A Jumonji (Jarid2) Protein Complex Represses cyclin D1 Expression by Methylation of Histone H3-K9

Abstract

Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification. Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification. Covalent modifications of histone tails, such as methylation, acetylation, and phosphorylation, have important roles in regulating chromatin dynamics (1Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6585) Google Scholar, 2Turner B.M. Bioessays. 2000; 22: 836-845Crossref PubMed Scopus (965) Google Scholar, 3Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7633) Google Scholar, 4Turner B.M. Nat Cell Biol. 2007; 9: 2-6Crossref PubMed Scopus (294) Google Scholar). Indeed, many residues in histone tails are modified. For instance, several lysine and arginine residues are methylated. With regard to lysine, methylation occurs on at least 5 residues of H3 (H3-K4, -K9, -K27, -K36, and -K79) and a single lysine residue of H4 (H4-K20). Lysine methylation is involved in a variety of biological events including transcriptional regulation, heterochromatin formation and X-chromosome inactivation (5Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (694) Google Scholar, 6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (533) Google Scholar, 7Martin C. Zhang Y. Nat Rev Mol. Cell. Biol. 2005; 6: 838-849Crossref PubMed Scopus (1591) Google Scholar). The effects of methylation are dependent on the particular lysine residue. For example, methylation of H3-K4 and H3-K9 is linked to transcriptional activation and repression, respectively. In addition, the biological effect of methylation can differ depending on the state of methylation (mono-, di-, or tri-methylation), even within the same lysine residue. In an earlier study, we identified the jumonji (jmj, Jarid2) gene and produced jmj mutant mice using a mouse gene trap strategy (8Takeuchi T. Yamazaki Y. Katoh-Fukui Y. Tsuchiya R. Kondo S. Motoyama J. Higashinakagawa T. Genes Dev. 1995; 9: 1211-1222Crossref PubMed Scopus (255) Google Scholar). In the Jmj protein, we found novel domains, which were subsequently named the JmjN and JmjC domains, and that several proteins conserved the domains (8Takeuchi T. Yamazaki Y. Katoh-Fukui Y. Tsuchiya R. Kondo S. Motoyama J. Higashinakagawa T. Genes Dev. 1995; 9: 1211-1222Crossref PubMed Scopus (255) Google Scholar). More than 100 proteins from many species including bacteria, fungi, plants, and animals have been shown to have the jmjC domain or both the jmjN and jmjC domains (9Balciunas D. Ronne H. Trends Biochem. Sci. 2000; 25: 274-276Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10Clissold P.M. Ponting C.P. Trends Biochem. Sci. 2001; 26: 7-9Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and form a Jmj family. All members of the Jmj family have a jmjC domain. Recently, many members have been shown to be histone demethylases for H3-K4, -K9, -K27, and -K36, and a jmjC is a catalytic domain of the enzymes (for review, see Refs. 11Takeuchi T. Watanabe Y. Takano-Shimizu T. Kondo S. Dev. Dyn. 2006; 235: 2449-2459Crossref PubMed Scopus (157) Google Scholar, 12Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (952) Google Scholar, 13Shi Y. Nat. Rev. Genet. 2007; 8: 829-833Crossref PubMed Scopus (474) Google Scholar). Jmj may not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. However, Jmj shows transcriptional repressor activity (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Importantly, Jmj binds to the promoter of cyclin D1, a gene encoding one of the G1 cyclins, and represses the transcriptional activity. The repression is necessary for negative regulation of the cardiac myocyte proliferation during development (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) as well as coordination of cell cycle exit, differentiation, and migration of neural progenitor cells in the hindbrain (15Takahashi M. Kojima M. Nakajima K. Suzuki-Migishima R. Takeuchi T. Dev. Biol. 2007; 