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Involvement of Histone H3 Lysine 9 (H3K9) Methyltransferase G9a in the Maintenance of HIV-1 Latency and Its Reactivation by BIX01294

2010; Elsevier BV; Volume: 285; Issue: 22 Linguagem: Inglês

10.1074/jbc.m110.103531

1083-351X

Kenichi Imai, Hiroaki Togami, Takashi Okamoto,

HIV/AIDS Research and Interventions

Elucidating the mechanism of human immunodeficiency virus, type 1 (HIV-1) provirus transcriptional silencing in latently infected cells is crucial for understanding the pathophysiological process of HIV-1 infection. It is well established that hypoacetylation of histone proteins by histone deacetylases is involved in the maintenance of HIV-1 latency by repressing viral transcription. Although histone methylation is involved in the organization of chromatin domains and plays a central epigenetic role in gene expression, the role of histone methylation in the maintenance of HIV-1 latency has not been clarified. Here we present evidence that histone H3 Lys9 (H3K9) methyltransferase G9a is responsible for transcriptional repression of HIV-1 by promoting repressive dimethylation at H3K9 and for the maintenance of viral latency. We observed that G9a significantly inhibited basal, as well as, the induced HIV-1 gene expression by tumor necrosis factor-α or Tat. Mutant G9a, however, lacking the SET domain responsible for the catalytic activity of histone methyltransferase, did not show such an effect. When G9a expression was knocked down by small interfering RNA, HIV-1 replication was augmented from cells transiently transfected with a full-length HIV-1 clone. Moreover, a specific inhibitor of G9a, BIX01294, could reactivate expression of HIV-1 from latently infected cells such as ACH-2 and OM10.1. Furthermore, chromatin immunoprecipitation assays revealed the presence of G9a and H3K9 dimethylation on nucleosome histones in the vicinity of the HIV-1 long terminal repeat promoter. These results suggest that G9a is responsible for the transcriptional quiescence of latent HIV-1 provirus and provide a molecular basis for understanding the mechanism by which HIV-1 latency is maintained. Elucidating the mechanism of human immunodeficiency virus, type 1 (HIV-1) provirus transcriptional silencing in latently infected cells is crucial for understanding the pathophysiological process of HIV-1 infection. It is well established that hypoacetylation of histone proteins by histone deacetylases is involved in the maintenance of HIV-1 latency by repressing viral transcription. Although histone methylation is involved in the organization of chromatin domains and plays a central epigenetic role in gene expression, the role of histone methylation in the maintenance of HIV-1 latency has not been clarified. Here we present evidence that histone H3 Lys9 (H3K9) methyltransferase G9a is responsible for transcriptional repression of HIV-1 by promoting repressive dimethylation at H3K9 and for the maintenance of viral latency. We observed that G9a significantly inhibited basal, as well as, the induced HIV-1 gene expression by tumor necrosis factor-α or Tat. Mutant G9a, however, lacking the SET domain responsible for the catalytic activity of histone methyltransferase, did not show such an effect. When G9a expression was knocked down by small interfering RNA, HIV-1 replication was augmented from cells transiently transfected with a full-length HIV-1 clone. Moreover, a specific inhibitor of G9a, BIX01294, could reactivate expression of HIV-1 from latently infected cells such as ACH-2 and OM10.1. Furthermore, chromatin immunoprecipitation assays revealed the presence of G9a and H3K9 dimethylation on nucleosome histones in the vicinity of the HIV-1 long terminal repeat promoter. These results suggest that G9a is responsible for the transcriptional quiescence of latent HIV-1 provirus and provide a molecular basis for understanding the mechanism by which HIV-1 latency is maintained. IntroductionIn eukaryotic cells, transcriptional activity of each gene is largely regulated at the epigenetic level, in which biochemical modifications of chromatin-associated proteins such as histones play critical roles (1Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). The DNA-associated histones conform an octamer configuration containing two copies of each core histone protein including H2A, H2B, H3, and H4. The N-terminal region of these histone proteins is unstructured and, thus, considered to be in a highly dynamic structure (3Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6795) Google Scholar). Histone tails protrude out from the globular center of the nucleosome where they may interact with other nuclear factors (2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar, 4Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). The N-terminal tails are subjective to a variety of post-translational modifications, such as phosphorylation, acetylation, methylation, and ubiquitination (4Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar, 6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (530) Google Scholar, 7Li B. Carey M. Workman J.L. Cell. 2007; 128: 707-719Abstract Full Text Full Text PDF PubMed Scopus (2643) Google Scholar). These modifications affect the affinity of other nuclear proteins in binding to the histone tail and, thus, regulate the