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• W1966092430 abstract "During mammalian cell division, DNA methylation patterns are transferred accurately to the newly synthesized DNA strand. This depends on maintenance DNA methyltransferase activity. DNA methylation can affect chromatin organization and gene expression by recruitment of histone deacetylases (HDACs). Here we show that the methyl-CpG binding protein, MeCP2, interacts directly with the maintenance DNA methyltransferase, Dnmt1. The region of MeCP2 that interacts with Dnmt1 corresponds to the transcription repressor domain which can also recruit HDACs via a corepressor, mSin3A. Dnmt1 can form complexes with HDACs as well as MeCP2. Surprisingly, the MeCP2-Dnmt1 complex does not contain the histone deacetylase, HDAC1. Thus, Dnmt1 takes the place of the mSin3A-HDAC1 complex, indicating that the MeCP2-interacting Dnmt1 does not bind to HDAC1. Further, we demonstrate that MeCP2 can form a complex with hemimethylated as well as fully methylated DNA. Immunoprecipitated MeCP2 complexes show DNA methyltransferase activity to hemimethylated DNA. These results suggest that Dnmt1 associates with MeCP2 in order to perform maintenance methylation in vivo. We propose that genome-wide and/or -specific local DNA methylation may be maintained by the Dnmt1-MeCP2 complexes, bound to hemimethylated DNA. Dnmt1 may be recruited to targeted regions via multiple steps that may or may not involve histone deacetylases. During mammalian cell division, DNA methylation patterns are transferred accurately to the newly synthesized DNA strand. This depends on maintenance DNA methyltransferase activity. DNA methylation can affect chromatin organization and gene expression by recruitment of histone deacetylases (HDACs). Here we show that the methyl-CpG binding protein, MeCP2, interacts directly with the maintenance DNA methyltransferase, Dnmt1. The region of MeCP2 that interacts with Dnmt1 corresponds to the transcription repressor domain which can also recruit HDACs via a corepressor, mSin3A. Dnmt1 can form complexes with HDACs as well as MeCP2. Surprisingly, the MeCP2-Dnmt1 complex does not contain the histone deacetylase, HDAC1. Thus, Dnmt1 takes the place of the mSin3A-HDAC1 complex, indicating that the MeCP2-interacting Dnmt1 does not bind to HDAC1. Further, we demonstrate that MeCP2 can form a complex with hemimethylated as well as fully methylated DNA. Immunoprecipitated MeCP2 complexes show DNA methyltransferase activity to hemimethylated DNA. These results suggest that Dnmt1 associates with MeCP2 in order to perform maintenance methylation in vivo. We propose that genome-wide and/or -specific local DNA methylation may be maintained by the Dnmt1-MeCP2 complexes, bound to hemimethylated DNA. Dnmt1 may be recruited to targeted regions via multiple steps that may or may not involve histone deacetylases. DNA methylation occurs at the fifth carbon position of cytosine in CpG dinucleotide sequences. In mammalian cells, 60–80% of CpG dinucleotides are methylated (1Razin A. Szyf M. Biochim. Biophys. Acta. 1984; 782: 331-342Crossref PubMed Scopus (258) Google Scholar). These modifications are considered to be important for development (2Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar, 3Shiota K. Yanagimachi R. Differentiation. 2002; 69: 162-166Crossref PubMed Scopus (61) Google Scholar), genomic imprinting (4Tucker K.L. Beard C. Dausmann J. Jackson-Grusby L. Laird P.W. Lei H. Li E. Jaenisch R. Genes Dev. 1996; 10: 1008-1020Crossref PubMed Scopus (239) Google Scholar), and X chromosome inactivation through gene silencing (5Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar, 6Li E. Nat. Rev. Genet. 2002; 3: 662-673Crossref PubMed Scopus (1584) Google Scholar). Aberrant DNA methylation has been observed in cancer cells (7Baylin S.B. Herman J.G. Trends Genet. 