Oligomerization and Binding of the Dnmt3a DNA Methyltransferase to Parallel DNA Molecules

Abstract

Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel. Because Dnmt3L does not have an RD interface, it prevents Dnmt3a oligomerization and binding of more than one DNA molecule. Both interfaces of Dnmt3a are necessary for the heterochromatic localization of the enzyme in cells. Overexpression of Dnmt3L in cells leads to the release of Dnmt3a from heterochromatic regions, which may increase its activity for methylation of euchromatic targets like the differentially methylated regions involved in imprinting. Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel. Because Dnmt3L does not have an RD interface, it prevents Dnmt3a oligomerization and binding of more than one DNA molecule. Both interfaces of Dnmt3a are necessary for the heterochromatic localization of the enzyme in cells. Overexpression of Dnmt3L in cells leads to the release of Dnmt3a from heterochromatic regions, which may increase its activity for methylation of euchromatic targets like the differentially methylated regions involved in imprinting. IntroductionThe coordinated gene expression during development and cell differentiation is regulated by epigenetic signals comprising DNA methylation, post-translational modification of histone tails, and non-coding RNAs (1Allis C.D. Jenuwein T. Reinberg D. Epigenetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2007Google Scholar). Among them, DNA methylation is a key epigenetic process involved in the control of gene activity (2Miranda T.B. Jones P.A. J. Cell. Physiol. 2007; 213: 384-390Crossref PubMed Scopus (501) Google Scholar, 3Lande-Diner L. Zhang J. Ben-Porath I. Amariglio N. Keshet I. Hecht M. Azuara V. Fisher A.G. Rechavi G. Cedar H. J. Biol. Chem. 2007; 282: 12194-12200Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), parental imprinting (4Delaval K. Feil R. Curr. Opin. Genet. Dev. 2004; 14: 188-195Crossref PubMed Scopus (315) Google Scholar, 5Hore T.A. Rapkins R.W. Graves J.A. Trends Genet. 2007; 23: 440-448Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 6Sha K. Annu Rev. Genomics Hum. Genet. 2008; 9: 197-216Crossref PubMed Scopus (67) Google Scholar), X-chromosome inactivation (7Heard E. Curr. Opin. Cell Biol. 2004; 16: 247-255Crossref PubMed Scopus (224) Google Scholar, 8Chang S.C. Tucker T. Thorogood N.P. Brown C.J. Front Biosci. 2006; 11: 852-866Crossref PubMed Scopus (81) Google Scholar, 9Yen Z.C. Meyer I.M. Karalic S. Brown C.J. Genomics. 2007; 90: 453-463Crossref PubMed Scopus (64) Google Scholar, 10Straub T. Becker P.B. Nat. Rev. Genet. 2007; 8: 47-57Crossref PubMed Scopus (164) Google Scholar), and maintenance of the genome integrity and stability through protection against endogenous retroviruses and transposons (11Howard G. Eiges R. Gaudet F. Jaenisch R. Eden A. Oncogene. 2008; 27: 404-408Crossref PubMed Scopus (264) Google Scholar). Aberrant DNA methylation patterns are associated with several human diseases, including cancer (12Jones P.A. Baylin S.B. Cell. 2007; 128: 683-692Abstract Full Text Full Text PDF PubMed Scopus (3601) Google Scholar, 13Egger G. Liang G. Aparicio A. Jones P.A. Nature. 2004; 429: 457-463Crossref PubMed Scopus (2474) Google Scholar, 14Feinberg A.P. Tycko B. Nat. Rev. Cancer. 2004; 4: 143-153Crossref PubMed Scopus (1774) Google Scholar, 15Rodenhiser D. Mann M. Cmaj. 2006; 174: 341-348Crossref PubMed Scopus (325) Google Scholar, 16Robertson K.D. Nat. Rev. Genet. 