nature of histone-protein complexes associated with each chromatin region. The ability of nuclear proteins to specifically associate with certain histone modifications is the basis of the histone code postulate (2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). According to this idea, nuclear proteins appear to function in activating or inhibiting transcription or, similarly, serve to maintain a specific chromatin structure.Human immunodeficiency virus, type 1 (HIV-1) 2The abbreviations used are: HIV-1human immunodeficiency virus type 15-aza-CdR5-aza-2′-deoxycytidineDNMTDNA methyltransferaseHDAChistone deacetylaseHMThistone methyltransferaseHP1heterochromatin protein 1SAHAsuberoylanilide hydroxamic acidSETSu(var)3-9 enhancer-of-zeste and trithoraxSuv39Hsuppressor of variegation 3-9 homologTNF-αtumor necrosis factor-αLTRlong terminal repeatsiRNAshort interference RNACMVcytomegalovirusChIPchromatin immunoprecipitationMMTVmurine mammary tumor virusELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinHTLV-1human T cell lymphotropic virus type 1. gene expression is the major determinant of viral replication leading to disease progression of acquired immunodeficiency syndrome (AIDS). After HIV-1 infection, integrated HIV-1 proviral DNA is incorporated into nucleosomes and the transcriptional activity of its long terminal repeat (LTR) is under the control of local nucleosomal structure (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 9Marcello A. Retrovirology. 2006; 3: 7Crossref PubMed Scopus (99) Google Scholar, 10Verdin E. J. Virol. 1991; 65: 6790-6799Crossref PubMed Google Scholar, 11Verdin E. Paras Jr., P. Van Lint C. EMBO J. 1993; 12: 3249-3259Crossref PubMed Scopus (405) Google Scholar). It has been suggested that epigenetic modifications of the nucleosomal structure (called "Nuc-1") near the viral mRNA start site may play regulatory roles in induction of LTR-driven transcription and viral expression (10Verdin E. J. Virol. 1991; 65: 6790-6799Crossref PubMed Google Scholar, 11Verdin E. Paras Jr., P. Van Lint C. EMBO J. 1993; 12: 3249-3259Crossref PubMed Scopus (405) Google Scholar). The compaction of HIV-1 proviral DNA and its permissiveness for viral transcription are directly dependent on histone post-translational modifications, such as acetylation and methylation (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 12Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (480) Google Scholar, 13Sheridan P.L. Mayall T.P. Verdin E. Jones K.A. Genes Dev. 1997; 11: 3327-3340Crossref PubMed Scopus (163) Google Scholar, 14Lusic M. Marcello A. Cereseto A. Giacca M. EMBO J. 2003; 22: 6550-6561Crossref PubMed Scopus (190) Google Scholar). These distinct modifications serve to recruit various regulatory protein complexes toward HIV-1 LTR and eventually up-regulate or down-regulate HIV-1 gene expression and viral replication. Activation of HIV-1 gene expression by cytokines and virally encoded transactivator Tat is also accompanied by histone acetylation, leading to loss or rearrangement of Nuc1 (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 9Marcello A. Retrovirology. 2006; 3: 7Crossref PubMed Scopus (99) Google Scholar, 10Verdin E. J. Virol. 1991; 65: 6790-6799Crossref PubMed Google Scholar, 11Verdin E. Paras Jr., P. Van Lint C. EMBO J. 1993; 12: 3249-3259Crossref PubMed Scopus (405) Google Scholar, 12Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (480) Google Scholar). In contrast to productively infected cells, latently infected cells harbor the proviral HIV-1 genome integrated into the silent chromatin allowing persistence of transcriptionally inactive proviruses (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 9Marcello A. Retrovirology. 2006; 3: 7Crossref PubMed Scopus (99) Google Scholar). Although evidence has been accumulated to elucidate the molecular mechanisms involved in viral promoter activation, as described above, the identity of cellular chromatin modifiers and the biochemical mechanism involved in the repression of the HIV-1 promoter is still unclear.Previous studies have shown that the presence of histone deacetylases (HDACs) at the vicinity of the HIV LTR is correlated with transcriptional repression leading to viral latency (15Coull J.J. Romerio F. Sun J.M. Volker J.L. Galvin K.M. Davie J.R. Shi Y. Hansen U. Margolis D.M. J. Virol. 2000; 74: 6790-6799Crossref PubMed Scopus (290) Google Scholar, 16Williams S.A. Chen L.F. Kwon H. Ruiz-Jarabo C.M. Verdin E. Greene W.C. EMBO J. 2006; 25: 139-149Crossref PubMed Scopus (374) Google Scholar, 17Tyagi M. Karn J. EMBO J. 2007; 26: 4985-4995Crossref PubMed Scopus (185) Google Scholar, 18Imai K. Okamoto T. J. Biol. Chem. 2006; 281: 12495-12505Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). HDAC1 mediates chromatin remodeling resulting in both LTR promoter activity and viral production repression. Negative transcription factors such as Ying Yang protein 1 (15Coull J.J. Romerio F. Sun J.M. Volker J.L. Galvin K.M. Davie J.R. Shi Y. Hansen U. Margolis D.M. J. Virol. 