2000; 16: 168-174Abstract Full Text Full Text PDF PubMed Scopus (1408) Google Scholar). The occurrence of DNA methylation is closely connected with chromatin organization by both histone acetylation and methylation, which are catalyzed by histone acetyltransferases (HDACs) 1The abbreviations used are: HDAC, histone deacetylase; CRID, co-repressor-interacting domain; TRD, transcription repressor domain; NLS, nuclear localization sequence; RFTS, replication foci targeting sequence; MBD, methyl-CpG binding domain; TBS, Tris-buffered saline; GST, glutathione S-transferase; PCNA, proliferating cell nuclear antigen and methyltransferases, respectively (8Burgers W.A. Fuks F. Kouzarides T. Trends Genet. 2002; 18: 275-277Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). A maintenance DNA methyltransferase, Dnmt1, preferentially methylates hemimethylated DNA after DNA replication (9Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 10Bestor T.H. Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1611) Google Scholar). Therefore, the patterns of DNA methylation in the genome are maintained by the maintenance DNA methyltransferase activity of Dnmt1, allowing inheritance of appropriate DNA methylation patterns. Targeted disruption of Dnmt1 causes partial loss of genomic methylation that leads to lethality (2Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar). The carboxyl-terminal region of Dnmt1 transfers methyl groups fromS-adenosyl-l-methionine to cytosines in CpG dinucleotides. It is thought that an N-terminal region of Dnmt1 regulates Dnmt1 function by protein-protein interactions with factors such as PCNA (11Chuang L.S. Ian H.I. Koh T.W. Ng H.H. Xu G. Li B.F. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar), DMAP1 (12Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (867) Google Scholar), HDAC1/2 (12Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (867) Google Scholar, 13Fuks F. Burgers W.A. Brehm A. Hughes-Davies L. Kouzarides T. Nat. Genet. 2000; 24: 88-91Crossref PubMed Scopus (823) Google Scholar), and Rb (14Robertson K.D. Ait-Si-Ali S. Yokochi T. Wade P.A. Jones P.L. Wolffe A.P. Nat. Genet. 2000; 25: 338-342Crossref PubMed Scopus (809) Google Scholar) (15Pradhan S. Kim G.D. EMBO J. 2002; 21: 779-788Crossref PubMed Scopus (93) Google Scholar), which are associated with DNA replication, histone deacetylation, and gene expression during the cell cycle. Methyl-CpG binding proteins (MBDs) bind DNA, including methylated CpG dinucleotides. MBDs are involved in chromatin organization and gene silencing via recruitment of HDACs (16Wade P.A. Bioessays. 2001; 23: 1131-1137Crossref PubMed Scopus (290) Google Scholar). Individual MBDs have specific methylated DNA binding properties and interact with transcriptional repressors and chromatin remodeling factors (5Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar). MeCP2, the first cloned Methyl-CpG binding protein, binds to single CpG dinucleotides that are symmetrically methylated (17Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1091) Google Scholar), whereas other MBDs have little or no binding activity to single symmetrically methylated CpG dinucleotides (18Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar). Thus, MeCP2 possesses higher affinity for all or most of the methylated CpG sites than other known MBDs. MeCP2 represses gene expression by recruiting mSin3A, which interacts with HDAC1 (19Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar,20Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2252) Google Scholar). The tissue distribution of MeCP2 is ubiquitous, and its expression is relatively abundant (21D'Esposito M. Quaderi N.A. Ciccodicola A. Bruni P. Esposito T. D'Urso M. Brown S.D. Mamm. Genome. 1996; 7: 533-535Crossref PubMed Scopus (107) Google Scholar, 22Coy J.F. Sedlacek Z. Bachner D. Delius H. Poustka A. Hum. Mol. Genet. 1999; 8: 1253-1262Crossref PubMed Scopus (124) Google Scholar). Mutations of the MeCP2 gene cause loss of body weight and a neurological disorder in the mouse, which is consistent with MECP2 mutations in Rett syndrome (23Guy J. Hendrich B. Holmes M. Martin J.E. Bird A. Nat. Genet. 2001; 27: 322-326Crossref PubMed Scopus (1229) Google Scholar, 24Chen R.Z. Akbarian S. Tudor M. Jaenisch R. Nat. Genet. 