2005; 6: 597-610Crossref PubMed Scopus (2043) Google Scholar, 17Esteller M. Nat. Rev. Genet. 2007; 8: 286-298Crossref PubMed Scopus (1684) Google Scholar). In mammals, DNA methylation patterns are established during embryogenesis by the action of the de novo DNA methyltransferases Dnmt3a and Dnmt3b, together with their regulatory factor, Dnmt3L (18Jurkowska R. Jurkowski T.P. Jeltsch A. Chembiochem. 2011; 12: 206-222Crossref PubMed Scopus (503) Google Scholar, 19Klose R.J. Bird A.P. Trends Biochem. Sci. 2006; 31: 89-97Abstract Full Text Full Text PDF PubMed Scopus (1880) Google Scholar). In addition, the Dnmt3a and Dnmt3b enzymes have a role in the preservation of DNA methylation at heterochromatin regions (20Dodge J.E. Okano M. Dick F. Tsujimoto N. Chen T. Wang S. Ueda Y. Dyson N. Li E. J. Biol. Chem. 2005; 280: 17986-17991Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 21Jeong S. Liang G. Sharma S. Lin J.C. Choi S.H. Han H. Yoo C.B. Egger G. Yang A.S. Jones P.A. Mol. Cell. Biol. 2009; 29: 5366-5376Crossref PubMed Scopus (149) Google Scholar). Disruption of the Dnmt3a or Dnmt3b gene in mice is lethal (22Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4408) Google Scholar), whereas Dnmt3L knock-out mice are viable and do not show discernable morphological abnormalities (23Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar, 24Bourc'his D. Bestor T.H. Nature. 2004; 431: 96-99Crossref PubMed Scopus (876) Google Scholar, 25Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar), indicating that Dnmt3a and Dnmt3b are functional in the absence of Dnmt3L.Dnmt3a and Dnmt3b are nuclear proteins, which stably associate with chromatin containing methylated DNA (21Jeong S. Liang G. Sharma S. Lin J.C. Choi S.H. Han H. Yoo C.B. Egger G. Yang A.S. Jones P.A. Mol. Cell. Biol. 2009; 29: 5366-5376Crossref PubMed Scopus (149) Google Scholar), including mitotic chromosomes, and localize to pericentromeric heterochromatin (26Bachman K.E. Rountree M.R. Baylin S.B. J. Biol. Chem. 2001; 276: 32282-32287Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 27Chen T. Tsujimoto N. Li E. Mol. Cell. Biol. 2004; 24: 9048-9058Crossref PubMed Scopus (196) Google Scholar, 28Ge Y.Z. Pu M.T. Gowher H. Wu H.P. Ding J.P. Jeltsch A. Xu G.L. J. Biol. Chem. 2004; 279: 25447-25454Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Dnmt3 proteins comprise two parts: a large multidomain N-terminal part of variable size, which has regulatory and targeting functions and a C-terminal catalytic part. The N-terminal part of Dnmt3a and Dnmt3b contains two defined domains: a cysteine-rich ADD (ATRX–DNMT3–DNMT3L) domain, which binds to H3 tails unmodified at K4 (29Otani J. Nankumo T. Arita K. Inamoto S. Ariyoshi M. Shirakawa M. EMBO Rep. 2009; 10: 1235-1241Crossref PubMed Scopus (275) Google Scholar, 30Zhang Y. Jurkowska R. Soeroes S. Rajavelu A. Dhayalan A. Bock I. Rathert P. Brandt O. Reinhardt R. Fischle W. Jeltsch A. Nucleic Acids Res. 2010; 38: 4246-4253Crossref PubMed Scopus (262) Google Scholar) and a PWWP domain, which binds to H3 trimethylated at K36 (31Dhayalan A. Rajavelu A. Rathert P. Tamas R. Jurkowska R.Z. Ragozin S. Jeltsch A. J. Biol. Chem. 2010; 285: 26114-26120Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar) and is essential for the heterochromatin localization of the enzymes (27Chen T. Tsujimoto N. Li E. Mol. Cell. Biol. 2004; 24: 