2000; 74: 6790-6799Crossref PubMed Scopus (290) Google Scholar), nuclear factor κB (NF-κB), p50 homodimer (16Williams S.A. Chen L.F. Kwon H. Ruiz-Jarabo C.M. Verdin E. Greene W.C. EMBO J. 2006; 25: 139-149Crossref PubMed Scopus (374) Google Scholar), and C-promoter binding factor (17Tyagi M. Karn J. EMBO J. 2007; 26: 4985-4995Crossref PubMed Scopus (185) Google Scholar) have been shown to mediate HDAC1 recruitment to the LTR and, consequently, inhibit transcription from the viral promoter. We also reported that activator protein 4 acts as a transcriptional repressor by recruiting HDAC molecules and is involved in the maintenance of viral latency (18Imai K. Okamoto T. J. Biol. Chem. 2006; 281: 12495-12505Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Others observed that drugs that inhibit HDAC activity such as trichostatin A, butyric acid, and valproic acid, could effectively induce HIV transcription in latently infected cells (12Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (480) Google Scholar, 13Sheridan P.L. Mayall T.P. Verdin E. Jones K.A. Genes Dev. 1997; 11: 3327-3340Crossref PubMed Scopus (163) Google Scholar, 19Lehrman G. Hogue I.B. Palmer S. Jennings C. Spina C.A. Wiegand A. Landay A.L. Coombs R.W. Richman D.D. Mellors J.W. Coffin J.M. Bosch R.J. Margolis D.M. Lancet. 2005; 366: 549-555Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 20Imai K. Ochiai K. Okamoto T. J. Immunol. 2009; 182: 3688-3695Crossref PubMed Scopus (96) Google Scholar). These studies suggest that epigenetic silencing is involved in the maintenance of HIV-1 transcriptional latency.In addition to histone acetylation, histone Lys methylation also plays an epigenetic role in the organization of chromatin domains and the regulation of gene expression (6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (530) Google Scholar, 21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). Methylation of histones at Lys and Arg residues play both positive and negative roles in transcriptional regulation. For example, methylation of at Lys4 and Arg17 of histone H3 is generally associated with active genes, whereas methylation of H3 at Lys9 (H3K9me) and Lys27 (H3K27me) has been associated with inactive genes (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). It was also noted that H3K9me exhibited more definitive transcriptional repression over H3K27me (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). The repressive methylation of H3K9 has been detected at the promoter regions of many silenced genes, together with increased DNA methylation and reduced histone acetylation (23Baylin S.B. Ohm J.E. Nat. Rev. Cancer. 2006; 6: 107-116Crossref PubMed Scopus (1379) Google Scholar, 24Yoo C.B. Jones P.A. Nat. Rev. Drug Discov. 2006; 5: 37-50Crossref PubMed Scopus (1113) Google Scholar, 25Feldman N. Gerson A. Fang J. Li E. Zhang Y. Shinkai Y. Cedar H. Bergman Y. Nat. Cell Biol. 2006; 8: 188-194Crossref PubMed Scopus (512) Google Scholar, 26Epsztejn-Litman S. Feldman N. Abu-Remaileh M. Shufaro Y. Gerson A. Ueda J. Deplus R. Fuks F. Shinkai Y. Cedar H. Bergman Y. Nat. Struct. Mol. Biol. 2008; 15: 1176-1183Crossref PubMed Scopus (344) Google Scholar, 27Dong K.B. Maksakova I.A. Mohn F. Leung D. Appanah R. Lee S. Yang H.W. Lam L.L. Mager D.L. Schübeler D. Tachibana M. Shinkai Y. Lorincz M.C. EMBO J. 2008; 27: 2691-2701Crossref PubMed Scopus (191) Google Scholar). Two recent reports (28Marban C. Suzanne S. Dequiedt F. de Walque S. Redel L. Van Lint C. Aunis D. Rohr O. EMBO J. 2007; 26: 412-423Crossref PubMed Scopus (283) Google Scholar, 29du Chéné I. Basyuk E. Lin Y.L. Triboulet R. Knezevich A. Chable-Bessia C. Mettling C. Baillat V. Reynes J. Corbeau P. Bertrand E. Marcello A. Emiliani S. Kiernan R. Benkirane M. EMBO J. 2007; 26: 424-435Crossref PubMed Scopus (248) Google Scholar) demonstrated that the histone methyltransferase (HMT) Suppressor of variegation 3–9 Homolog (Suv39H) 1, which is primarily involved in Lys9 trimethylation of histone H3 (H3K9me3), is responsible for HIV-1 transcriptional silencing. Although H3K9 methylation is known to play a crucial role in chromatin-mediated transcriptional silencing, the molecular mechanism of H3K9 methylation and another HMT on HIV-1 gene repression has yet to be clarified.In addition to Suv39H1, there are at least four other mammalian H3K9 HMTs, including Suv39H2, a close relative of Suv39H1, G9a, G9a-like protein (GLP)/EuHMTase1, and SETDB1/ERG-associated protein with SET domain (ESET) (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (951) Google Scholar, 32Rea 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 (2152) Google Scholar, 33Shi Y. Sawada J. Sui G. el Affar B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (630) Google Scholar, 34Schultz D.C. Ayyanathan K. Negorev D. Maul G.G. Rauscher 3rd, F.J. Genes Dev. 2002; 16: 919-932Crossref PubMed Scopus (868) Google Scholar). These proteins commonly contain a SET (Suv39, enhancer of zeste, trithorax) domain that is responsible for catalytic action (6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (530) Google Scholar, 21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). Among these HMTs, G9a is a key enzyme responsible for H3K9 dimethylation (H3K9me2) in mammals as disruption of the G9a gene resulted in a drastic decrease in H3K9 methylation primarily in the silenced region within euchromatin (30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (951) Google Scholar, 35Rice J.C. Briggs S.D. Ueberheide B. Barber C.M. Shabanowitz J. Hunt D.F. Shinkai Y. Allis C.D. Mol. Cell. 2003; 12: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 36Tachibana 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 (594) Google Scholar). Thus, G9a has been implicated in silencing the gene expression (25Feldman N. Gerson A. Fang J. Li E. Zhang Y. Shinkai Y. Cedar H. Bergman Y. Nat. Cell Biol. 2006; 8: 188-194Crossref PubMed Scopus (512) Google Scholar, 26Epsztejn-Litman S. Feldman N. Abu-Remaileh M. Shufaro Y. Gerson A. Ueda J. Deplus R. Fuks F. Shinkai Y. Cedar H. Bergman Y. Nat. Struct. Mol. Biol. 2008; 15: 1176-1183Crossref PubMed Scopus (344) Google Scholar, 27Dong K.B. Maksakova I.A. Mohn F. Leung D. Appanah R. Lee S. Yang H.W. Lam L.L. Mager D.L. Schübeler D. Tachibana M. Shinkai Y. Lorincz M.C. EMBO J. 2008; 27: 2691-2701Crossref PubMed Scopus (191) Google Scholar). Interestingly, in vitro experiments revealed that G9a exhibited a 10–20-fold stronger HMT activity toward H3K9 compared with Suv39H1 (30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar).In this study we investigated the role of G9a in HIV-1 gene expression. We demonstrate that G9a is responsible for the maintenance of chromatin-mediated HIV-1 silencing through histone modification of H3K9me2. Biological and therapeutic implications are discussed.DISCUSSIONThe ability of HIV-1 to establish latent infection is considered momentous for AIDS progression (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 9Marcello A. Retrovirology. 2006; 3: 7Crossref PubMed Scopus (99) Google Scholar). Elucidating the transcriptional silencing mechanism of HIV-1 provirus in latently infected cells is crucial in understanding the pathophysiological process of HIV-1 infection and to further develop novel therapies. Viral latency involves certain chromatin modifications, in particular those of histone proteins, leading to proviral quiescence. It is well established (15Coull J.J. Romerio F. Sun J.M. Volker J.L. Galvin K.M. Davie J.R. Shi Y. Hansen U. Margolis D.M. J. Virol. 2000; 74: 6790-6799Crossref PubMed Scopus (290) Google Scholar, 16Williams S.A. Chen L.F. Kwon H. Ruiz-Jarabo C.M. Verdin E. Greene W.C. EMBO J. 2006; 25: 139-149Crossref PubMed Scopus (374) Google Scholar, 17Tyagi M. Karn J. EMBO J. 2007; 26: 4985-4995Crossref PubMed Scopus (185) Google Scholar, 18Imai K. Okamoto T. J. Biol. Chem. 2006; 281: 12495-12505Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) that proviral quiescence is crossly associated with HDAC recruitment to HIV-1 LTR. In addition, although H3K9 trimethylation is triggered by Suv39H1 and is known to be associated with gene silencing (6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (530) Google Scholar, 21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar), the role of histone methylation by G9a in the maintenance of HIV-1 latency, however, has not yet been elucidated. Because the extent of histone methylation by another HMT G9a is much greater that Suv39H1 (30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar), we explored the effect of H3K9me2 on the transcriptional activity of latent HIV-1 proviral chromatin. We found that G9a is overexpressed, HIV-1 gene expression was greatly suppressed, whereas G9a knockdown mediated by siRNA strikingly augmented HIV-1 transcription, thus, vital replication. Moreover, when cell lines latently infected with HIV-1 were treated with BIX01294, a specific inhibitor of G9a, H3K9 dimethylation was down-regulated and reactivated HIV-1 transcription and viral replication in the latently infected cells. These findings were confirmed by ChIP assays. These results suggest that G9a is responsible for the maintenance of transcriptional quiescence of latent HIV-1 provirus.Although accumulating evidence suggests that HDACs are critical regulators of HIV-1 latency, du Chéné et al. (29du Chéné I. Basyuk E. Lin Y.L. Triboulet R. Knezevich A. Chable-Bessia C. Mettling C. Baillat V. Reynes J. Corbeau P. Bertrand E. Marcello A. Emiliani S. Kiernan R. Benkirane M. EMBO J. 2007; 26: 424-435Crossref PubMed Scopus (248) Google Scholar) observed that knockdown of HDAC1 had only a marginal effect on the basal transcriptional activity of latent HIV-1. However, by using a general HDAC inhibitor, trichostatin A, HIV-1 promoter activity was stimulated nearly 40-fold indicating that HDAC-mediated repression of the HIV-1 promoter involves more than one HDAC species (29du Chéné I. Basyuk E. Lin Y.L. Triboulet R. Knezevich A. Chable-Bessia C. Mettling C. Baillat V. Reynes J. Corbeau P. Bertrand E. Marcello A. Emiliani S. Kiernan R. Benkirane M. EMBO J. 2007; 26: 424-435Crossref PubMed Scopus (248) Google Scholar). Considering the same authors observed that knockdown of heterochromatin protein 1 (HP1) γ, a heterochromatin packaging protein that specifically recognizes tri- or dimethylated histone H3, could stimulate HIV-1 transcription over 70-fold (29du Chéné I. Basyuk E. Lin Y.L. Triboulet R. Knezevich A. Chable-Bessia C. Mettling C. Baillat V. Reynes J. Corbeau P. Bertrand E. Marcello A. Emiliani S. Kiernan R. Benkirane M. EMBO J. 2007; 26: 424-435Crossref PubMed Scopus (248) Google Scholar), it was suggested that histone methylation plays a major role in controlling the transcriptional activity of the latent HIV-1 provirus.Regarding the role of histone methylation on HIV-1 provirus transcription, Suv39H1 and its associating factors, such as HP1 proteins, were found to be accumulated in the vicinity of latent HIV-1 proviral DNA and conform the repressive