lysine 9 of H3 (18O'Carroll D. Scherthan H. Peters A.H. Opravil S. Haynes A.R. Laible G. Rea S. Schmid M. Lebersorger A. Jerratsch M. Sattler L. Mattei M.G. Denny P. Brown S.D. Schweizer D. Jenuwein T. Mol. Cell. Biol. 2000; 20: 9423-9433Crossref PubMed Scopus (250) Google Scholar), 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 (4Wei Y. Mizzen C.A. Cook R.G. Gorovsky M.A. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7480-7484Crossref PubMed Scopus (357) Google Scholar). 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 and5A). 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 H3in 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 and6A). Because the first position of the catalytic core motif in the mG9a SET domain (899R φφNHLC905) is arginine, which also exists in the core motif of the active HMTase CLR4 (406R φφNHSC412), 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 φφNHSC326) with arginine produced a hyperactive enzyme that possessed more than 20 times higher activity than the original (17Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2199) Google Scholar) supported this possibility. In addition to the higher enzymatic activity, we found that G9a could catalyze the methylation of lysines 9 and 27 in H3Nin 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." @default.
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• W2092794927 title "SET Domain-containing Protein, G9a, Is a Novel Lysine-preferring Mammalian Histone Methyltransferase with Hyperactivity and Specific Selectivity to Lysines 9 and 27 of Histone H3" @default.
• W2092794927 cites W145348279 @default.
• W2092794927 cites W1506012016 @default.
• W2092794927 cites W1509466986 @default.
• W2092794927 cites W1511815964 @default.
• W2092794927 cites W1544900705 @default.
• W2092794927 cites W1575147957 @default.
• W2092794927 cites W1603793127 @default.
• W2092794927 cites W160853306 @default.
• W2092794927 cites W1912766779 @default.
• W2092794927 cites W1968343429 @default.
• W2092794927 cites W1970569586 @default.
• W2092794927 cites W1973704051 @default.
• W2092794927 cites W1978197643 @default.
• W2092794927 cites W1992690684 @default.
• W2092794927 cites W1999284470 @default.
• W2092794927 cites W2001653133 @default.
• W2092794927 cites W2002698073 @default.
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• W2092794927 cites W2010487837 @default.
• W2092794927 cites W2017920584 @default.
• W2092794927 cites W2018635260 @default.
• W2092794927 cites W2035516904 @default.
• W2092794927 cites W2040375560 @default.
• W2092794927 cites W2059851697 @default.
• W2092794927 cites W2061805545 @default.
• W2092794927 cites W2064767087 @default.
• W2092794927 cites W2066607735 @default.
• W2092794927 cites W2070948501 @default.
• W2092794927 cites W2072744580 @default.
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• W2092794927 cites W2163191416 @default.
• W2092794927 cites W2174739588 @default.
• W2092794927 cites W2190911698 @default.
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