303: 549-560Crossref PubMed Scopus (20) Google Scholar). However, the mechanisms by which Jmj represses transcription remain unknown. In the present study, we examined these mechanisms and found that Jmj protein complexes had histone methyltransferase activity toward histone H3-K9. Further study revealed that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, suggesting the Jmj family proteins have a novel function in transcriptional control. Plasmids and Transient Transfection—The following expression vectors were used: mammalian expression vectors for only FLAG epitope (pFLAG), FLAG-Jmj (pFLAG-Jmj), myc-G9a, FLAG-GLP (16Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (603) Google Scholar), FLAG-SETDB1 (17Ichimura T. Watanabe S. Sakamoto Y. Aoto T. Fujita N. Nakao M. J. Biol. Chem. 2005; 280: 13928-13935Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) (a kind gift from Dr. Nakao), myc-SET7, myc-Suv39h1 (kind gifts from Dr. Urano), and bacterial expression vectors for glutathione S-transferase (GST) 3The abbreviations used are: GST, glutathione S-transferase; PBS, phosphate-buffered saline; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; ChIP, chromatin immunoprecipitation assay; CBB, Coomassie Brilliant Blue; HMT, histone methyltransferase.-histone H3 N-tail mutants (18Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). In addition, cDNA for only FLAG epitope or FLAG-Jmj was inserted into pCAG-IRES-Puro vector (a kind gift from Dr. Niwa), and designated to be pFLAG-IP or pFLAG-Jmj-IP, respectively. These vectors were used for selection of transfectants. Transfection into 293T and NIH3T3 was performed using the Trans-Fast (Promega) and FuGENE HD (Roche Applied Science), respectively. The procedure was performed according to the manufacturer's instructions. Antibodies—We used antibodies against FLAG (M2, Sigma), T7 (Novagen), Myc (9E10, Santa Cruz Biotechnology) epitopes, G9a (16Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (603) Google Scholar), GLP (C-7–5, MBL), Jmj (NB100–2214, Novus), SETDB1 (ab12317, abcam), histone H3 (ab1791, abcam), histone H3-K9me1–3 (me1, 07–450; me2, 07–441; me3, 07–442; Upstate), mouse IgG (Sigma), and guinea pig IgG (Santa Cruz Biotechnology) for immunoprecipitation, Western blotting, immunostaining, and ChIP assay. Histone Methyltransferase (HMT) Assay—The 293T cells were transfected with pFLAG or pFLAG-jmj vectors. Cell extracts and immunoprecipitated complex with FLAG were prepared using the same method described under “Protein-Protein Interaction.” The immunoprecipitated complex was washed and incubated at 30 °C for 60 min in a HMT reaction buffer (50 mm Tris-HCl pH 8.5, 20 mm KCl, 10 mm MgCl2, 10 mm 2-ME, 250 mm sucrose) containing 250 nCi of S-adenosyl-[methyl-14C]-l-methionine and 5–10 μg of histone substrate. The substrate was core histones (Roche Applied Science) or GST-histone H3-tail fusion proteins (18Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). The reactions were terminated by the addition of 2× SDS sample loading buffer and boiling for 5 min. The reaction products were separated by 15% SDS-PAGE and visualized by CBB staining. The gels were incubated in Enlight solution (MoBiTec), dried, and then the methylated products were detected by autoradiography. Protein-Protein Interaction—NIH3T3 cells were transfected with various expression vectors. Cell extracts were prepared with Nonidet P-40 lysis buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40) containing Complete protease inhibitor mixture (Roche Applied Science) from NIH3T3 transfectants or mouse embryos at embryonic day 14.5. The extracts were incubated with 5–10 μg of anti-FLAG, anti-G9a, or anti-GLP antibodies, and immunoprecipitated with protein G-Sepharose (GE Healthcare). Immunoprecipitates were analyzed by Western blot analysis. ChIP Assay—The assays were performed using a ChIP Assay kit (Upstate) according to the manufacturer's instructions and as previously