or transcriptionally silent heterochromatin (28Marban C. Suzanne S. Dequiedt F. de Walque S. Redel L. Van Lint C. Aunis D. Rohr O. EMBO J. 2007; 26: 412-423Crossref PubMed Scopus (283) Google Scholar, 29du Chéné I. Basyuk E. Lin Y.L. Triboulet R. Knezevich A. Chable-Bessia C. Mettling C. Baillat V. Reynes J. Corbeau P. Bertrand E. Marcello A. Emiliani S. Kiernan R. Benkirane M. EMBO J. 2007; 26: 424-435Crossref PubMed Scopus (248) Google Scholar). There have been a number of interesting observations regarding the chromatin configurations and the transcriptional status of latent HIV-1 proviral DNA. For example, du Chéné et al. (29du Chéné I. Basyuk E. Lin Y.L. Triboulet R. Knezevich A. Chable-Bessia C. Mettling C. Baillat V. Reynes J. Corbeau P. Bertrand E. Marcello A. Emiliani S. Kiernan R. Benkirane M. EMBO J. 2007; 26: 424-435Crossref PubMed Scopus (248) Google Scholar) and Marban et al. (28Marban C. Suzanne S. Dequiedt F. de Walque S. Redel L. Van Lint C. Aunis D. Rohr O. EMBO J. 2007; 26: 412-423Crossref PubMed Scopus (283) Google Scholar) reported that HP1γ was associated with the latent HIV-1 proviral DNA and when HP1γ was knocked down, latent HIV-1 was reactivated. In contrast, Mateescu et al. (51Mateescu B. Bourachot B. Rachez C. Ogryzko V. Muchardt C. EMBO Rep. 2008; 9: 267-272Crossref PubMed Scopus (45) Google Scholar) reported that H3K9 methylation and HP1β, but not HP1γ, were associated with latent HIV-1 proviral DNA upon ChIP analyses and that knockdown of HP1β reactivated latent HIV-1 gene expression, whereas HP1γ knockdown suppressed HIV-1 transcription. Thus, the effect of HP1 in the maintenance of HIV-1 latency is still controversial.Another HMT catalyst, G9a, primarily located at the silenced euchromatin (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (951) Google Scholar, 35Rice J.C. Briggs S.D. Ueberheide B. Barber C.M. Shabanowitz J. Hunt D.F. Shinkai Y. Allis C.D. Mol. Cell. 2003; 12: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 47Kubicek S. O'Sullivan R.J. August E.M. Hickey E.R. Zhang Q. Teodoro M.L. Rea S. Mechtler K. Kowalski J.A. Homon C.A. Kelly T.A. Jenuwein T. Mol. Cell. 2007; 25: 473-481Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar) is considered to have distinct target genes. It is possible that Suv39H and G9a have distinct roles in chromatin modification by having distinct molecular partners, thus, recognizing different modalities of distinct histone modifications (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar, 30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (951) Google Scholar, 32Rea 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 (2152) Google Scholar, 35Rice J.C. Briggs S.D. Ueberheide B. Barber C.M. Shabanowitz J. Hunt D.F. Shinkai Y. Allis C.D. Mol. Cell. 2003; 12: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). In addition, a recent report by Gazzar et al. (52El Gazzar M. Yoza B.K. Chen X. Hu J. Hawkins G.A. McCall C.E. J. Biol. Chem. 2008; 283: 32198-32208Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) demonstrated that G9a is involved in endotoxin tolerance and that G9a knock-down was correlated with the loss of Suv39H binding to the TNF-α promoter, and not vice versa, suggesting that Suv39H appears to be located downstream of G9a. Thus, it is suggested that when the gene, such as HIV-1 provirus, is silenced by the recruitment of G9a and, subsequently, with dimethylation of histone H3 (H3K9me2), which was then recognized by a heterochromatin protein complex containing HP1 and HDACs (33Shi Y. Sawada J. Sui G. el Affar B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (630) Google Scholar, 52El Gazzar M. Yoza B.K. Chen X. Hu J. Hawkins G.A. McCall C.E. J. Biol. Chem. 2008; 283: 32198-32208Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 53Ogawa H. Ishiguro K. Gaubatz S. Livingston D.M. Nakatani Y. Science. 2002; 296: 1132-1136Crossref PubMed Scopus (622) Google Scholar, 54Roopra A. Qazi R. Schoenike B. Daley T.J. Morrison J.F. Mol. Cell. 2004; 14: 727-738Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 55Sampath S.C. Marazzi I. Yap K.L. Sampath S.C. Krutchinsky A.N. Mecklenbräuker I. Viale A. Rudensky E. Zhou M.M. Chait B.T. Tarakhovsky A. Mol. Cell. 2007; 27: 596-608Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), followed by recruitment of Suv39H thus converting the silent euchromatin to a heterochromatin. G9a-mediated chromatin silencing might have a critical role in establishment of the latent HIV-1 provirus. In fact, a G9a-specific inhibitor, BIX01294, could efficiently reactivate the latent HIV-1 genes in a wide variety of cells (Fig. 4).In addition to histone modifications, the spatial distribution of genes within the nucleus might also contribute to the transcriptional control. For example, a recent report demonstrated an interesting correlation between transcriptional repression of the HIV-1 provirus and its spatial interaction with a pericentromeric heterochromatin region located in certain chromosomes, such as chromosome 12 in multiple distinctive Jurkat-derived cell clones where HIV-1 proviral DNAs are latently infected (56Dieudonné M. Maiuri P. Biancotto C. Knezevich A. Kula A. Lusic M. Marcello A. EMBO J. 2009; 28: 2231-2243Crossref PubMed Scopus (53) Google Scholar). Furthermore, as euchromatins are known to be dispersed within the nuclear core, certain portions of the nucleus, where G9a is predominantly present, could provide an intranuclear environment allowing reversible silencing (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 56Dieudonné M. Maiuri P. Biancotto C. Knezevich A. Kula A. Lusic M. Marcello A. EMBO J. 2009; 28: 2231-2243Crossref PubMed Scopus (53) Google Scholar). Nevertheless, further studies are needed to clarify the possible mechanism that links histone and DNA methylation. At present, it is well established that the latent HIV-1 provirus is associated with DNA methylation in its vicinity (57Ishida T. Hamano A. Koiwa T. Watanabe T. Retrovirology. 