9048-9058Crossref PubMed Scopus (196) Google Scholar, 28Ge Y.Z. Pu M.T. Gowher H. Wu H.P. Ding J.P. Jeltsch A. Xu G.L. J. Biol. Chem. 2004; 279: 25447-25454Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31Dhayalan A. Rajavelu A. Rathert P. Tamas R. Jurkowska R.Z. Ragozin S. Jeltsch A. J. Biol. Chem. 2010; 285: 26114-26120Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). The C-terminal parts of Dnmt3 enzymes resemble prokaryotic DNA-(cytosine C5)-methyltransferases and harbor the active centers of the enzymes and are active in an isolated form (32Gowher H. Jeltsch A. J. Biol. Chem. 2002; 277: 20409-20414Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Dnmt3L is catalytically inactive, directly interacts with the catalytic domains of Dnmt3a and Dnmt3b and stimulates their activity both in vivo and in vitro (25Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 33Chedin F. Lieber M.R. Hsieh C.L. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16916-16921Crossref PubMed Scopus (375) Google Scholar, 34Chen Z.X. Mann J.R. Hsieh C.L. Riggs A.D. Chédin F. J. Cell. Biochem. 2005; 95: 902-917Crossref PubMed Scopus (168) Google Scholar, 35Suetake I. Shinozaki F. Miyagawa J. Takeshima H. Tajima S. J. Biol. Chem. 2004; 279: 27816-27823Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 36Gowher H. Liebert K. Hermann A. Xu G. Jeltsch A. J. Biol. Chem. 2005; 280: 13341-13348Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 37Kareta M.S. Botello Z.M. Ennis J.J. Chou C. Chédin F. J. Biol. Chem. 2006; 281: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar).The structure of a complex consisting of the C-terminal parts of Dnmt3a and Dnmt3L (Dnmt3a-C/Dnmt3L-C) revealed a linear heterotetramer, in which the two Dnmt3L subunits are positioned at the edges and two Dnmt3a molecules in the center (Fig. 1A) (38Jia D. Jurkowska R.Z. Zhang X. Jeltsch A. Cheng X. Nature. 2007; 449: 248-251Crossref PubMed Scopus (606) Google Scholar, 39Cheng X. Blumenthal R.M. Structure. 2008; 16: 341-350Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). The Dnmt3a C-terminal domain provides two interfaces for protein-protein contacts: one hydrophobic FF interface at the Dnmt3a-C/3L-C contact formed by the stacking interaction of four phenylalanine residues (two from each subunit) and one polar RD interface at the Dnmt3a-C/3a-C contact mediated by a hydrogen bonding network between arginine and aspartate residues. The RD interface forms the DNA binding site, while both interfaces are essential for cofactor S-adenosyl-l-methionine binding and catalytic activity of the enzyme (38Jia D. Jurkowska R.Z. Zhang X. Jeltsch A. Cheng X. Nature. 2007; 449: 248-251Crossref PubMed Scopus (606) Google Scholar, 40Jurkowska R.Z. Anspach N. Urbanke C. Jia D. Reinhardt R. Nellen W. Cheng X. Jeltsch A. Nucleic Acids Res. 2008; 36: 6656-6663Crossref PubMed Scopus (93) Google Scholar). We demonstrate here that Dnmt3a-C oligomerizes in a reversible manner. Initial dimer formation occurs through the FF interface, further oligomerization via the RD interface of the FF dimers. Because each RD interface constitutes an independent DNA binding site, Dnmt3a-C oligomers can bind to two or more DNA molecules, oriented next to each other, roughly in parallel. Dnmt3L prevents this oligomerization, because it lacks the RD interface, and it therefore precludes binding of Dnmt3a to more than one DNA molecule. The formation of