DNA Recognition by the Methyl-CpG Binding Domain of MeCP2

Abstract

The methyl-CpG binding domain (MBD) of the transcriptional repressor MeCP2 has been proposed to recognize a single symmetrically methylated CpG base pair via hydrophobic patches on an otherwise positively charged DNA binding surface. We have tested this binding model by analysis of mutant derivatives of the MeCP2 MBD in electrophoretic mobility shift assays complemented by NMR structural analysis. Exposed arginine side chains on the binding face, in particular Arg-111, were found to be critical for binding. Arg-111 was found to interact with the conserved aspartate side chain Asp-121, which is proposed to orientate the arginine side chain to allow specific contacts with the DNA. The conformational flexibility of the disordered B-C loop region, which forms part of the binding face, was also shown to be important. In contrast, mutation of the exposed hydrophobic side chains had a less severe effect on DNA binding. This suggests that the Arg-111 side chain may contribute to sequence-specific recognition of the CpG site rather than simply making nonspecific contacts with the phosphate backbone. The majority of missense mutations within the MBD found in the human genetic disorder Rett syndrome were shown or predicted to affect folding of the domain rather than the DNA recognition event directly. The methyl-CpG binding domain (MBD) of the transcriptional repressor MeCP2 has been proposed to recognize a single symmetrically methylated CpG base pair via hydrophobic patches on an otherwise positively charged DNA binding surface. We have tested this binding model by analysis of mutant derivatives of the MeCP2 MBD in electrophoretic mobility shift assays complemented by NMR structural analysis. Exposed arginine side chains on the binding face, in particular Arg-111, were found to be critical for binding. Arg-111 was found to interact with the conserved aspartate side chain Asp-121, which is proposed to orientate the arginine side chain to allow specific contacts with the DNA. The conformational flexibility of the disordered B-C loop region, which forms part of the binding face, was also shown to be important. In contrast, mutation of the exposed hydrophobic side chains had a less severe effect on DNA binding. This suggests that the Arg-111 side chain may contribute to sequence-specific recognition of the CpG site rather than simply making nonspecific contacts with the phosphate backbone. The majority of missense mutations within the MBD found in the human genetic disorder Rett syndrome were shown or predicted to affect folding of the domain rather than the DNA recognition event directly. methyl-CpG binding domain heteronuclear spin quantum correlation electrophoretic mobility shift assay Methylation of cytosine residues in the dinucleotide sequence CpG within mammalian DNA is associated with transcriptional repression. A family of methyl-CpG binding proteins, which are thought to mediate the biological consequences of DNA methylation, has been identified and characterized (1Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar), of which the founder member is the transcriptional repressor MeCP2. These proteins contain a conservedmethyl-CpG binding domain (MBD),1 which is 70–75 amino acids in length and exhibits 50–70% similarity between the proteins. The MBD of MeCP2 is necessary and sufficient for DNA binding in vitro and allows MeCP2 to recognize a single symmetrically methylated CpG dinucleotide in diverse sequence contexts (2Nan X. Meehan R.R. Bird A. Nucleic Acids Res. 1993; 25: 4886-4892Crossref Scopus (493) Google Scholar). Of the other MBD-containing proteins, MBD1 and MBD2 also bind to single symmetrically methylated CpG dinucleotides in vitro and act as transcriptional repressors (3Ng H.-H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 53-61Crossref Scopus (775) Google Scholar, 4Ng H.-H. Jeppesen P. Bird A. Mol. Cell. Biol. 2000; 20: 1394-1406Crossref PubMed Scopus (220) Google Scholar). Repression by MeCP2 can act at a distance from a promoter and involves the targeting of histone deacetylases via a C-terminal transcription repression domain, which interacts with the corepressor mSin3 (5Nan 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), although a deacetylase-independent mechanism may also contribute to repression (5Nan 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, 6Kaludov N.K. Wolffe A.P. Nucleic Acids Res. 2000; 28: 1921-1928Crossref PubMed Scopus (121) Google Scholar, 7Yu F. Thiesen J. Strätling W.H. Nucleic Acids Res. 2000; 28: 2201-2206Crossref PubMed Scopus (89) Google Scholar). Targeting of histone deacetylases is probably also important in transcriptional repression by MBD1 and MBD2 (3Ng H.-H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 53-61Crossref Scopus (775) Google Scholar, 4Ng H.