2006; 3: 69Crossref PubMed Scopus (77) Google Scholar, 58Kauder S.E. Bosque A. Lindqvist A. Planelles V. Verdin E. PLoS Pathog. 2009; 5: e1000495Crossref PubMed Scopus (295) Google Scholar, 59Blazkova J. Trejbalova K. Gondois-Rey F. Halfon P. Philibert P. Guiguen A. Verdin E. Olive D. Van Lint C. Hejnar J. Hirsch I. PLoS Pathog. 2009; 5: e1000554Crossref PubMed Scopus (252) Google Scholar). In addition, because G9a is known to associate with DNMT (26Epsztejn-Litman S. Feldman N. Abu-Remaileh M. Shufaro Y. Gerson A. Ueda J. Deplus R. Fuks F. Shinkai Y. Cedar H. Bergman Y. Nat. Struct. Mol. Biol. 2008; 15: 1176-1183Crossref PubMed Scopus (344) Google Scholar, 52El Gazzar M. Yoza B.K. Chen X. Hu J. Hawkins G.A. McCall C.E. J. Biol. Chem. 2008; 283: 32198-32208Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 60Estève P.O. Chin H.G. Smallwood A. Feehery G.R. Gangisetty O. Karpf A.R. Carey M.F. Pradhan S. Genes Dev. 2006; 20: 3089-3103Crossref PubMed Scopus (407) Google Scholar) and G9a knockout cells revealed significantly reduced DNA methylation (26Epsztejn-Litman S. Feldman N. Abu-Remaileh M. Shufaro Y. Gerson A. Ueda J. Deplus R. Fuks F. Shinkai Y. Cedar H. Bergman Y. Nat. Struct. Mol. Biol. 2008; 15: 1176-1183Crossref PubMed Scopus (344) Google Scholar, 27Dong K.B. Maksakova I.A. Mohn F. Leung D. Appanah R. Lee S. Yang H.W. Lam L.L. Mager D.L. Schübeler D. Tachibana M. Shinkai Y. Lorincz M.C. EMBO J. 2008; 27: 2691-2701Crossref PubMed Scopus (191) Google Scholar, 60Estève P.O. Chin H.G. Smallwood A. Feehery G.R. Gangisetty O. Karpf A.R. Carey M.F. Pradhan S. Genes Dev. 2006; 20: 3089-3103Crossref PubMed Scopus (407) Google Scholar, 61Xin Z. Tachibana M. Guggiari M. Heard E. Shinkai Y. Wagstaff J. J. Biol. Chem. 2003; 278: 14996-15000Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), it is possible that G9a indirectly induces DNA methylation. Thus, molecular actions of G9a in regulating the transcriptional activity of the HIV-1 promoter need to be further explored. IntroductionIn eukaryotic cells, transcriptional activity of each gene is largely regulated at the epigenetic level, in which biochemical modifications of chromatin-associated proteins such as histones play critical roles (1Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). The DNA-associated histones conform an octamer configuration containing two copies of each core histone protein including H2A, H2B, H3, and H4. The N-terminal region of these histone proteins is unstructured and, thus, considered to be in a highly dynamic structure (3Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6795) Google Scholar). Histone tails protrude out from the globular center of the nucleosome where they may interact with other nuclear factors (2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar, 4Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). The N-terminal tails are subjective to a variety of post-translational modifications, such as phosphorylation, acetylation, methylation, and ubiquitination (4Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2126) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar, 6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (530) Google Scholar, 7Li B. Carey M. Workman J.L. Cell. 2007; 128: 707-719Abstract Full Text Full Text PDF PubMed Scopus (2643) Google Scholar). These modifications affect the affinity of other nuclear proteins in binding to the histone tail and, thus, regulate the nature of histone-protein complexes associated with each chromatin region. The ability of nuclear proteins to specifically associate with certain histone modifications is the basis of the histone code postulate (2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). According to this idea, nuclear proteins appear to function in activating or inhibiting transcription or, similarly, serve to maintain a specific chromatin structure.Human immunodeficiency virus, type 1 (HIV-1) 2The abbreviations used are: HIV-1human immunodeficiency virus type 15-aza-CdR5-aza-2′-deoxycytidineDNMTDNA methyltransferaseHDAChistone deacetylaseHMThistone methyltransferaseHP1heterochromatin protein 1SAHAsuberoylanilide hydroxamic acidSETSu(var)3-9 enhancer-of-zeste and trithoraxSuv39Hsuppressor of variegation 3-9 homologTNF-αtumor necrosis factor-αLTRlong terminal repeatsiRNAshort interference RNACMVcytomegalovirusChIPchromatin immunoprecipitationMMTVmurine mammary tumor virusELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinHTLV-1human T cell lymphotropic virus type 1. gene expression is the major determinant of viral replication leading to disease progression of acquired immunodeficiency syndrome (AIDS). After HIV-1 infection, integrated HIV-1 proviral DNA is incorporated into nucleosomes and the transcriptional activity of its long terminal repeat (LTR) is under the control of local nucleosomal structure (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 9Marcello A. Retrovirology. 