Dnmt3a oligomers is required for the localization of the enzyme to heterochromatin, and binding of Dnmt3L changes the subnuclear localization of Dnmt3a, which could be one mechanism describing how Dnmt3L stimulates the methylation of imprinted loci.DISCUSSIONThe Dnmt3a C-terminal domain employs two interfaces for protein/protein contacts in the structure of the Dnmt3a/Dnmt3L heterotetrameric complex: one FF interface and one RD interface. We show here that the FF interface also supports self-interaction of Dnmt3a-C and that Dnmt3a-C dimers can form protein oligomers using their RD interfaces. Since each RD interface constitutes a potential DNA binding site, oligomerization of Dnmt3a creates several DNA binding sites. Our modeling of a Dnmt3a oligomer predicted binding to several DNA molecules oriented next to each other roughly in parallel (Fig. 1 and supplemental Fig. S2B). We document this unusual mode of DNA binding to Dnmt3a-C oligomers by binding experiments performed in solution (FRET) and imaging (SFM). Binding of two DNA molecules in parallel orientation so far has been observed only with few proteins (one noticeable example being RecA (50Bell C.E. Mol. Microbiol. 2005; 58: 358-366Crossref PubMed Scopus (79) Google Scholar)) and such DNA binding mode was not anticipated for Dnmt3a.Dnmt3a has been shown to bind tightly to chromatin (21Jeong S. Liang G. Sharma S. Lin J.C. Choi S.H. Han H. Yoo C.B. Egger G. Yang A.S. Jones P.A. Mol. Cell. Biol. 2009; 29: 5366-5376Crossref PubMed Scopus (149) Google Scholar). It is engaged in two interactions with the H3 tail, mediated by its ADD domain binding to the end of the H3 tail unmodified at K4 (29Otani J. Nankumo T. Arita K. Inamoto S. Ariyoshi M. Shirakawa M. EMBO Rep. 2009; 10: 1235-1241Crossref PubMed Scopus (275) Google Scholar, 30Zhang Y. Jurkowska R. Soeroes S. Rajavelu A. Dhayalan A. Bock I. Rathert P. Brandt O. Reinhardt R. Fischle W. Jeltsch A. Nucleic Acids Res. 2010; 38: 4246-4253Crossref PubMed Scopus (262) Google Scholar), and by its PWWP domain binding to H3K36me3, which was found to be essential for heterochromatic localization (31Dhayalan A. Rajavelu A. Rathert P. Tamas R. Jurkowska R.Z. Ragozin S. Jeltsch A. J. Biol. Chem. 2010; 285: 26114-26120Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). In addition, Dnmt3a polymerizes on the DNA (38Jia D. Jurkowska R.Z. Zhang X. Jeltsch A. Cheng X. Nature. 2007; 449: 248-251Crossref PubMed Scopus (606) Google Scholar, 40Jurkowska R.Z. Anspach N. Urbanke C. Jia D. Reinhardt R. Nellen W. Cheng X. Jeltsch A. Nucleic Acids Res. 2008; 36: 6656-6663Crossref PubMed Scopus (93) Google Scholar) and we show here that Dnmt3a oligomers bind to several DNA molecules oriented in parallel. Since heterochromatin is defined as a region of very high DNA density, the ability of Dnmt3a oligomers to bind to parallel DNA close to each other may contributes to the targeting of the enzyme to such DNA dense regions. This hypothesis is supported by our finding that heterochromatic localization of Dnmt3a was lost with non-oligomerizing Dnmt3a mutants affected at the FF interface and in the presence of Dnmt3L, which disrupts oligomerization and multiple DNA binding because it lacks an RD interface. The structure of heterochromatic DNA is not known. However, at least one model of the 30 nm chromatin fiber, the helical ribbon model (51Dorigo B. Schalch T. Kulangara A. S. Science. 