described (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). DNA was analyzed by PCR using specific primers to mouse cyclin D1 promoter, P1 (-33): 5′-CCCAGTTTGGAGAGAAGCAG-3′ and 5′-ACTCCCCTGTAGTCCGTGTG-3′, P2 (-591): 5′-CC-AGCGAGGAGGAATAGATG-3′ and 5′-AGCGTCCCTGTCTTCTTTCA-3′, P3 (-982): 5′-TCA-CCTTATCGGCTCACAAGT-3′ and 5′-AGACACGATAGGCTCCTTCC-3′, P4 (-2707): 5′-TGAAATCCGCTCAGGGTAAC-3′ and 5′-GGACTTGGCTGTTTCTGCTC-3′, mouse cyclin D1 exon 3: 5′-TGGAACTGCTTCTGGTGAAC-3′ and 5′-ATCCGCCTCTGGCATTTTGG-3′, and mouse amylase 2.1:5′-TCAGTTGTAATTCTCCTTGTACGG-3′ and 5′-CCTCCCATCTGAAGTATGTGGGTC-3′. NIH3T3 cells transfected with pFLAG-IP or pFLAG-jmj-IP were selected in the presence of 1–2 μg/ml puromycin (Sigma). For in vivo ChIP experiments, cell extracts were prepared from the hearts of normal mice. A PCR primer for P3 set was used. All ChIP were performed at least and the same results were by cells were used for the and transfected using according to the manufacturer's instructions. pFLAG-IP or pFLAG-jmj-IP was transfected The transfectants for expression vectors were selected in the presence of 1–2 μg/ml puromycin and used for the ChIP and analysis. for mouse G9a and GLP were and GLP and showed effects on and GLP, negative were also used. was using cDNA was performed with and primer the manufacturer's instructions The (Roche Applied Science) and were used. PCR was with the following and cyclin and 5′-ATCCGCCTCTGGCATTTTGG-3′, and and The other products including primer were by and For of the of or were used as the instructions of Applied the of sample was used as the control. and were in phosphate-buffered and then in in for immunostaining, or for 30 min to in Nonidet P-40 in for staining. were was performed as described previously T. Kojima M. Nakajima K. Kondo S. Dev. PubMed Scopus (89) Google Scholar). For of G9a and GLP, the same antibodies as for the ChIP were used. The procedure was described in a study J. K. Kojima M. Kondo S. Takeuchi T. Dev. PubMed Scopus Google Scholar). for were analyzed using least a with of of were The Jmj Histone Methyltransferase toward Histone we showed that a protein complex including Jmj protein binds to the cyclin D1 promoter and represses the transcriptional activity. The repression is for repression of cell proliferation of cardiac during development (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, the mechanisms of transcriptional repression by Jmj remain unknown. the we examined the that Jmj is involved in of the A Jmj protein complex was from cells transfected with a FLAG-Jmj expression and we examined the of the complex with to histone in and methylation of several histone residues are well as histone modifications involved in transcriptional repression (for review, see B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6585) Google Scholar). We not histone activity not the other histone methyltransferase showed that the Jmj complex had histone methyltransferase activity toward histone H3 we examined the residues of histone H3 by the Jmj For we used a normal H3 residues and several mutant in which or were for as were detected in and and in but were not detected in and in showed that the Jmj complex not to These results that the Jmj complex histone methyltransferase activity to H3-K9 in We histone methyltransferase activity of the Jmj complex to H3-K9 in cyclin D1 is a of Jmj (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). because Jmj represses cell proliferation (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, M. Kojima M. Takeuchi T. Biochem. 