2006; 3: 7Crossref PubMed Scopus (99) Google Scholar, 10Verdin E. J. Virol. 1991; 65: 6790-6799Crossref PubMed Google Scholar, 11Verdin E. Paras Jr., P. Van Lint C. EMBO J. 1993; 12: 3249-3259Crossref PubMed Scopus (405) Google Scholar). It has been suggested that epigenetic modifications of the nucleosomal structure (called "Nuc-1") near the viral mRNA start site may play regulatory roles in induction of LTR-driven transcription and viral expression (10Verdin E. J. Virol. 1991; 65: 6790-6799Crossref PubMed Google Scholar, 11Verdin E. Paras Jr., P. Van Lint C. EMBO J. 1993; 12: 3249-3259Crossref PubMed Scopus (405) Google Scholar). The compaction of HIV-1 proviral DNA and its permissiveness for viral transcription are directly dependent on histone post-translational modifications, such as acetylation and methylation (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 12Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (480) Google Scholar, 13Sheridan P.L. Mayall T.P. Verdin E. Jones K.A. Genes Dev. 1997; 11: 3327-3340Crossref PubMed Scopus (163) Google Scholar, 14Lusic M. Marcello A. Cereseto A. Giacca M. EMBO J. 2003; 22: 6550-6561Crossref PubMed Scopus (190) Google Scholar). These distinct modifications serve to recruit various regulatory protein complexes toward HIV-1 LTR and eventually up-regulate or down-regulate HIV-1 gene expression and viral replication. Activation of HIV-1 gene expression by cytokines and virally encoded transactivator Tat is also accompanied by histone acetylation, leading to loss or rearrangement of Nuc1 (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 9Marcello A. Retrovirology. 2006; 3: 7Crossref PubMed Scopus (99) Google Scholar, 10Verdin E. J. Virol. 1991; 65: 6790-6799Crossref PubMed Google Scholar, 11Verdin E. Paras Jr., P. Van Lint C. EMBO J. 1993; 12: 3249-3259Crossref PubMed Scopus (405) Google Scholar, 12Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (480) Google Scholar). In contrast to productively infected cells, latently infected cells harbor the proviral HIV-1 genome integrated into the silent chromatin allowing persistence of transcriptionally inactive proviruses (8Colin L. Van Lint C. Retrovirology. 2009; 6: 111Crossref PubMed Scopus (182) Google Scholar, 9Marcello A. Retrovirology. 2006; 3: 7Crossref PubMed Scopus (99) Google Scholar). Although evidence has been accumulated to elucidate the molecular mechanisms involved in viral promoter activation, as described above, the identity of cellular chromatin modifiers and the biochemical mechanism involved in the repression of the HIV-1 promoter is still unclear.Previous studies have shown that the presence of histone deacetylases (HDACs) at the vicinity of the HIV LTR is correlated with transcriptional repression leading to viral latency (15Coull J.J. Romerio F. Sun J.M. Volker J.L. Galvin K.M. Davie J.R. Shi Y. Hansen U. Margolis D.M. J. Virol. 2000; 74: 6790-6799Crossref PubMed Scopus (290) Google Scholar, 16Williams S.A. Chen L.F. Kwon H. Ruiz-Jarabo C.M. Verdin E. Greene W.C. EMBO J. 2006; 25: 139-149Crossref PubMed Scopus (374) Google Scholar, 17Tyagi M. Karn J. EMBO J. 2007; 26: 4985-4995Crossref PubMed Scopus (185) Google Scholar, 18Imai K. Okamoto T. J. Biol. Chem. 2006; 281: 12495-12505Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). HDAC1 mediates chromatin remodeling resulting in both LTR promoter activity and viral production repression. Negative transcription factors such as Ying Yang protein 1 (15Coull J.J. Romerio F. Sun J.M. Volker J.L. Galvin K.M. Davie J.R. Shi Y. Hansen U. Margolis D.M. J. Virol. 2000; 74: 6790-6799Crossref PubMed Scopus (290) Google Scholar), nuclear factor κB (NF-κB), p50 homodimer (16Williams S.A. Chen L.F. Kwon H. Ruiz-Jarabo C.M. Verdin E. Greene W.C. EMBO J. 2006; 25: 139-149Crossref PubMed Scopus (374) Google Scholar), and C-promoter binding factor (17Tyagi M. Karn J. EMBO J. 2007; 26: 4985-4995Crossref PubMed Scopus (185) Google Scholar) have been shown to mediate HDAC1 recruitment to the LTR and, consequently, inhibit transcription from the viral promoter. We also reported that activator protein 4 acts as a transcriptional repressor by recruiting HDAC molecules and is involved in the maintenance of viral latency (18Imai K. Okamoto T. J. Biol. Chem. 2006; 281: 12495-12505Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Others observed that drugs that inhibit HDAC activity such as trichostatin A, butyric acid, and valproic acid, could effectively induce HIV transcription in latently infected cells (12Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (480) Google Scholar, 13Sheridan P.L. Mayall T.P. Verdin E. Jones K.A. Genes Dev. 1997; 11: 3327-3340Crossref PubMed Scopus (163) Google Scholar, 19Lehrman G. Hogue I.B. Palmer S. Jennings C. Spina C.A. Wiegand A. Landay A.L. Coombs R.W. Richman D.D. Mellors J.W. Coffin J.M. Bosch R.J. Margolis D.M. Lancet. 2005; 366: 549-555Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 20Imai K. Ochiai K. Okamoto T. J. Immunol. 