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• W2097778167 abstract "DNA methylation is interpreted by a family of methyl-CpG binding domain (MBD) proteins that repress transcription through recruitment of corepressors that modify chromatin. To compare in vivo binding of MeCP2 and MBD2, we analyzed immunoprecipitated chromatin from primary human cells. Genomic sites occupied by the two MBD proteins were mutually exclusive. As MeCP2 was unable to colonize sites vacated by depletion of MBD2, we tested the hypothesis that methyl-CpG alone is insufficient to direct MeCP2 binding. In vitro selection for MeCP2 bound DNA-enriched fragments containing A/T bases ([A/T]≥4) adjacent to methyl-CpG. [A/T]≥4 was found to be essential for high-affinity binding at selected sites and at known MeCP2 target regions in the Bdnf and Dlx6 genes. MBD2 binding, however, did not require an A/T run. The unexpected restriction of MeCP2 to a defined subset of methyl-CpG sites will facilitate identification of genomic targets that are relevant to Rett Syndrome. DNA methylation is interpreted by a family of methyl-CpG binding domain (MBD) proteins that repress transcription through recruitment of corepressors that modify chromatin. To compare in vivo binding of MeCP2 and MBD2, we analyzed immunoprecipitated chromatin from primary human cells. Genomic sites occupied by the two MBD proteins were mutually exclusive. As MeCP2 was unable to colonize sites vacated by depletion of MBD2, we tested the hypothesis that methyl-CpG alone is insufficient to direct MeCP2 binding. In vitro selection for MeCP2 bound DNA-enriched fragments containing A/T bases ([A/T]≥4) adjacent to methyl-CpG. [A/T]≥4 was found to be essential for high-affinity binding at selected sites and at known MeCP2 target regions in the Bdnf and Dlx6 genes. MBD2 binding, however, did not require an A/T run. The unexpected restriction of MeCP2 to a defined subset of methyl-CpG sites will facilitate identification of genomic targets that are relevant to Rett Syndrome. About 70% of CpG dinucleotides in the mammalian genome are methylated at position five of the cytosine ring (Bird, 2002Bird A. DNA methylation patterns and epigenetic memory.Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (4995) Google Scholar). This epigenetic DNA modification affects gene expression and genome integrity (Eden et al., 2003Eden A. Gaudet F. Waghmare A. Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation.Science. 2003; 300: 455Crossref PubMed Scopus (1008) Google Scholar) and is required for normal mouse development (Jackson-Grusby et al., 2001Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Jaenisch R. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation.Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (542) Google Scholar, Li et al., 1992Li E. Bestor T.H. Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3075) Google Scholar). Mechanistically, DNA methylation is thought to elicit its effects by interfering with binding of proteins to their cognate binding sites (Watt and Molloy, 1988Watt F. Molloy P.L. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus late promoter.Genes Dev. 1988; 2: 1136-1143Crossref PubMed Scopus (364) Google Scholar) or by creating a binding site for proteins that recognize methyl-CpG (Boyes and Bird, 1991Boyes J. Bird A. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein.Cell. 1991; 64: 1123-1134Abstract Full Text PDF PubMed Scopus (561) Google Scholar, Lewis et al., 1992Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, Meehan et al., 1989Meehan R.R. Lewis J.D. McKay S. Kleiner E.L. Bird A.P. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs.Cell. 1989; 58: 499-507Abstract Full Text PDF PubMed Scopus (503) Google Scholar). A family of methyl-CpG binding proteins has been characterized comprising MBD1, MBD2, MBD3, MeCP2, and MBD4, each of which contains a conserved methyl-CpG binding domain or MBD (Hendrich and Bird, 1998Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1009) Google Scholar, Nan et al., 1993Nan X. Meehan R.R. Bird A. Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.Nucleic Acids Res. 1993; 21: 4886-4892Crossref PubMed Scopus (466) Google Scholar). MBD1, MBD2, and MeCP2 are associated with transcriptional repression and chromatin remodeling activities (Feng and Zhang, 2001Feng Q. Zhang Y. The MeCP1 complex represses transcription through preferential binding, remodeling, and deacetylating methylated nucleosomes.Genes Dev. 2001; 15: 827-832PubMed Google Scholar, Jones et al., 1998Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2142) Google Scholar, Klose and Bird, 2004Klose R.J. Bird A.P. MeCP2 behaves as an elongated monomer that does not stably associate with the Sin3a chromatin remodeling complex.J. Biol. Chem. 2004; 279: 46490-46496Crossref PubMed Scopus (72) Google Scholar, Nan et al., 1998Nan X. Ng H.-H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.Nature. 1998; 393: 386-389Crossref PubMed Scopus (2625) Google Scholar, Ng et al., 1999Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex.Nat. Genet. 1999; 23: 58-61PubMed Scopus (0) Google Scholar, Sarraf and Stancheva, 2004Sarraf S.A. Stancheva I. Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly.Mol. Cell. 2004; 15: 595-605Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Mammalian MBD3 does not bind specifically to methylated DNA, and MBD4 is a DNA repair protein (Hendrich et al., 1999Hendrich B. Hardeland U. Ng H.H. Jiricny J. Bird A. The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites.Nature. 1999; 401: 301-304Crossref PubMed Scopus (497) Google Scholar, Millar et al., 2002Millar C.B. Guy J. Sansom O.J. Selfridge J. MacDougall E. Hendrich B. Keightley P.D. Bishop S.M. Clarke A.R. Bird A. Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice.Science. 2002; 297: 403-405Crossref PubMed Scopus (249) Google Scholar), although recent evidence suggests that MBD4 may also act as a transcriptional repressor (Kondo et al., 2005Kondo E. Gu Z. Horii A. Fukushige S. The thymine DNA glycosylase MBD4 represses transcription and is associated with methylated p16INK4a and hMLH1 genes.Mol. Cell. Biol. 2005; 25: 4388-4396Crossref PubMed Scopus (89) Google Scholar). The biomedical relevance of MBD proteins became apparent with the discovery that the human neurodevelopmental disorder Rett syndrome is caused by mutations in the MECP2 gene (Amir et al., 1999Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3519) Google Scholar). Mutational profiling of Rett syndrome patients identified a significant fraction of point mutations that inactivated the MBD itself (Kriaucionis and Bird, 2003Kriaucionis S. Bird A. DNA methylation and Rett syndrome.Hum. Mol. Genet. 2003; 12 Spec No 2: R221-R227Crossref PubMed Scopus (98) Google Scholar). The DNA and protein features that determine the specificity of the MBD for methyl-CpG sites have been examined (Free et al., 2001Free A. Wakefield R.I. Smith B.O. Dryden D.T. Barlow P.N. Bird A.P. DNA recognition by the methyl-CpG binding domain of MeCP2.J. Biol. Chem. 2001; 276: 3353-3360Crossref PubMed Scopus (83) Google Scholar, Meehan et al., 1992Meehan R.R. Lewis J.D. Bird A.P. Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA.Nucleic Acids Res. 1992; 20: 5085-5092Crossref PubMed Scopus (392) Google Scholar, Nan et al., 1993Nan X. Meehan R.R. Bird A. Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.Nucleic Acids Res. 1993; 21: 4886-4892Crossref PubMed Scopus (466) Google Scholar, Yusufzai and Wolffe, 2000Yusufzai T.M. Wolffe A.P. Functional consequences of Rett syndrome mutations on human MeCP2.Nucleic Acids Res. 2000; 28: 4172-4179Crossref PubMed Scopus (108) Google Scholar), and the three-dimensional structure of the domain, both alone (Ohki et al., 1999Ohki I. Shimotake N. Fujita N. Nakao M. Shirakawa M. Solution structure of the methyl-CpG-binding domain of the methylation-dependent transcriptional repressor MBD1.EMBO J. 1999; 18: 6653-6661Crossref PubMed Scopus (89) Google Scholar, Wakefield et al., 1999Wakefield R.I. Smith B.O. Nan X. Free A. Soteriou A. Uhrin D. Bird A.P. Barlow P.N. The solution structure of the domain from MeCP2 that binds to methylated DNA.J. Mol. Biol. 1999; 291: 1055-1065Crossref PubMed Scopus (153) Google Scholar) and in complex with methylated DNA (Ohki et al., 2001Ohki I. Shimotake N. Fujita N. Jee J. Ikegami T. Nakao M. Shirakawa M. Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.Cell. 2001; 105: 487-497Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), has been solved by NMR analysis. The MBD core consists of an α/β sandwich, with specific residue clusters mediating contacts with each 5-methylcytosine moiety in the major groove (Ohki et al., 2001Ohki I. Shimotake N. Fujita N. Jee J. Ikegami T. Nakao M. Shirakawa M. Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.Cell. 2001; 105: 487-497Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Evidence that nuclear factors associate with methylated DNA in vivo came initially from studies examining the accessibility of CpG dinucleotides to cleavage by restriction endonucleases (Antequera et al., 1989Antequera F. Macleod D. Bird A.P. Specific protection of methylated CpGs in mammalian nuclei.Cell. 1989; 58: 509-517Abstract Full Text PDF PubMed Scopus (154) Google Scholar). Methylated CpG dinucleotides were refractory to digestion, whereas nonmethylated sites were relatively accessible. Immunofluorescence confirmed that MBD proteins colocalize with sites of dense DNA methylation in living cells, in particular mouse satellite DNA, which is concentrated at pericentromeric heterochromatin (Hendrich and Bird, 1998Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1009) Google Scholar, Nan et al., 1996Nan X. Tate P. Li E. Bird A. DNA methylation specifies chromosomal localization of MeCP2.Mol. Cell. Biol. 1996; 16: 414-421Crossref PubMed Scopus (272) Google Scholar). Several studies have since shown that MeCP2 associates with a variety of methylated gene sequences in vivo, but not with the same sequences when they are nonmethylated (El-Osta et al., 2002El-Osta A. Kantharidis P. Zalcberg J.R. Wolffe A.P. Precipitous release of methyl-CpG binding protein 2 and histone deacetylase 1 from the methylated human multidrug resistance gene (MDR1) on activation.Mol. Cell. Biol. 2002; 22: 1844-1857Crossref PubMed Scopus (172) Google Scholar, Ghoshal et al., 2002Ghoshal K. Datta J. Majumder S. Bai S. Dong X. Parthun M. Jacob S.T. Inhibitors of histone deacetylase and DNA methyltransferase synergistically activate the methylated metallothionein I promoter by activating the transcription factor MTF-1 and forming an open chromatin structure.Mol. Cell. Biol. 2002; 22: 8302-8319Crossref PubMed Scopus (140) Google Scholar, Gregory et al., 2001Gregory R.I. Randall T.E. Johnson C.A. Khosla S. Hatada I. O’Neill L.P. Turner B.M. Feil R. DNA methylation is linked to deacetylation of histone H3, but not H4, on the imprinted genes Snrpn and U2af1-rs1.Mol. Cell. Biol. 2001; 21: 5426-5436Crossref PubMed Scopus (115) Google Scholar, Nan et al., 1996Nan X. Tate P. Li E. Bird A. DNA methylation specifies chromosomal localization of MeCP2.Mol. Cell. Biol. 1996; 16: 414-421Crossref PubMed Scopus (272) Google Scholar, Nguyen et al., 2001Nguyen C.T. Gonzales F.A. Jones P.A. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation.Nucleic Acids Res. 2001; 29: 4598-4606Crossref PubMed Scopus (194) Google Scholar, Rietveld et al., 2002Rietveld L.E. Caldenhoven E. Stunnenberg H.G. In vivo repression of an erythroid-specific gene by distinct corepressor complexes.EMBO J. 2002; 21: 1389-1397Crossref PubMed Scopus (34) Google Scholar). Likely MBD protein target sites in human cancer cells have been identified by characterizing DNA fragments that were immunoprecipitated with MBD protein-specific antibodies (Ballestar et al., 2003Ballestar E. Paz M.F. Valle L. Wei S. Fraga M.F. Espada J. Cigudosa J.C. Huang T.H. Esteller M. Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer.EMBO J. 2003; 22: 6335-6345Crossref PubMed Scopus (274) Google Scholar, Koch and Stratling, 2004Koch C. Stratling W.H. DNA binding of methyl-CpG-binding protein MeCP2 in human MCF7 cells.Biochemistry. 