2001; 27: 327-331Crossref PubMed Scopus (1042) Google Scholar). This indicates that MeCP2 is not essential for development but participates in epigenetic control of neuronal function. Dnmt1 does not contain a methyl-CpG binding domain common to the MBD family (18Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar), raising the question of how this enzyme is recruited to hemimethylated DNA and how it replicates the methylation pattern. We tested the hypothesis that the pattern of DNA methylation is replicated through recruitment of Dnmt1 by MeCP2, which binds to the methylated cytosine of the template DNA. Here we show that Dnmt1 interacts with MeCP2 directly, and this interaction is mainly dependent upon the transcription repressor domain (TRD) of MeCP2. The Dnmt1-MeCP2 complex does not include HDAC1 but has maintenance methyltransferase activity. Moreover, MeCP2 can preferentially associate with both hemimethylated and fully methylated DNA under a physiological salt concentration condition. These results suggest that both Dnmt1 and MeCP2 may contribute to maintenance of DNA methylation during DNA replication by multiple regulatory machineries including various protein-protein interactions. We subcloned rat Dnmt1 cDNA (25Kimura H. Takeda T. Tanaka S. Ogawa T. Shiota K. Biochem. Biophys. Res. Commun. 1998; 253: 495-501Crossref PubMed Scopus (8) Google Scholar) into pCS2+MT (26Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar) and then cloned Myc-tagged rat Dnmt1 cDNA and rat Dnmt1 cDNA into pcDNA3 to produce pcDNA3-Myc-Dnmt1. We cloned rat MeCP2 cDNA and internal deletion constructs (ΔMBD, ΔCRID, and ΔTRD) into pCMV2-FLAG (Sigma). Individual domains of rat MeCP2 were cloned into pGEX 4T-3 (Amersham Biosciences) by PCR using appropriate sets of primers. We cloned mouse HDAC1 cDNA into pHM (Roche Molecular Biochemicals) using PCR from pCI-HDAC1-myc (gift from Dr. C. Seiser) to produce pHM-HA-mouse HDAC1. We verified all constructs by DNA sequencing. We maintained cell lines in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. For transfection of 293T, we seeded 4 × 105 cells before the procedure and transfected them with DNA (21 μg) using a calcium phosphate co-precipitation method (27Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). The culture medium was changed 6 h after transfection, and the cells were incubated for an additional 42 h before harvest. We transiently transfected 293T cells in culture dishes (60-mm diameter) with appropriate expression vectors including pME18S SRα-Rb (gift from Dr. T. Yamamoto) and harvested cells 48 h after transfection. The cells were pelleted by centrifugation at 5,000 rpm at 4 °C. Nonidet P-40 buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 10% glycerol, 1 mm dithiothreitol, 0.5 mm EDTA, 0.5 mm EGTA, 0.5 mm NaF, 1 mmNa3VO4, 1% aprotinin, 5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride) was added to the cell pellets, and the cells were lysed by pipetting and then placed for 30 min on ice. Cleared whole cell extracts were prepared by centrifugation at 15,000 rpm for 30 min at 4 °C. The cleared extracts were then subjected to immunoprecipitation for 1 h on ice. Immunoprecipitation and Western blot analysis were performed using anti-Myc (9E10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-FLAG (M2; Sigma), anti-Rb (C-15; Santa Cruz Biotechnology), or anti-HA antibody (12CA5; Roche Molecular Biochemicals). Protein G-Sepharose beads (Amersham Biosciences) were added, and the mixture was rotated for 1 h at 4 °C. Beads were washed three times with Nonidet P-40 buffer (1 ml). The precipitates or cleared extracts were then subjected to Western blot analysis. Blots were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skim milk. We incubated the membrane at room temperature for 1 h with appropriate antibodies described above in TBS containing 0.1% Tween 20 and 0.5% skim milk. The blots were then incubated for 1 h at room temperature with secondary antibody and washed in TBS containing 0.1% Tween 20. Immunoreactive bands were revealed using an ECL Kit (PerkinElmer Life Sciences) according to the manufacturer's instructions. For reprobing, blotted membrane was treated with stripping buffer (62.5 mm Tris-Cl, pH 6.8, 2% SDS, 100 mm 2-mercaptoethanol) for 30 min at 50 °C. The membrane was washed three times with TBS containing 0.1% Tween 20 for 30 min and then blocked for 1 h at room temperature with TBS containing 0.1% Tween 20 and 