2004; PubMed Scopus Google Scholar), an parallel of the DNA regions in a that to the model of the Dnmt3a oligomers binding to parallel DNA the unusual DNA binding mode of Dnmt3a could a mechanism of heterochromatic have shown that Dnmt3L disrupts the reversible oligomerization of Dnmt3a-C and it with the binding to several DNA molecules to We the of Dnmt3L on the subnuclear localization of more than of the cells lost the heterochromatic localization of Dnmt3a in the presence of Dnmt3L, not Dnmt3a was with the Dnmt3L which lost its ability to with This a mode of action of Dnmt3L in DNA methylation In to the activity of Dnmt3a, it be involved in the release of Dnmt3a from dense heterochromatin to it to at imprinted differentially methylated regions, which are in The expression of Dnmt3L in cells may Dnmt3a from heterochromatin to increase Dnmt3a and DNA methylation activity for the of DNA methylation IntroductionThe coordinated gene expression during development and cell differentiation is regulated by epigenetic signals comprising DNA methylation, post-translational modification of histone tails, and non-coding RNAs (1Allis C.D. Jenuwein T. Reinberg D. Epigenetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2007Google Scholar). Among them, DNA methylation is a key epigenetic process involved in the control of gene activity (2Miranda T.B. Jones P.A. J. Cell. Physiol. 2007; 213: 384-390Crossref PubMed Scopus (501) Google Scholar, 3Lande-Diner L. Zhang J. Ben-Porath I. Amariglio N. Keshet I. Hecht M. Azuara V. Fisher A.G. Rechavi G. Cedar H. J. Biol. Chem. 2007; 282: 12194-12200Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), parental imprinting (4Delaval K. Feil R. Curr. Opin. Genet. Dev. 2004; 14: 188-195Crossref PubMed Scopus (315) Google Scholar, 5Hore T.A. Rapkins R.W. Graves J.A. Trends Genet. 2007; 23: 440-448Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 6Sha K. Annu Rev. Genomics Hum. Genet. 2008; 9: 197-216Crossref PubMed Scopus (67) Google Scholar), X-chromosome inactivation (7Heard E. Curr. Opin. Cell Biol. 2004; 16: 247-255Crossref PubMed Scopus (224) Google Scholar, 8Chang S.C. Tucker T. Thorogood N.P. Brown C.J. Front Biosci. 2006; 11: 852-866Crossref PubMed Scopus (81) Google Scholar, 9Yen Z.C. Meyer I.M. Karalic S. Brown C.J. Genomics. 2007; 90: 453-463Crossref PubMed Scopus (64) Google Scholar, 10Straub T. Becker P.B. Nat. Rev. Genet. 2007; 8: 47-57Crossref PubMed Scopus (164) Google Scholar), and maintenance of the genome integrity and stability through protection against endogenous retroviruses and transposons (11Howard G. Eiges R. Gaudet F. Jaenisch R. Eden A. Oncogene. 2008; 27: 404-408Crossref PubMed Scopus (264) Google Scholar). Aberrant DNA methylation patterns are associated with several human diseases, including cancer (12Jones P.A. Baylin S.B. Cell. 2007; 128: 683-692Abstract Full Text Full Text PDF PubMed Scopus (3601) Google Scholar, 13Egger G. Liang G. Aparicio A. Jones P.A. Nature. 2004; 429: 457-463Crossref PubMed Scopus (2474) Google Scholar, 14Feinberg A.P. Tycko B. Nat. Rev. Cancer. 2004; 4: 143-153Crossref PubMed Scopus (1774) Google Scholar, 15Rodenhiser D. Mann M. Cmaj. 2006; 174: 341-348Crossref PubMed Scopus (325) Google Scholar, 16Robertson K.D. Nat. Rev. Genet. 2005; 6: 597-610Crossref PubMed Scopus (2043) Google Scholar, 17Esteller M. Nat. Rev. Genet. 