2004; 43: 5011-5021Crossref PubMed Scopus (28) Google Scholar, Sarraf and Stancheva, 2004Sarraf S.A. Stancheva I. Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly.Mol. Cell. 2004; 15: 595-605Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). A similar study of mouse brain chromatin using an anti-MeCP2 antibody identified the mouse Dlx5/Dlx6 genes as targets for MeCP2-mediated repression (Horike et al., 2005Horike S. Cai S. Miyano M. Cheng J.F. Kohwi-Shigematsu T. Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome.Nat. Genet. 2005; 37: 31-40Crossref PubMed Scopus (273) Google Scholar). Given their common DNA binding domains, different MBD proteins might be expected to compete for binding to methyl-CpG sites in the genome. About 9% of MeCP2 sites in a cancer cell line, however, were not immunoprecipitated by antibodies against MBD1 or MBD2 (Ballestar et al., 2003Ballestar E. Paz M.F. Valle L. Wei S. Fraga M.F. Espada J. Cigudosa J.C. Huang T.H. Esteller M. Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer.EMBO J. 2003; 22: 6335-6345Crossref PubMed Scopus (274) Google Scholar). In the present study using primary human cells, we find that the great majority of MeCP2 bound genomic fragments do not bind MBD1 or MBD2. Moreover, MeCP2 is unable to colonize most sites that are vacated by depletion of MBD2. To explain this specificity, we entertained the hypothesis that methyl-CpG is necessary, but not sufficient, for MeCP2 binding. Artificial selection of MeCP2 binding sites in vitro using Methyl-SELEX demonstrated that MeCP2 requires an A/T run of four or more base pairs ([A/T]≥4) adjacent to the methyl-CpG for efficient DNA binding. We propose that the presence of an A/T run is essential at biologically relevant MeCP2 binding sites and may help to predict MeCP2 interaction sites. In order to identify genomic loci occupied by MeCP2 in vivo, we performed chromatin immunoprecipitation (ChIP) with MeCP2-specific antibodies. We chose to study the MRC-5 human lung embryonic fibroblast cell line, as it displays characteristics of untransformed cells and has a normal diploid karyotype. At least three methyl-CpG binding proteins, MeCP2, MBD2, and MBD1, are detectable in nuclear extracts of MRC-5 cells (data not shown). To identify specific DNA sites bound by MeCP2, we used a “ChIP-and-clone” approach. Growing MRC-5 cells were formaldehyde fixed, and isolated chromatin was sonicated to generate DNA fragments of 250–750 bp. MeCP2-associated chromatin was then immunoprecipitated with MeCP2-specific antibodies, recovered, and adaptor sequences added. Primers complementary to the adaptor sequences were used to amplify DNA fragments, which were then cloned, and 98 plasmids were sequenced. As a control for random effects, sonicated genomic DNA was also cloned and sequenced (Tables S1 and S3 available in the Supplemental Data with this article online). Blast analysis indicated that 57% of MeCP2 binding sites mapped to intergenic sequences and 28% to introns and exons within annotated genes (Tables S1 and S3). To ensure that the recovered loci were genuine MeCP2 targets, we repeated the ChIP but assayed with primers that were specific for each of 24 representative single-copy sequences from the first experiment (Figure 1A and Figure S1C). MeCP2 was reproducibly immunoprecipitated at loci identified in the initial ChIP-and-clone screen but rarely (one out of 18 fragments tested) at loci recovered from cloning of random genomic DNA (Figure S1B). When cells were pretreated with 5-azacytidine, the MeCP2 ChIP signal was lost, demonstrating that binding is dependent on DNA methylation (Figure 1A , lanes MeCP2*). Bisulfite sequencing of eight of these loci independently confirmed that all these sites contain highly methylated CpGs in MRC-5 cells (Figure S2). Surprisingly, ChIP of the same fixed chromatin sample with antibodies against MBD1 and MBD2 (Figure 1A) showed that only one of 12 