2000; PubMed Scopus Google Scholar), are cell which Jmj is to the hearts of jmj mutant embryos by ChIP because the mutant embryonic day and the is we examined by of function but not of We examined the effects of expression of Jmj on histone methylation of cyclin D1 promoter using ChIP in the promoter were We not methylation (mono-, di-, and of H3-K9 in the vector FLAG was was transfected the other or or me2, of H3-K9 was in by Jmj expression was also detected in the but the were for H3-K9me1–3 were also in These in and in vivo showed that the Jmj complex histone methyltransferase activity toward H3-K9. Jmj to the H3-K9 Histone G9a and GLP and to the cyclin D1 histone methyltransferase activity in the Jmj complex and the of domains for histone methyltransferases such as in Jmj that the complex H3-K9 histone we examined Jmj protein with H3-K9 methyltransferases GLP, and we examined H3-K9 methyltransferases to Jmj in NIH3T3 the expression of Jmj is or be in cell such as NIH3T3 FLAG-Jmj expression vector was into NIH3T3 cells by We found that Jmj bound to G9a and GLP, but not to SETDB1 The binding was by negative of only FLAG or immunoprecipitation using for IgG experiments, not and In the of not be because the was or the against not we examined binding and Jmj. However, not be we the binding of Jmj to G9a and GLP in embryos at embryonic day the were than for binding G9a and GLP We not binding Jmj and SETDB1 in the embryos These results showed that Jmj binds to G9a and GLP the H3-K9 methyltransferases We also examined G9a or GLP binds to cyclin D1 promoter and histone H3-K9 in the cyclin D1 promoter is methylated in ChIP using mouse embryos at that for the binding of G9a and GLP to the cyclin D1 promoter and H3-K9 in the cyclin D1 promoter were However, these were not detected in the cyclin D1 exon These results are with the that G9a and GLP H3-K9 in the cyclin D1 promoter in we examined the effect of Jmj on the binding of H3-K9 methyltransferases to the cyclin D1 shown Jmj protein binds to cyclin D1 promoter in 293T cells (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) Expression of jmj was not detected in cell We not binding of H3-K9 methyltransferases GLP, and to cyclin D1 promoter jmj was not and However, binding of G9a and GLP to cyclin D1 promoter was detected Jmj was and for SETDB1 and were These results show that the expression of Jmj recruited G9a and GLP to cyclin D1 of G9a and GLP H3-K9 and of cyclin D1 Expression by the Jmj examined the effects of of G9a and GLP on H3-K9 methylation by Jmj in an to the roles of G9a and GLP in the Jmj we that Jmj expression H3-K9 methylation in the cyclin D1 promoter even were transfected of G9a by was for see and showed effects for in the P2 we both G9a and GLP. Transfection of for GLP results in a in GLP and also G9a is with in GLP cells (16Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (603) Google Scholar). G9a protein because GLP is for the of G9a In of GLP the G9a protein but not the and In the of G9a and GLP, for H3-K9 methylation by jmj expression in The promoter of gene was used for a negative because the gene shows specific and was to be in the NIH3T3 were detected In addition, we performed ChIP assays using an that H3 of modifications to the that the in histone methylation, in histone were detected the the not in histone complex represses cyclin D1 expression by G9a or GLP. NIH3T3 cells were transfected with FLAG or FLAG-Jmj expression vectors and also for G9a or GLP. The transfectants for expression vectors were selected in the presence of The of G9a, GLP, and cyclin D1 in NIH3T3 cells were by The are as the of and with to the we the effects of of on repression of cyclin D1 by Jmj. The of of G9a, GLP, and cyclin D1 were by Jmj expression the of cyclin D1 were transfected cyclin D1, However, of GLP or both G9a and GLP, but not for only G9a, increased the and to the was by of of G9a and In these 5 and we used and the same These results that Jmj H3-K9 methylation of the cyclin D1 promoter and represses cyclin D1 expression by of GLP or both G9a and GLP. Jmj binds to the cyclin D1 promoter and represses cyclin D1 The repression cell proliferation in cardiac (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In the present study, we analyzed the of the transcriptional repression by Jmj. results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP. G9a, GLP, and