2007; 8: 286-298Crossref PubMed Scopus (1684) Google Scholar). In mammals, DNA methylation patterns are established during embryogenesis by the action of the de novo DNA methyltransferases Dnmt3a and Dnmt3b, together with their regulatory factor, Dnmt3L (18Jurkowska R. Jurkowski T.P. Jeltsch A. Chembiochem. 2011; 12: 206-222Crossref PubMed Scopus (503) Google Scholar, 19Klose R.J. Bird A.P. Trends Biochem. Sci. 2006; 31: 89-97Abstract Full Text Full Text PDF PubMed Scopus (1880) Google Scholar). In addition, the Dnmt3a and Dnmt3b enzymes have a role in the preservation of DNA methylation at heterochromatin regions (20Dodge J.E. Okano M. Dick F. Tsujimoto N. Chen T. Wang S. Ueda Y. Dyson N. Li E. J. Biol. Chem. 2005; 280: 17986-17991Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 21Jeong S. Liang G. Sharma S. Lin J.C. Choi S.H. Han H. Yoo C.B. Egger G. Yang A.S. Jones P.A. Mol. Cell. Biol. 2009; 29: 5366-5376Crossref PubMed Scopus (149) Google Scholar). Disruption of the Dnmt3a or Dnmt3b gene in mice is lethal (22Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4408) Google Scholar), whereas Dnmt3L knock-out mice are viable and do not show discernable morphological abnormalities (23Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar, 24Bourc'his D. Bestor T.H. Nature. 2004; 431: 96-99Crossref PubMed Scopus (876) Google Scholar, 25Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar), indicating that Dnmt3a and Dnmt3b are functional in the absence of Dnmt3L.Dnmt3a and Dnmt3b are nuclear proteins, which stably associate with chromatin containing methylated DNA (21Jeong S. Liang G. Sharma S. Lin J.C. Choi S.H. Han H. Yoo C.B. Egger G. Yang A.S. Jones P.A. Mol. Cell. Biol. 2009; 29: 5366-5376Crossref PubMed Scopus (149) Google Scholar), including mitotic chromosomes, and localize to pericentromeric heterochromatin (26Bachman K.E. Rountree M.R. Baylin S.B. J. Biol. Chem. 2001; 276: 32282-32287Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 27Chen T. Tsujimoto N. Li E. Mol. Cell. Biol. 2004; 24: 9048-9058Crossref PubMed Scopus (196) Google Scholar, 28Ge Y.Z. Pu M.T. Gowher H. Wu H.P. Ding J.P. Jeltsch A. Xu G.L. J. Biol. Chem. 2004; 279: 25447-25454Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Dnmt3 proteins comprise two parts: a large multidomain N-terminal part of variable size, which has regulatory and targeting functions and a C-terminal catalytic part. The N-terminal part of Dnmt3a and Dnmt3b contains two defined domains: a cysteine-rich ADD (ATRX–DNMT3–DNMT3L) domain, which binds to H3 tails unmodified at K4 (29Otani J. Nankumo T. Arita K. Inamoto S. Ariyoshi M. Shirakawa M. EMBO Rep. 2009; 10: 1235-1241Crossref PubMed Scopus (275) Google Scholar, 30Zhang Y. Jurkowska R. Soeroes S. Rajavelu A. Dhayalan A. Bock I. Rathert P. Brandt O. Reinhardt R. Fischle W. Jeltsch A. Nucleic Acids Res. 2010; 38: 4246-4253Crossref PubMed Scopus (262) Google Scholar) and a PWWP domain, which binds to H3 trimethylated at K36 (31Dhayalan A. Rajavelu A. Rathert P. Tamas R. Jurkowska R.Z. Ragozin S. Jeltsch A. J. Biol. Chem. 2010; 285: 26114-26120Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar) and is essential for the heterochromatin localization of the enzymes (27Chen T. Tsujimoto N. Li E. Mol. Cell. Biol. 2004; 24: 9048-9058Crossref PubMed Scopus (196) Google Scholar, 28Ge Y.Z. Pu M.T. Gowher H. Wu H.P. Ding J.P. Jeltsch A. Xu G.L. J. Biol. Chem. 2004; 279: 25447-25454Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31Dhayalan A. Rajavelu A. Rathert