5% skim milk. The membrane was subjected to antibody reactions as described above. We expressed GST and GST fusion proteins inEscherichia coli XL1-blue using the pGEX vector system and purified protein from crude bacterial lysates according to the manufacturer's instructions (Amersham Biosciences). We carried outin vitro transcription/translation using the TNT system (Promega). Myc-Dnmt1 was translated in vitro from pCS2+MT-Dnmt1. We performed pull-down assays by preincubating GST or GST fusion protein and glutathione-Sepharose beads in binding buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 0.1% Tween 20, 0.5% skim milk) at room temperature. After 10 min, in vitrotranslated proteins were added, and the mixture was incubated for 1 h at 4 °C. The beads were then washed four times in the ice-cold binding buffer without 0.5% skim milk. Pelleted beads were suspended in 2× SDS-PAGE sample buffer. Bound proteins were resolved by SDS-PAGE and subjected to autoradiography. Alternatively, we transiently transfected 293T cells in culture dishes (100-mm diameter) with pcDNA3 (30 μg) or pcDNA3-Myc-Dnmt1 (30 μg), and the cells were harvested 48 h after the transfection and then lysed. GST or GST fusion protein was added into the lysate and incubated for 1 h on ice. Subsequently, glutathione-Sepharose beads were added and incubated with a rotary for 1 h at 4 °C. The beads were pelleted by centrifugation and washed with ice-cold Nonidet P-40 buffer three times. Bound proteins were resolved by SDS-PAGE and subjected to Western blot analysis using anti-Myc antibody (9E10). We transiently transfected 293T cells in culture dishes (90-mm diameter) with pCMV2-FLAG vector or pCMV2-FLAG-MeCP2 (30 μg) and collected cells 48 h post-transfection. We lysed cells in Nonidet P-40 buffer for 30 min at 4 °C and removed debris by centrifugation. The cleared lysates (total protein amount 1 mg) were then subjected to each DNA binding reaction for 1 h at 4 °C using 6 μg (30 μmol) of double-stranded oligonucleotides corresponding to E-cadherin CpG nucleotide sequences (28Rhee I. Jair K.W. Yen R.W. Lengauer C. Herman J.G. Kinzler K.W. Vogelstein B. Baylin S.B. Schuebel K.E. Nature. 2000; 404: 1003-1007Crossref PubMed Scopus (373) Google Scholar) (Sigma Genosys Japan KK). These were either nonmethylated, hemimethylated (on either the sense strand or on the antisense strand), or fully methylated, and all were biotinylated at the 5′-ends. The sense strand has the sequence 5′-CCAGCCCGGCCCGACCCGACCGCACCCGGCGC-3′ (portion of methylated cytosines are underlined). Methylated oligonucleotides were generated at specific portions by using methylated precursors. Streptavidin-magnetic beads (Roche Molecular Biochemicals) were added to collect the biotinylated oligonucleotides with binding proteins, and incubation was continued with a rotary for 1 h at 4 °C. The beads were washed three times with Nonidet P-40 buffer (1 ml) and were subjected to Western blot analysis using anti-FLAG antibody (M2). We transfected 293T cells with either Myc-Dnmt1 and FLAG-MeCP2 expression vectors, empty vector, and FLAG-MeCP2 expression vectors, empty vector alone, or FLAG-Dnmt1 expression vector. Cells were lysed using Nonidet P-40 buffer as described above. After immunoprecipitation using anti-FLAG antibody, beads were washed three times with Nonidet P-40 buffer, and then the precipitates were assayed for methyltransferase activity in a 100-μl reaction solution containing a 32-bp hemimethylated oligonucleotide substrate (500 ng (25 pmol)), which is the same as described above (28Rhee I. Jair K.W. Yen R.W. Lengauer C. Herman J.G. Kinzler K.W. Vogelstein B. Baylin S.B. Schuebel K.E. Nature. 2000; 404: 1003-1007Crossref PubMed Scopus (373) Google Scholar),S-adenosyl-l-[methyl-3H]methionine (2 μl; 77 Ci/mmol; Amersham Biosciences), 50 mm Tris-Cl, pH 7.5, 5 mm EDTA, 50% glycerol, 5 mmdithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. After incubation for 1 h at 37 °C or 4 °C, we removed unincorporated nuclides with Sephadex G-50 spin column (AmershamBiosciences) and determined incorporation of radioactivity in ASCII solvent (Amersham Biosciences) by liquid scintillation counting (13Fuks F. Burgers W.A. Brehm A. Hughes-Davies L. Kouzarides T. Nat. Genet. 