MeCP2 bound fragments was immunoprecipitated by the anti-MBD2 antibody, and none were precipitated by anti-MBD1 antibody. We conclude that in these primary human cells, MeCP2 binds to unique loci that, for the most part, do not attract the MBD2 and MBD1. Given that MeCP2 and MBD2 each recognize methyl-CpG, we wondered if MBD2 would take over these sequences in the absence of MeCP2. To investigate this possibility, we first depleted MeCP2 in MRC-5 cells by transfection with a morpholino oligonucleotide (MeCP2-MO) directed against its translational initiation codon (Stancheva et al., 2003Stancheva I. Collins A.L. Van den Veyver I.B. Zoghbi H. Meehan R.R. A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos.Mol. Cell. 2003; 12: 425-435Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). A related oligonucleotide with five mismatches (MeCP2-mis-MO) served as a control. MeCP2 was efficiently depleted after two sequential MeCP2-MO transfections, as indicated by the loss of MeCP2 protein from the nuclear extracts, but treatment with MeCP2-mis-MO had no effect (Figure 1B). Levels of MBD2 or the unrelated protein PCNA were unaffected by either morpholino. Depletion of MeCP2 led to loss of DNA bound MeCP2, as anti-MeCP2 antibodies now weakly precipitated only five of the original 24 loci, the other 19 loci appearing unbound by this assay. Treatment with the control MeCP2-mis-MO allowed normal recovery of all 24 sites by ChIP. To find out if MBD2 could occupy vacated MeCP2 binding sites, chromatin from MeCP2-MO-treated cells was immunoprecipitated with anti-MBD2 antibodies, and the loci were tested for altered occupancy (Figure 1C and Figure S1C). In MeCP2-deficient cells, 13 of the 24 tested loci were now bound by MBD2. Having established that MBD2 could colonize methylated sites vacated by MeCP2, we next asked the converse question: could MeCP2 move into sites vacated by MBD2? To address this, we isolated loci from the MRC-5 genome that were bound by MBD2 using the ChIP-and-clone methodology. Cloned loci were sequenced, located on the human genome sequence assembly (Tables S2 and S3), and verified by PCR amplification of an independent MBD2 ChIP fraction using locus-specific primers (Figure S1A). To determine if these loci could be colonized by MeCP2, we depleted MBD2 in MRC-5 cells, again using a morpholino approach (Figure 1B). After two sequential transfections with MBD2 morpholino (MBD2-MO), MBD2 protein was undetectable in nuclear extracts from these cells. The effect was specific, as levels of MeCP2 or the unrelated protein PCNA were unaltered. As expected, depletion of MBD2 greatly reduced the recovery of MBD2-associated loci by ChIP with anti-MBD2 antibodies (Figure 1D and Figure S1D). Only five of the 25 loci gave a residual signal, whereas fragments were recovered normally after treatment with a mismatched morpholino (MBD2-mis-MO) that had no effect on MBD2 abundance. To assess whether MeCP2 was able to take over MBD2 sites, ChIP experiments were carried out in parallel on the MBD2-MO-treated samples by using MeCP2-specific antibodies. Depletion of MBD2 resulted in MeCP2 occupancy at only three of the 25 sites vacated by MBD2. These data indicate that MeCP2 is reluctant to replace MBD2. The specificity of MeCP2 for a subset of methylated sites in the genome could be due to a requirement for additional DNA sequences at its genomic binding sites. According to this hypothesis, methyl-CpG would be necessary, but not sufficient, for DNA binding of MeCP2. To establish the molecular determinants for methyl-CpG-dependent DNA recognition by MeCP2, we employed a modified form of the in vitro DNA binding site selection technique SELEX (He et al., 1996He Y.Y. Stockley P.G. Gold L. In vitro evolution of the DNA binding sites of Escherichia coli methionine repressor, MetJ.J. Mol. Biol. 1996; 255: 55-66Crossref PubMed Scopus (37) Google Scholar, Klug and Famulok, 1994Klug S.J. Famulok M. All you wanted to know about SELEX.Mol. Biol. Rep. 1994; 20: 97-107Crossref PubMed Scopus (215) Google