P. Tamas R. Jurkowska R.Z. Ragozin S. Jeltsch A. J. Biol. Chem. 2010; 285: 26114-26120Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). The C-terminal parts of Dnmt3 enzymes resemble prokaryotic DNA-(cytosine C5)-methyltransferases and harbor the active centers of the enzymes and are active in an isolated form (32Gowher H. Jeltsch A. J. Biol. Chem. 2002; 277: 20409-20414Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Dnmt3L is catalytically inactive, directly interacts with the catalytic domains of Dnmt3a and Dnmt3b and stimulates their activity both in vivo and in vitro (25Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 33Chedin F. Lieber M.R. Hsieh C.L. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16916-16921Crossref PubMed Scopus (375) Google Scholar, 34Chen Z.X. Mann J.R. Hsieh C.L. Riggs A.D. Chédin F. J. Cell. Biochem. 2005; 95: 902-917Crossref PubMed Scopus (168) Google Scholar, 35Suetake I. Shinozaki F. Miyagawa J. Takeshima H. Tajima S. J. Biol. Chem. 2004; 279: 27816-27823Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 36Gowher H. Liebert K. Hermann A. Xu G. Jeltsch A. J. Biol. Chem. 2005; 280: 13341-13348Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 37Kareta M.S. Botello Z.M. Ennis J.J. Chou C. Chédin F. J. Biol. Chem. 2006; 281: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar).The structure of a complex consisting of the C-terminal parts of Dnmt3a and Dnmt3L (Dnmt3a-C/Dnmt3L-C) revealed a linear heterotetramer, in which the two Dnmt3L subunits are positioned at the edges and two Dnmt3a molecules in the center (Fig. 1A) (38Jia D. Jurkowska R.Z. Zhang X. Jeltsch A. Cheng X. Nature. 2007; 449: 248-251Crossref PubMed Scopus (606) Google Scholar, 39Cheng X. Blumenthal R.M. Structure. 2008; 16: 341-350Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). The Dnmt3a C-terminal domain provides two interfaces for protein-protein contacts: one hydrophobic FF interface at the Dnmt3a-C/3L-C contact formed by the stacking interaction of four phenylalanine residues (two from each subunit) and one polar RD interface at the Dnmt3a-C/3a-C contact mediated by a hydrogen bonding network between arginine and aspartate residues. The RD interface forms the DNA binding site, while both interfaces are essential for cofactor S-adenosyl-l-methionine binding and catalytic activity of the enzyme (38Jia D. Jurkowska R.Z. Zhang X. Jeltsch A. Cheng X. Nature. 2007; 449: 248-251Crossref PubMed Scopus (606) Google Scholar, 40Jurkowska R.Z. Anspach N. Urbanke C. Jia D. Reinhardt R. Nellen W. Cheng X. Jeltsch A. Nucleic Acids Res. 2008; 36: 6656-6663Crossref PubMed Scopus (93) Google Scholar). We demonstrate here that Dnmt3a-C oligomerizes in a reversible manner. Initial dimer formation occurs through the FF interface, further oligomerization via the RD interface of the FF dimers. Because each RD interface constitutes an independent DNA binding site, Dnmt3a-C oligomers can bind to two or more DNA molecules, oriented next to each other, roughly in parallel. Dnmt3L prevents this oligomerization, because it lacks the RD interface, and it therefore precludes binding of Dnmt3a to more than one DNA molecule. The formation of Dnmt3a oligomers is required for the localization of the enzyme to heterochromatin, and binding of Dnmt3L changes the subnuclear localization of Dnmt3a, which could be one mechanism describing how Dnmt3L stimulates the methylation of imprinted