2000; 24: 88-91Crossref PubMed Scopus (823) Google Scholar). The majority of MeCP2 is concentrated on heterochromatin in the genome (29Nan X. Tate P. Li E. Bird A. Mol. Cell. Biol. 1996; 16: 414-421Crossref PubMed Scopus (285) Google Scholar). Lysis under high salt concentration conditions results in extraction of all MeCP2 in the cell (17Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1091) Google Scholar). However, a high salt extraction also disrupts weaker affinity interactions. The extraction conditions used here take advantage of excluding MeCP2 associated with tightly condensed chromatin such as heterochromatin and of selecting MeCP2 associated with relatively loose chromatin such as during DNA replication. In previous reports, an interaction of Dnmt1 with PCNA (11Chuang L.S. Ian H.I. Koh T.W. Ng H.H. Xu G. Li B.F. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar), DMAP1 (12Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (867) Google Scholar), HDACs (19Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar), and Rb (15Pradhan S. Kim G.D. EMBO J. 2002; 21: 779-788Crossref PubMed Scopus (93) Google Scholar) has been shown under the same physiological salt condition. To test an interaction of Dnmt1 with MeCP2, we co-transfected 293T cells with plasmid encoding Myc-tagged, full-length rat Dnmt1 (Myc-Dnmt1) and FLAG-tagged, full-length rat MeCP2 (FLAG-MeCP2). Then we lysed the cells using a lysis buffer with a typical physiological salt concentration and carried out immunoprecipitation with the anti-FLAG antibody, followed by Western blot analysis using the anti-Myc antibody. Dnmt1 and MeCP2 interactedin vivo (Fig. 1, Aand B, lanes 4 and 8), and Dnmt1 was not immunoprecipitated in the absence of MeCP2 (Fig. 1,A and B, lanes 3 and7). Endogenous MeCP2 was detectable under the same salt condition in mouse primary cells using an anti-MeCP2 antibody (data not shown). We then determined whether MeCP2 can bind directly to Dnmt1in vitro by means of a GST pull-down assay using seven kinds of bacterially expressed and purified GST fusion MeCP2 proteins; GST 1–76, 77–161 (MBD), 162–206 (CRID), 207–310 (TRD), 311–403, 404–491, and wild type (residues 1–491) (Fig. 1 C). Three separate domains of MeCP2, as well as full-length MeCP2 protein, were able to bind to in vitro translated full-length Dnmt1 (Fig. 1 D, lanes 4–6 and 9). These associations were specific because Dnmt1 failed to bind to GST alone (Fig. 1 D, lane 2). To confirm that the GST-fused individual domains of MeCP2 and full-length MeCP2 can bind to Dnmt1 prepared from mammalian cells, we transfected 293T cells with an empty vector or Myc-Dnmt1 expression vector. We lysed the cells and carried out GST pull-down assays using the lysate, followed by Western blot analysis using the anti-Myc antibody. Three domains (MBD, CRID, and TRD) of MeCP2 and full-length MeCP2 bound full-length Dnmt1 (Fig. 1 E). These associations were specific because GST alone failed to bind to Dnmt1 (Fig. 1 E, lane 4). Dnmt1 was not detectable from the lysate transfected with empty vector (Fig. 1 E, odd-numbered lanes). These results indicate that Dnmt1 binds directly to MeCP2 in vitro (Fig. 1, D and E). Moreover, three different functional domains of MeCP2 could bind to Dnmt1 (Fig. 1,D (lanes 4–6) and E(lanes 8, 10, and 12)). Dnmt1 possesses regulatory domains in the N-terminal region (10Bestor T.H. Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1611) Google Scholar), which can form complexes with many proteins (e.g. DMAP1 (12Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (867) Google Scholar), PCNA (11Chuang L.S. Ian H.I. Koh T.W. Ng H.H. Xu G. Li B.F. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar), Rb (14Robertson K.D. Ait-Si-Ali S. Yokochi T. Wade P.A. Jones P.L. Wolffe A.P. Nat. Genet. 