2009; 182: 3688-3695Crossref PubMed Scopus (96) Google Scholar). These studies suggest that epigenetic silencing is involved in the maintenance of HIV-1 transcriptional latency.In addition to histone acetylation, histone Lys methylation also plays an epigenetic role in the organization of chromatin domains and the regulation of gene expression (6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (530) Google Scholar, 21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). Methylation of histones at Lys and Arg residues play both positive and negative roles in transcriptional regulation. For example, methylation of at Lys4 and Arg17 of histone H3 is generally associated with active genes, whereas methylation of H3 at Lys9 (H3K9me) and Lys27 (H3K27me) has been associated with inactive genes (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). It was also noted that H3K9me exhibited more definitive transcriptional repression over H3K27me (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). The repressive methylation of H3K9 has been detected at the promoter regions of many silenced genes, together with increased DNA methylation and reduced histone acetylation (23Baylin S.B. Ohm J.E. Nat. Rev. Cancer. 2006; 6: 107-116Crossref PubMed Scopus (1379) Google Scholar, 24Yoo C.B. Jones P.A. Nat. Rev. Drug Discov. 2006; 5: 37-50Crossref PubMed Scopus (1113) Google Scholar, 25Feldman N. Gerson A. Fang J. Li E. Zhang Y. Shinkai Y. Cedar H. Bergman Y. Nat. Cell Biol. 2006; 8: 188-194Crossref PubMed Scopus (512) Google Scholar, 26Epsztejn-Litman S. Feldman N. Abu-Remaileh M. Shufaro Y. Gerson A. Ueda J. Deplus R. Fuks F. Shinkai Y. Cedar H. Bergman Y. Nat. Struct. Mol. Biol. 2008; 15: 1176-1183Crossref PubMed Scopus (344) Google Scholar, 27Dong K.B. Maksakova I.A. Mohn F. Leung D. Appanah R. Lee S. Yang H.W. Lam L.L. Mager D.L. Schübeler D. Tachibana M. Shinkai Y. Lorincz M.C. EMBO J. 2008; 27: 2691-2701Crossref PubMed Scopus (191) Google Scholar). Two recent reports (28Marban C. Suzanne S. Dequiedt F. de Walque S. Redel L. Van Lint C. Aunis D. Rohr O. EMBO J. 2007; 26: 412-423Crossref PubMed Scopus (283) Google Scholar, 29du Chéné I. Basyuk E. Lin Y.L. Triboulet R. Knezevich A. Chable-Bessia C. Mettling C. Baillat V. Reynes J. Corbeau P. Bertrand E. Marcello A. Emiliani S. Kiernan R. Benkirane M. EMBO J. 2007; 26: 424-435Crossref PubMed Scopus (248) Google Scholar) demonstrated that the histone methyltransferase (HMT) Suppressor of variegation 3–9 Homolog (Suv39H) 1, which is primarily involved in Lys9 trimethylation of histone H3 (H3K9me3), is responsible for HIV-1 transcriptional silencing. Although H3K9 methylation is known to play a crucial role in chromatin-mediated transcriptional silencing, the molecular mechanism of H3K9 methylation and another HMT on HIV-1 gene repression has yet to be clarified.In addition to Suv39H1, there are at least four other mammalian H3K9 HMTs, including Suv39H2, a close relative of Suv39H1, G9a, G9a-like protein (GLP)/EuHMTase1, and SETDB1/ERG-associated protein with SET domain (ESET) (21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (951) Google Scholar, 32Rea 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 (2152) Google Scholar, 33Shi Y. Sawada J. Sui G. el Affar B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (630) Google Scholar, 34Schultz D.C. Ayyanathan K. Negorev D. Maul G.G. Rauscher 3rd, F.J. Genes Dev. 2002; 16: 919-932Crossref PubMed Scopus (868) Google Scholar). These proteins commonly contain a SET (Suv39, enhancer of zeste, trithorax) domain that is responsible for catalytic action (6Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (530) Google Scholar, 21Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 22Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (329) Google Scholar). Among these HMTs, G9a is a key enzyme responsible for H3K9 dimethylation (H3K9me2) in mammals as disruption of the G9a gene resulted in a drastic decrease in H3K9 methylation primarily in the silenced region within euchromatin (30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (951) Google Scholar, 35Rice J.C. Briggs S.D. Ueberheide B. Barber C.M. Shabanowitz J. Hunt D.F. Shinkai Y. Allis C.D. Mol. Cell. 2003; 12: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 36Tachibana 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 (594) Google Scholar). Thus, G9a has been implicated in silencing the gene expression (25Feldman N. Gerson A. Fang J. Li E. Zhang Y. Shinkai Y. Cedar H. Bergman Y. Nat. Cell Biol. 2006; 8: 188-194Crossref PubMed Scopus (512) Google Scholar, 26Epsztejn-Litman S. Feldman N. Abu-Remaileh M. Shufaro Y. Gerson A. Ueda J. Deplus R. Fuks F. Shinkai Y. Cedar H. Bergman Y. Nat. Struct. Mol. Biol. 2008; 15: 1176-1183Crossref PubMed Scopus (344) Google Scholar, 27Dong K.B. Maksakova I.A. Mohn F. Leung D. Appanah R. Lee S. Yang H.W. Lam L.L. Mager D.L. Schübeler D. Tachibana M. Shinkai Y. Lorincz M.C. EMBO J. 2008; 27: 2691-2701Crossref PubMed Scopus (191) Google Scholar). Interestingly, in vitro experiments revealed that G9a exhibited a 10–20-fold stronger HMT activity toward H3K9 compared with Suv39H1 (30Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar).In this study we investigated the role of G9a in HIV-1 gene expression. We demonstrate that G9a is responsible for the maintenance of chromatin-mediated HIV-1 silencing through histone modification of H3K9me2. Biological and therapeutic implications are discussed.