Scholar). Methyl-SELEX involved generating double-stranded DNA fragments that have a fixed central CpG in the context of an HpaII methyltransferase/restriction endonuclease site flanked by random DNA sequence (Figure 2A ). The initial pool of DNA fragments was methylated by using M.HpaII methyltransferase and tested for complete methylation by loss of sensitivity to cleavage by HpaII restriction endonuclease. We used the N-terminal half of human MeCP2 (amino acids 1–205) for DNA binding, as it forms a discrete DNA-protein complex more reproducibly than the full-length protein (1–486) in our hands. The protein was mixed with the starting DNA, and DNA-protein complex was recovered after an electrophoretic mobility shift assay (EMSA). DNA from the complex was amplified, remethylated, and once more bound to MeCP2 (1–205). After eight cycles of binding and amplification, we observed that the efficiency of complex formation increased ∼5-fold compared with the random starting DNA (Figure 2B). The sequence of 88 selected fragments and 86 unselected fragments was determined (see Unselected Methyl-SELEX Fragments and Mecp2 Methyl-SELEX Fragments in the Supplemental Data). Analysis of these sequences revealed the frequent presence of an A/T run of four or more bases ([A/T]≥4) close to the methyl-CpG site in the enriched sample. When sequences were aligned with the A/T run to the right of the methyl-CpG (Figure 3A ), an [A/T]≥4 motif appeared to cluster in two vertical stripes located one to three base pairs or six to nine base pairs from the methylated CCGG site. In contrast, when the MBD of MBD2 was used in the same Methyl-SELEX experiment, there was no enrichment of an [A/T]≥4 motif, but instead, sequences containing more than one methylated CCGG site were recovered (Figure S3 and MBD2 Methyl-SELEX Fragments in the Supplemental Data). To determine whether the DNA fragments in the MeCP2-selected pool had an increased affinity for MeCP2, we analyzed five of the selected fragments (S1–S5) and two unselected fragments (U6 and U7) by EMSA (Figures 3B and 3C). Fragments with an A/T run within three bases (S1 and S2) or eight bases (S3 and S4) of the methyl-CpG bound efficiently to MeCP2 (1–205), whereas two unselected fragments that lacked an [A/T]≥4 motif did not show detectable complexes. Within the selected pool, six out of 88 fragments contained no A/T run. EMSA analysis using one of these sequences (S5) showed low affinity for MeCP2, suggesting that this was a contaminant (Figure 3C). To directly test the importance of the A/T-rich sequences for MeCP2 binding, we placed C/G pairs within the A/T runs of fragments S1 and S3 by mutagenesis (Figure 4A ). In each case, the mutations drastically reduced the MeCP2 binding affinity of these probes to levels seen in unselected DNA (Figure 4B). As all probes were methylated at a fixed central CCGG, we tested whether the two nucleotides flanking the CpG contributed to the observed binding. Changing CCGG in probe A1 to GCGC (methylated by M.HhaI methyltransferase) did not affect the high-affinity binding to MeCP2 (Figure 4B). If an A/T-rich run adjacent to methyl-CpG is sufficient for efficient MeCP2 binding, it should be possible to engineer a high-affinity MeCP2 binding site by using these criteria alone. Therefore, probe A2 was constructed in which an [A/T]≥4 run was placed three base pairs away from the central CpG within the context of a random flanking nucleotide sequence. The engineered DNA sequence bound MeCP2 as efficiently as the positively selected Methyl-SELEX fragments, and mutation of the A/T run again abolished high-affinity binding (Figure 4B). An [A/T]≥4 sequence adjacent to a methyl-CpG is therefore necessary and sufficient for high-affinity MeCP2 binding. The unexpected DNA sequence specificity of MeCP2 raises the possibility that MBD2, whose binding sites in the genome are largely distinct from those of MeCP2, might exhibit comparable selectivity. Full-length MBD2 did not form complexes with DNA under conditions used in our EMSA