2000; 25: 338-342Crossref PubMed Scopus (809) Google Scholar), and RFTS (30Leonhardt H. Page A.W. Weier H.U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (832) Google Scholar)). We next examined which domain of Dnmt1 binds to MeCP2 by GST pull-down assay using several Dnmt1 constructs with serial deletions from the C terminus. A bipartite nuclear localization sequence has previously been identified in the N-terminal region (NLS; residues 171–201, rat Dnmt1) (Fig. 2 A) (30Leonhardt H. Page A.W. Weier H.U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (832) Google Scholar). All Dnmt1 constructs could be localized in the nucleus (Fig. 2 A). Lysates from 293T cells transfected with the mutant Dnmt1 constructs were provided to the assay. Most Dnmt1 fragments (residues 1–1,622, 1–1,414, 1–1,203, 1–1,014, 1–807, 1–608, 1–428, and 1–327) bound to GST-full-length MeCP2, and the three individual domains of MeCP2 (Fig. 2 B). The binding activity of fragment 1–327 to MBD was weaker than others. In contrast, Dnmt1 constructs 1–263 and 1–201 bound incompletely to the MeCP2 protein (Fig. 2 B). These results suggest that residues 264–326 of Dnmt1 are necessary for the interaction with MeCP2. The MBD, CRID, and TRD domains of MeCP2 can bind independently to Dnmt1 (Fig. 2 B). To examine which domain plays a central role for an interaction with Dnmt1 in vivo, we generated a series of FLAG-tagged internal deletion mutants of MeCP2 (FLAG-MeCP2, -ΔMBD, -ΔCRID, and -ΔTRD), all of which localize in the nucleus (Fig. 3 A). We co-transfected 293T cells with Myc-Dnmt1 and with empty vector, FLAG-MeCP2, FLAG-ΔMBD, FLAG-ΔCRID, or FLAG-ΔTRD. We lysed the cells and carried out immunoprecipitation with the anti-FLAG antibody, followed by Western blot analysis using the anti-Myc antibody. Full-length Dnmt1 and MeCP2 interacted as in Fig. 1 A (Fig. 3 B,lanes 1 and 2, column 1). Dnmt1 interacted specifically with ΔMBD and ΔCRID (Fig. 3 B, lanes 3 and 4), but the interaction of Dnmt1 with ΔTRD decreased strikingly (Fig. 3 B, lane 5, column 4). These results indicate that the TRD plays a central role for an interaction of Dnmt1 with MeCP2 in vivo. Deletion of MBD reduced the binding activity to Dnmt1 (Fig. 3 B,lane 3, column 2). This may suggest that MBD also contributes for an interaction of MeCP2 and Dnmt1. A previous study has reported that Dnmt1 associates with HDAC1 (13Fuks F. Burgers W.A. Brehm A. Hughes-Davies L. Kouzarides T. Nat. Genet. 2000; 24: 88-91Crossref PubMed Scopus (823) Google Scholar). MeCP2 also interacts with HDAC1 by forming a corepressor complex including mSin3A (19Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar, 20Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2252) Google Scholar). In our system, Dnmt1, MeCP2, and Rb all interacted with HDAC1 (Fig. 3 C). To examine whether a Dnmt1-MeCP2 complex includes HDAC1, we co-transfected 293T cells with plasmids containing FLAG-MeCP2, Myc-Dnmt1, and HA-HDAC1. Immunoprecipitates using anti-FLAG antibody from lysates including FLAG-MeCP2, Myc-Dnmt1, and HA-HDAC1 did not include HA-HDAC1 (Fig. 3 D, lane 7), whereas immunoprecipitates from lysates with FLAG-MeCP2 and HA-HDAC1 included HA-HDAC1 (Fig. 3 D, lane 6). These results suggest that an interaction of Dnmt1 with MeCP2 is stronger than that of mSin3A with MeCP2, and the Dnmt1-MeCP2 complex excludes HDAC1 from both Dnmt1-HDAC1 and MeCP2-mSin3A-HDAC1 components. If the interaction of Dnmt1 with MeCP2 is involved in replicating patterns of methylation, MeCP2 would be predicted to bind to hemimethylated DNA as well as to fully methylated DNA. We used synthetic biotinylated oligonucleotides corresponding to the in vivo CpG island of the E-cadherin gene (28Rhee I. Jair K.W. Yen R.W. Lengauer C. Herman J.G. Kinzler K.W. Vogelstein B. Baylin S.B. Schuebel K.E. Nature. 2000; 404: 1003-1007Crossref PubMed Scopus (373) Google Scholar) to investigate this. We confirmed that hemimethylated double-stranded oligonucleotides do not form a hairpin loop structure in the methylated strand using DNA electrophoresis under nondenaturing conditions and by oligonucleotide secondary structure predictions using Vector NTI software (data not shown). By means of an in vitro DNA pull-down assay (Fig. 4 A), we found that MeCP2 bound not only to fully methylated oligonucleotides but also to hemimethylated ones (Fig. 4 B). These associations were specific because MeCP2 failed to bind to the nonmethylated oligonucleotides of the same sequence (Fig. 4 B) (17Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1091) Google