-H. Jeppesen P. Bird A. Mol. Cell. Biol. 2000; 20: 1394-1406Crossref PubMed Scopus (220) Google Scholar). Mammalian MBD3 is found in a histone deacetylase complex, the Mi-2 or NuRD complex (8Zhang Y. Ng H.-H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (935) Google Scholar), but does not bind specifically to methylated DNA in vitro and is not localized to methylated chromosomal regions in vivo (1Hendrich B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar). MBD4 is probably a methyl-CpG-TpG mismatch-specific glycosylase (9Hendrich B. Hardeland U. Ng H.-H. Jiricny J. Bird A. Nature. 1999; 401: 301-304Crossref PubMed Scopus (528) Google Scholar). Recently, an indication of the role of MeCP2 in human development has been provided by the linking of mutations in the MeCP2 gene to the human neurodevelopmental disorder Rett syndrome (10Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3859) Google Scholar). This disorder is a childhood-onset regressive disease that causes loss of speech and hand movement, coupled with autistic behavior, microencephaly, and growth retardation and affects one in every 10,000–15,000 live female births (11Hagberg B. Aicardi J. Dias K. Ramos O. Ann. Neurol. 1983; 14: 471-479Crossref PubMed Scopus (1174) Google Scholar, 12Hendrich B. Curr. Biol. 2000; 10: 60-63Abstract Full Text Full Text PDF PubMed Google Scholar). Mutations found in the DNA of Rett syndrome patients include frameshift mutations generating truncations within the MBD, the transcription repression domain, or the C terminus of MeCP2, nonsense mutations preceding and immediately downstream of the MBD or within the transcription repression domain, or most interestingly, missense mutations within the MBD and transcription repression domain domains (10Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3859) Google Scholar, 13Amir R.E. Van den Veyver I.B. Schultz R. Malicki D.M. Tran C.Q. Dahle E.J. Philippi A. Timar L. Percy A.K. Motil K.J. Lichtarge O. Smith E.O. Glaze D.G. Zoghbi H.Y. Ann. Neurol. 2000; 47: 670-679Crossref PubMed Scopus (293) Google Scholar, 14Bienvenu T. Carrie A. de Roux P. P. L. A. M. M. J. Mol. Genet. 2000; PubMed Scopus Google Scholar, H. J. A. H. M. H. M. D. A. Mol. Genet. 2000; PubMed Scopus Google Scholar, P. F. F. Mol. Genet. 2000; PubMed Scopus Google Scholar, M. Zhang X. R.E. Zoghbi H.Y. Francke U. J. Genet. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, F. P. Zhang M. J. Genet. 2000; PubMed Scopus Google Scholar). is of particular to this of mutations have on the of the MeCP2 to of methylated DNA binding and transcriptional repression. We have the of the MeCP2 MBD by NMR Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). The domain has a that forms a an on one of the and a C-terminal and on the other the of the a loop B-C between the and with the exposed of the and and to a binding for methylated DNA. on this and the of the DNA binding by NMR a model was proposed in which a conserved hydrophobic of the side chains of and recognize the of the methylated cytosine residues in the Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). A NMR on the MBD of the repressor MBD1 M. M. J. 1999; 18: PubMed Scopus Google a that the MBD-containing proteins are to recognize DNA in an These also proposed a model for DNA recognition and that mutations in the conserved and charged side chains within the binding recognition in mobility shift In this have mutant proteins in mobility to the model for DNA recognition by the MBD domain of MeCP2. In have the critical role of residues and the loop on the DNA binding of the in recognition of methylated DNA. In contrast, that the conserved hydrophobic side chains in the binding a less role in the We have also the that missense mutations within the MeCP2 MBD associated with Rett syndrome have on the folding of the domain and on DNA binding and have structural on the MBD to for the of a of This to that the majority of missense mutations within the MeCP2 MBD associated with Rett syndrome affect the of the domain to interact with methylated CpG dinucleotides via a of the domain rather than mutation of side chains in DNA The MeCP2 MBD was from the X. Bird A.P. Nat. Genet. PubMed Scopus Google the and and the was with and to This residues of MeCP2 with the sequence The of MeCP2 is that as the for recognition of methyl-CpG (2Nan X. Meehan R.R. Bird A. Nucleic Acids Res. 1993; 25: 4886-4892Crossref Scopus (493) Google and is to that for of the NMR of the MBD Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google that the C-terminal residues of the MBD, the was with and as a to the of a of was of and at to of the were with and for were in and in binding were by and was by at for were with binding with binding and with of binding were with binding and with of binding by of binding the majority of the in the of The were of and the was as by were by the assay the of for NMR were in with and and as from the was as that binding and was The from the were and to a of A symmetrically methylated at a single site on and B. Bird A. Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google was with an to the of the pair was proteins were with as a nonspecific in in a of at for was and the were on at for in at were in and the were by by at for were a DNA to the by to were were on a in at in a phosphate to and effect were on a NMR were with the the at the of with the of an J. Google Scholar, Van T. PubMed Scopus Google was to and for The for MBD of MeCP2 was Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). on mutant MeCP2 MBD proteins, was to the majority of and of the mutant were from effect on the The shift for were is the shift and and are the in between and mutant The of heteronuclear from with a and a was to the of the MBD mutant J. PubMed Scopus Google Scholar). We a of mutations within the MBD that charged and hydrophobic and side chains predicted to be important for the of the within the domain These mutant proteins were for binding to a methylated a symmetrically methylated CpG dinucleotide at and DNA of and this the MBD is in the mutant proteins a of binding from to of the the for the not Of the mutations a in DNA the and or were of particular is at the of the disordered B-C which otherwise to and Asp-121, which had predicted to be in with arginine side chains on the DNA binding Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), was shown to be critical for as mutation to or the DNA The mutation was also to a in binding was found to be to in of Rett syndrome T. Carrie A. de Roux P. P. L. A. M. M. J. Mol. Genet. 2000; PubMed Scopus Google Scholar, P. F. F. Mol. Genet. 2000; PubMed Scopus Google Scholar), and the in binding by this may contribute to the in the MBD of MeCP2 and DNA binding of the acids in the are by single with for MeCP2. amino acids are in and residues in of Rett syndrome are by of the residues within the of the MBD is side chains that contribute to the hydrophobic are by of the amino within the MBD family is shift shift of the in the MeCP2 MBD with DNA binding is as the of DNA at a of by not acids in the are by single with for MeCP2. amino acids are in and residues in of Rett syndrome are by The of the residues within the of the MBD is side chains that contribute to the hydrophobic are by of the amino within the MBD family is The shift of the in the MeCP2 MBD with DNA Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google DNA binding is as the of DNA at a of by in a not a mutation of within the disordered B-C loop to had a effect on DNA binding This is in to the of other mutations within this loop the The of the with a is predicted to of the B-C loop the MBD domain to DNA. the of the effect of this on a analysis with the mutant binding was at the that binding was than that of the MBD, which binding at that flexibility in the B-C loop is a critical of DNA recognition by the MBD, the of this to the of DNA and contacts with the phosphate Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). NMR analysis of the of the mutant in the loop from residues with the structural of this loop as a of the mutation In contrast, the of the of the MBD that the as analysis of the of the loop in the mutant that is disordered that the specific rather than a in is also that the a with the DNA in the with that a of between the B-C loop and the DNA M. M. J. 1999; 18: PubMed Scopus Google Scholar, Wolffe A.P. 2000; PubMed Scopus (121) Google Scholar). that an conformational in this of the DNA to the binding the that was critical for DNA binding and had been that this side chain an important with the Arg-111 or side chains Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), the mutant was with the domain by NMR analysis of the shift between the and mutant proteins for residues the domain that this mutation causes in the of the B-C loop and the C terminus of the domain, which on of of which is a the Arg-111 not be in the mutant by the shift was not the Arg-111 side chain were by the mutation and from an shift to a in the by the majority of arginine side chain This suggests that the of is with Arg-111 and that the effect of the and mutations on DNA binding may be to the of this the critical role of Arg-111 in DNA binding that the the arginine side chain that can interact with the DNA We the Arg-111 of the MeCP2 MBD to and the effect of the mutation on DNA binding. The mutant for methylated DNA at the tested although the domain with in the of Arg-111 and the B-C loop We also that the mutant other MBD derivatives to not These are the most severe for single MBD missense mutation and that the provided by the Arg-111 side chain is for the of the domain with DNA and the charged also suggests that the binding for the and may in part be to on the of this arginine side A arginine side that of also from the DNA binding of the domain, is conserved in the MBD and a shift DNA binding Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). This