assay, but a fragment corresponding to the MBD bound equally to sites with and without the adjacent A/T run (Figure 4C). MBD2 is therefore indifferent to the presence or absence of [A/T]≥4, in agreement with results obtained in Methyl-SELEX using MBD2 (Figure S3 and MBD2 Methyl-SELEX Fragments in the Supplemental Data). Examination of DNA fragments obtained from MRC-5 cells by ChIP and clone showed that CpGs followed by [A/T]≥4 were somewhat enriched among the MeCP2 ChIP sequences. Within MeCP2 ChIP fragments, most of which contained multiple CpGs, 50% of CpGs (523 in total) were followed by an A/T run compared to 33.5% of CpGs (435) in MBD2 ChIP fragments. Considering only sequences that contained a single CpG, 11 out of 12 MeCP2 bound loci contained an [A/T]≥4 motif within eight base pairs of the CpG (Figure 4D). In one MeCP2 bound fragment (CS76), an [A/T]6 motif was 11 base pairs away from the CpG (Figure 4D). Independent ChIP and PCR demonstrated that CS76 is weakly precipitated by anti-MeCP2 antibodies compared with the other fragments (data not shown) and thus may represent a relatively low-affinity binding site in vivo. These findings confirm that the DNA binding requirements for MeCP2 that were established by Methyl-SELEX are relevant in vivo. Methylated CpG sites are symmetrical, and therefore, MeCP2 can theoretically bind in either of two orientations. The presence of an adjacent A/T run, however, potentially confers asymmetry to the MeCP2 binding site. To test for directional binding of MeCP2, we bound MeCP2 to the S1 probe and performed DNase I footprinting analysis. The zone of protection was not centered on the methyl-CpG but extended to cover the adjacent A/T run (Figure 4E). The A/T run therefore confers directional binding of MeCP2 at a methyl-CpG site. It was noted in early studies that MeCP2 contains a domain that is shared with proteins that bind the minor groove of A/T-rich DNA (Lewis et al., 1992Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, Nan et al., 1993Nan X. Meehan R.R. Bird A. Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.Nucleic Acids Res. 1993; 21: 4886-4892Crossref PubMed Scopus (466) Google Scholar). The effect of this domain on MeCP2 function remains unknown, but close sequence similarity with an AT hook domain of HMGA1 (formerly HMG I/Y) and across MeCP2 orthologs is striking (Aravind and Landsman, 1998Aravind L. Landsman D. AT-hook motifs identified in a wide variety of DNA-binding proteins.Nucleic Acids Res. 1998; 26: 4413-4421Crossref PubMed Scopus (368) Google Scholar) (Figure 5A ). We asked whether the AT hook might be responsible for the A/T run-dependent binding of MeCP2 by mutating two conserved arginine amino acids within the MeCP2 AT hook to glycine (Figure 5A). Equivalent mutations within the AT hook of HMGA1 significantly inhibited the recovery of binding to heterochromatin in photobleaching experiments (Harrer et al., 2004Harrer M. Luhrs H. Bustin M. Scheer U. Hock R. Dynamic interaction of HMGA1a proteins with chromatin.J. Cell Sci. 2004; 117: 3459-3471Crossref PubMed Scopus (81) Google Scholar). When wt and mutant MeCP2 proteins were assayed by EMSA for binding to the S1 and S1-mutated DNA fragments, however, we were surprised to find that loss of the AT hook domain had no effect on the affinity or specificity of MeCP2 for probes that contained an A" @default.
• W2097778167 created "2016-06-24" @default.
• W2097778167 creator A5031443094 @default.
• W2097778167 creator A5034245512 @default.
• W2097778167 creator A5049681235 @default.
• W2097778167 creator A5052906260 @default.
• W2097778167 creator A5073071237 @default.
• W2097778167 creator A5080164627 @default.
• W2097778167 date "2005-09-01" @default.
• W2097778167 modified "2023-10-18" @default.
• W2097778167 title "DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG" @default.
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• W2097778167 doi "https://doi.org/10.1016/j.molcel.2005.07.021" @default.
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