Scholar). This interaction led us to consider that the Dnmt1-MeCP2 complex could be associated with maintenance DNA methyltransferase activity. To test this idea, we performed transfections with either FLAG-MeCP2 and empty vector or FLAG-MeCP2 and Myc-Dnmt1 expression vectors. We lysed the cells, immunoprecipitated FLAG-MeCP2 with anti-FLAG antibody from the lysates, and then assayed the immunoprecipitates for maintenance methyltransferase activity. Immune complexes from cells transfected with FLAG-MeCP2 and Myc-Dnmt1 expression vectors possessed higher methyltransferase activity than cells transfected with FLAG-MeCP2 expression and empty vectors (Fig. 4 C, columns 1 and 2). Immunoprecipitates of Dnmt1 were also assayed as a positive control and indicated sufficient enzymatic activity in this system (Fig. 4 C, columns 3 and 4). Methyltransferase activity of Dnmt1 to hemimethylated DNA is ∼50-fold higher than de novomethyltransferase activity (9Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar). Thus, these results indicate that MeCP2-interacting Dnmt1 has significant maintenance DNA methyltransferase activity and that MeCP2 does not vanish Dnmt1 enzymatic activity. When this enzymatic reaction was also tested at 4 °C, maintenance DNA methyltransferase activity was equal to that of cells transfected with an empty vector (data not shown). DNA pull-down assay and methyltransferase assay were performed using the same hemimethylated double-stranded oligonucleotides (see “Experimental Procedures”). These results indicate that hemimethylated double-stranded oligonucleotides do not become fully methylated by endogenous MeCP2 under the DNA pull-down condition. Our data indicate a direct interaction between Dnmt1 and MeCP2. This interaction prevents the association of HDAC1 with MeCP2. MeCP2 associates with hemimethylated as well as fully methylated DNA in vitro. The MeCP2-Dnmt1 complex possesses maintenance DNA methyltransferase activity that is dependent upon Dnmt1, demonstrating that the Dnmt1-MeCP2 complex could be involved in maintenance of DNA methylation. Histone deacetylase activity associates with Rb (31Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1080) Google Scholar), Dnmt1 (13Fuks F. Burgers W.A. Brehm A. Hughes-Davies L. Kouzarides T. Nat. Genet. 2000; 24: 88-91Crossref PubMed Scopus (823) Google Scholar), and MBD proteins such as MeCP2 (19Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar), MBD1 (32Ng H.H. Jeppesen P. Bird A. Mol. Cell. Biol. 2000; 20: 1394-1406Crossref PubMed Scopus (220) Google Scholar), and MBD2 (33Bird A.P. Wolffe A.P. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar). Thus, Rb, Dnmt1, and MBDs complexes have histone deacetylase activity. Our findings suggest that histone deacetylation at least partly mediates various nucleosomal events such as chromatin integrity and gene regulation. We propose that histone deacetylase is not essential for maintenance DNA methyltransferase activity in the Dnmt1-MeCP2 complex. An N-terminal domain of Dnmt1 interacts with cell cycle-related molecules such as PCNA (11Chuang L.S. Ian H.I. Koh T.W. Ng H.H. Xu G. Li B.F. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar) and Rb (14Robertson K.D. Ait-Si-Ali S. Yokochi T. Wade P.A. Jones P.L. Wolffe A.P. Nat. Genet. 2000; 25: 338-342Crossref PubMed Scopus (809) Google Scholar, 15Pradhan S. Kim G.D. EMBO J. 2002; 21: 779-788Crossref PubMed Scopus (93) Google Scholar). The RFTS domain of Dnmt1 supports foci formation in the nucleus (30Leonhardt H. Page A.W. Weier H.U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (832) Google Scholar). The MeCP2 interaction domain of Dnmt1 is near the RFTS, and deletion of the RFTS (amino acids 328–378) reduces, but does not eliminate, interaction with MeCP2 (Fig. 2). These results suggest that interaction of the two may be supported by the RFTS. Subcellular localization of Dnmt1 may be regulated by interplay of the MeCP2 interaction domain and RFTS. The connection between DNA methylation and histone acetylation will mainly affect chromatin organization by maintaining a stable epigenetic state and remodeling chromatin, but the enzymatic activity of DNA