is found in a of Rett syndrome (10Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3859) Google Scholar). We a mutant MBD the mutation and effect on binding to that of the Arg-111 binding to methylated DNA at a of and had on the of the domain the the mutation not affect the shift of the that interacts with Arg-111 rather than We also that mutation of to had a less effect on binding than the at this that the of this arginine side chain does not contribute to the DNA although the binding of the mutant probably for as in Rett this side chain is not of to Arg-111 in binding binding by hydrophobic and Rett of the MeCP2 acids are by single with for MeCP2. amino acids are in and residues in of Rett syndrome are by of the residues within the of the MBD is side chains that contribute to the hydrophobic are by of the amino within the MBD family is shift shift of the in the MeCP2 MBD with DNA binding is as the of DNA at the as by acids are by single with for MeCP2. amino acids are in and residues in of Rett syndrome are by The of the residues within the of the MBD is side chains that contribute to the hydrophobic are by of the amino within the MBD family is The shift of the in the MeCP2 MBD with DNA Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google DNA binding is as the of DNA at the as by in a is to that the in a shift from the in the mutant was in the C by an in In the of the to the was not at or in the of the had or to a in In is that to This is one of hydrophobic side chains within the DNA binding of MBD proteins that proposed to an site for the of a methylated CpG dinucleotide Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). of the of in to binding M. M. J. 1999; 18: PubMed Scopus Google Scholar), although is by a in the The shift of may be by the mutation to the of the side In MeCP2 the side chain also to the hydrophobic Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), and that mutation of this side chain to the DNA binding We and of MeCP2 to and the binding of the mutant proteins to methylated DNA mutant proteins binding although in this was than the and the majority of the was at the and mutant had as severe an effect on binding as of the arginine or to a charged of the hydrophobic a severe effect on but with methylated DNA was The mutation had structural on the domain to NMR not with and binding to although the and side chains contribute to the with methylated is in this of the recognition The role of side chains as Arg-111 to a with the flexibility of the B-C loop is important in binding to the the methylated interact with the residues in the DNA binding and the for methylated DNA is a to be by the of the the which has a effect on DNA binding is found in of Rett We other missense mutations within the MBD identified in the of MeCP2 mutations in Rett syndrome R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3859) Google to also DNA binding. acids and have side chains that contribute to the hydrophobic of the MBD domain Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google and are to and in specific Rett syndrome of the mutation have been identified R.E. Van den Veyver I.B. Schultz R. Malicki D.M. Tran C.Q. Dahle E.J. Philippi A. Timar L. Percy A.K. Motil K.J. Lichtarge O. Smith E.O. Glaze D.G. Zoghbi H.Y. Ann. Neurol. 2000; 47: 670-679Crossref PubMed Scopus (293) Google Scholar, P. F. F. Mol. Genet. 2000; PubMed Scopus Google F. P. Zhang M. J. Genet. 2000; PubMed Scopus Google Scholar), and and mutations have also been found in Rett syndrome patients T. Carrie A. de Roux P. P. L. A. M. M. J. Mol. Genet. 2000; PubMed Scopus Google Scholar, F. P. Zhang M. J. Genet. 2000; PubMed Scopus Google Scholar). The was found to bind methylated DNA the although a complex at not shift the to The that a of the to the may have been and NMR of of mutant proteins the domain to be of the is that the mutant in particular may be in the assay by the of DNA and of by the at which the is and by the effect of the a of the mutation in the of the MeCP2 in which mobility shift were at to binding of the mutant the MBD Wolffe A.P. 2000; PubMed Scopus (121) Google Scholar). The of the and proteins in vivo at is to in DNA for the In contrast, a which has been found in Rett syndrome (10Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3859) Google Scholar, 13Amir R.E. Van den Veyver I.B. Schultz R. Malicki D.M. Tran C.Q. Dahle E.J. Philippi A. Timar L. Percy A.K. Motil K.J. Lichtarge O. Smith E.O. Glaze D.G. Zoghbi H.Y. Ann. Neurol. 2000; 47: 670-679Crossref PubMed Scopus (293) Google Scholar, 14Bienvenu T. Carrie A. de Roux P. P. L. A. M. M. J. Mol. Genet. 2000; PubMed Scopus Google Scholar, P. F. F. Mol. Genet. 2000; PubMed Scopus Google Scholar, F. P. Zhang M. J. Genet. 