Jmj G9a, GLP, and Jmj form one G9a and GLP form a complex in many including NIH3T3 and 293T which were used in study (16Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (603) Google Scholar). of G9a or GLP in of both G9a and GLP, at least in cells (16Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (603) Google Scholar). of GLP in a in G9a protein but not G9a in and NIH3T3 cells (16Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (603) Google Scholar) and suggesting that binding of G9a to GLP is for of G9a transcriptional complexes such as and both G9a and GLP H. K. S. Y. Science. 2002; PubMed Scopus Google Scholar, Y. J. H. Y. Y. Nature. 2003; PubMed Scopus Google Scholar, H. Sci. S. PubMed Scopus Google Scholar). These results suggest that the G9a and GLP complex is than and Jmj bound to the the of Jmj in the is a of Jmj family members of Jmj family proteins have been to be histone demethylases T. Watanabe Y. Takano-Shimizu T. Kondo S. Dev. Dyn. 2006; 235: 2449-2459Crossref PubMed Scopus (157) Google Scholar, 12Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (952) Google Scholar, 13Shi Y. Nat. Rev. Genet. 2007; 8: 829-833Crossref PubMed Scopus (474) Google Scholar). Jmj shows an that of histone by a protein is Jmj histone demethylase the is because the for binding to in jmjC are not conserved T. Watanabe Y. Takano-Shimizu T. Kondo S. Dev. Dyn. 2006; 235: 2449-2459Crossref PubMed Scopus (157) Google Scholar). Jmj G9a and GLP to the cyclin D1 promoter and an showed that Jmj can to the cyclin D1 promoter Jmj a that G9a and GLP to the cyclin D1 is that methylation of H3-K9 in the cyclin D1 promoter by Jmj expression not be detected in P1 which are in the to the Jmj (14Toyoda M. Shirato H. Nakajima K. Kojima M. Takahashi M. Kubota M. Suzuki-Migishima R. Motegi Y. Yokoyama M. Takeuchi T. Dev. Cell. 2003; 5: 85-97Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). is that the of ChIP was in the because is and the of H3-K9 antibodies are not is also that the Jmj complex binds to the Jmj but the complex methylates H3-K9 in the and the methylation is critical for the repression of cyclin D1 expression. of G9a and showed that GLP for the of G9a in in and were in the GLP protein was not (16Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (603) Google Scholar, M. Sugimoto K. M. Ueda J. Ohta T. M. Fukuda M. Takeda N. H. H. Shinkai Y. Genes Dev. 2002; PubMed Scopus Google Scholar). However, in and in the cyclin D1 promoter were in both G9a and GLP NIH3T3 but not in G9a cells that GLP can for the of G9a for at least cyclin D1 gene in NIH3T3 We produced cardiac mice using the cardiac not show during and of and GLP were detected in at M. M. and Y. These suggest of G9a and GLP, and cell or regulation of H3-K9 methylation by these be to the function of GLP is cell or is that members of the the or of GLP are of Jmj Histone addition to protein lacks conserved residues critical for histone demethylation in the JmjC domain, suggesting of the activity T. Watanabe Y. Takano-Shimizu T. Kondo S. Dev. Dyn. 2006; 235: 2449-2459Crossref PubMed Scopus (157) Google Scholar). is a gene for the mouse mutant S. C. S. Sci. S. PubMed Scopus Google Scholar). as a with such as and represses transcriptional in the of these Genes Dev. 2001; PubMed Scopus Google Scholar, Mol. Cell. Biol. 2002; 22: PubMed Scopus Google Scholar, J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). present Jmj suggest the that is also involved in other histone histone demethylation and represses the gene expression through these The and roles of JmjC of Jmj and remain unknown. However, these JmjC domains can to methylated is that the JmjC domains the of histone The regulation regulation of histone methylation. In show a novel function of Jmj family proteins in transcriptional and mechanisms cyclin D1, which in the cell cycle and is involved in several We Nakao and of for and and the NIH3T3 cell We also Suzuki-Migishima for