methyltransferase itself may be regulated by molecules such as MeCP2 that bind to DNA. Some studies suggest that the Dnmt1 enzymatic reaction may take place at classical replication foci (30Leonhardt H. Page A.W. Weier H.U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (832) Google Scholar), but little is known about the molecular mechanism of how CpG methylation is maintained. The discovery that Dnmt1 directly interacts with MeCP2 will help us to understand this issue. In proliferating cells, expression of Dnmt1 is induced as DNA synthesis approaches (34Szyf M. Bozovic V. Tanigawa G. J. Biol. Chem. 1991; 266: 10027-10030Abstract Full Text PDF PubMed Google Scholar). Recent studies and our data imply a mechanism by which DNA methylation is maintained as described below (Fig. 5). First, Dnmt1 is recruited to replication foci by DMAP1 and/or PCNA (11Chuang L.S. Ian H.I. Koh T.W. Ng H.H. Xu G. Li B.F. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar, 12Rountree M.R. Bachman K.E. Baylin S.B. Nat. Genet. 2000; 25: 269-277Crossref PubMed Scopus (867) Google Scholar). When replication forks with the PCNA clamp structure progress (35Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar), MeCP2 bound to symmetrically methylated DNA is released. Free MeCP2 reassociates with hemimethylated, newly synthesized DNA. Dnmt1 interacts with MeCP2, recognizes the complementary nonmethylated CpG dinucleotides, and transfers a methyl group by the enzymatic reaction. Then MeCP2 may change its conformation promptly. Finally, MeCP2 would bind to fully methylated CpG dinucleotides to maintain gene silencing and chromatin structures by recruitment of HDACs (33Bird A.P. Wolffe A.P. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar). In our model of maintenance of DNA methylation, these properties of DNA binding activity and interaction with some molecules may be regulated by post-translational modifications such as phosphorylation during DNA replication. In this regard, we found that both Dnmt1 and MeCP2 are phosphorylated at multiple sites in vivo. 2H. Kimura and K. Shiota, unpublished data. MeCP2 possesses at least two kinds of modification states in vivo.2 Thus, Dnmt1 might require MeCP2 to carry out genome-wide and/or local maintenance DNA methylation. Although mutations of DNA methyltransferase and methyl-CpG-binding protein family genes affect loss of DNA methylation in development (36Lei H. Oh S.P. Okano M. Juttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar) and/or cellular function (23Guy J. Hendrich B. Holmes M. Martin J.E. Bird A. Nat. Genet. 2001; 27: 322-326Crossref PubMed Scopus (1229) Google Scholar, 24Chen R.Z. Akbarian S. Tudor M. Jaenisch R. Nat. Genet. 2001; 27: 327-331Crossref PubMed Scopus (1042) Google Scholar), little is understood regarding which protein complexes are involved in those processes. Our discovery implicates that Dnmt1 and MeCP2 may interplay in related pathways in both DNA methylation and histone modification. An interaction of Dnmt1 and MeCP2 is dependent upon the TRD in the C-terminal region of MeCP2in vivo. Therefore, the MBD of MeCP2 within the complex could bind hemimethylated DNA, targeting Dnmt1 activity to MeCP2-responsive genes. One important question that remains to be answered about the Dnmt1-MeCP2 interaction is whether there is a specific target for the genome or other molecules to be involved in DNA methylation and chromatin remodeling. Future studies of the functions of complexes formed by DNA methyltransferases and methyl-CpG binding domain proteins will be required to determine which complexes are responsible for tissue-specific organization and remodeling of chromatin structure and gene regulation by DNA methylation and histone modification in normal development and epigenetic diseases. We thank Drs. C. Seiser and T. Yamamoto for plasmids. We also thank Drs. Tom Curran and Jared Ordway for critical reading of the manuscript and helpful comments." @default.
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• W1966092430 title "Methyl-CpG-binding Protein, MeCP2, Is a Target Molecule for Maintenance DNA Methyltransferase, Dnmt1" @default.
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