2000; PubMed Scopus Google Scholar), had for the methylated This has also been shown in the of MeCP2 Wolffe A.P. 2000; PubMed Scopus (121) Google and is as the side chain is on the side of the domain from the DNA binding and has role in or may be in between the MBD and other domains of MeCP2 or other proteins, which affect the of the In this have amino side chains within the DNA binding of the MeCP2 MBD that an important to the recognition of methylated DNA. is from assays that the arginine which is conserved in the MBD a critical role in DNA as mutation in an MBD although has for DNA. Arg-111 to be in an with the conserved aspartate side chain Asp-121, which is also to and as was Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), this may orientate the arginine that is in the to bind DNA. the severe effect of Arg-111, is that DNA is a specific one a in the methylated CpG recognition sequence rather than a nonspecific one with the phosphate backbone. of via a of this is an important of DNA binding by the of the M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, mutation of to found to in of Rett an MBD with but for methylated a at had a effect on DNA binding. We have also an important role for flexibility in the disordered B-C loop in DNA binding. mutations within the loop not affect a which is predicted to the conformational flexibility in this of the for DNA The mutation is by NMR to affect the of the loop as but to the of the MBD This is with the that the B-C loop may to the of the DNA as the that may be by this is the critical side chain of Arg-111, although the of in the loop does not have a severe effect as the of the the mutant has for DNA at of than is that a nonspecific is with DNA by in the loop as and Arg-111 has a specific role to The hydrophobic residues exposed on the DNA binding face, and in MeCP2, have been shown to contribute to the of binding to methylated DNA. in to from the model Smith X. A. A. D. Bird A.P. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, M. M. J. 1999; 18: PubMed Scopus Google Scholar), to be and for methylated DNA is are severe on binding can be which the phosphate are at and although binding is not This suggests that for the exposed in the of the DNA may be a of hydrophobic within the this and the that a charged Arg-111, to be critical for DNA is also that the of the arginine side chain may contribute to recognition of the methylated have the that Rett missense mutations within the MBD have on DNA binding In were found to to a in binding but in one was this a effect of a in This is the Rett syndrome mutations tested and in binding structural effect on the A of the mutation in the of MeCP2 has that may an Wolffe A.P. 2000; PubMed Scopus (121) Google Scholar), but the analysis in that does not a of structural In contrast, NMR analysis of the mutant MBD in the of the domain, that is to the the and mutations affect the and of the domain, to NMR as for side chains that contribute to the hydrophobic in particular DNA binding in the that the domain is not The to found in a Rett syndrome also a in DNA binding but a which has been found in Rett not to affect DNA binding The most for the Rett syndrome of this mutation is that the side which is on the side of the MBD domain from the DNA binding is important for between this domain and the of the MeCP2 this other Rett mutations have been found within the These include at or and R.E. Van den Veyver I.B. Schultz R. Malicki D.M. Tran C.Q. Dahle E.J. Philippi A. Timar L. Percy A.K. Motil K.J. Lichtarge O. Smith E.O. Glaze D.G. Zoghbi H.Y. Ann. Neurol. 2000; 47: 670-679Crossref PubMed Scopus (293) Google Scholar, H. J. A. H. M. H. M. D. A. Mol. Genet. 2000; PubMed Scopus Google P. F. F. Mol. Genet. 2000; PubMed Scopus Google Scholar), which probably have structural on the domain between the A and and at the terminus of the aspartate to mutations at and the C terminus of the have also been found in Rett patients R.E. Van den Veyver I.B. Schultz R. Malicki D.M. Tran C.Q. Dahle E.J. Philippi A. Timar L. Percy A.K. Motil K.J. Lichtarge O. Smith E.O. Glaze D.G. Zoghbi H.Y. Ann. Neurol. 2000; 47: 670-679Crossref PubMed Scopus (293) Google Scholar, F. P. Zhang M. J. Genet. 2000; PubMed Scopus Google although the is conserved in the MBD mutation is predicted to have a severe effect on the of the domain In to R.E. Van den Veyver I.B. Schultz R. Malicki D.M. Tran C.Q. Dahle E.J. Philippi A. Timar L. Percy A.K. Motil K.J. Lichtarge O. Smith E.O. Glaze D.G. Zoghbi H.Y. Ann. Neurol. 2000; 47: 670-679Crossref PubMed Scopus (293) Google Scholar), is not in in DNA binding. the between the MBD and other domains of the other be by the and that in the missense mutations found in Rett syndrome may in structural in the domain rather than specifically targeting side chains in DNA the the be that the mutation at within the DNA the MBD probably with of the mutations identified at CpG be to the by of the site mutations in this as the loop mutation and the severe in an in vivo model We for with NMR and for DNA and for and and for on the