Methyl Cpg Binding Proteins: Coupling Chromatin Architecture to Gene Regulation

Methyl CpG binding proteins: coupling chromatin architecture to gene regulation

Paul A Wade*,1

1Emory University School of Medicine, Department of Pathology and Laboratory Medicine, Woodru Memorial Research Building ± Room 7105B, 1639 Pierce Drive, Atlanta, GA 30322, USA

A correlation between DNA methylation and transcrip- been substantiated via mutation of the methyltransfer- tional silencing has existed for many years. Recently, ase enzymes themselves which leads to developmental substantial progress has been reported in the search for defects and death prior to or shortly after birth (Okano proteins that interpret the regulatory information et al., 1999; Li et al., 1992). Several excellent recent inherent in DNA methylation and translate this informa- reviews describe the DNA methyltransferases and their tion into functional states, resulting in the identi®cation biology (Bestor, 2000; Robertson and Wole, 2000; of a family of highly conserved proteins, the MBD Hendrich and Bird, 2000), this review will focus instead family. Direct connections between these proteins and on proteins that function downstream of the DNA histone modi®cation enzymes have emerged as a common methylation signal. While higher plants clearly possess theme, implying that DNA methylation exerts its eects sophisticated gene regulatory circuits featuring DNA primarily through repressive chromatin architecture. methylation, this review will focus on methyl CpG Recent structural determinations of the DNA binding binding proteins in animals. domain of two MBD family members, MeCP2 and Formally, DNA methylation might lead to transcrip- MBD1, provide a framework to model the interactions of tional repression through multiple mechanisms. Methy- this family with DNA. Comparative sequence analysis lation is known to interfere with the ability of some and experimental DNA binding data can be interpreted transcription factors to bind their cognate recognition using this structural framework allowing one to contrast sequences. It might also result in structural eects on the members of the MBD family with each other and to nucleosomes themselves or eects on nucleosome predict the properties of new family members. The positioning, nucleosome stability, or assembly of higher identi®cation of mutations in MeCP2, the founding order chromatin structures. However, work over the member of this family, as causal for the neurological last decade has led to the accumulation of a body of developmental disorder Rett Syndrome provides addi- data suggesting that the functional properties of tional information regarding amino acid residues crucial methylated DNA result primarily from the action of to the functions of this interesting protein family. a conserved family of proteins that selectively bind Oncogene (2001) 20, 3166 ± 3173. methylated CpG dinucleotides (Bird and Wole, 1999).

Keywords: DNA methylation; MeCP2; Rett Syndrome; histone deacetylase; chromatin MeCP2 ± the prototype methyl CpG binding protein

The prototype methyl CpG binding protein is MeCP2, a polypeptide capable of binding selectively to a single Methylation is a common form of DNA modi®cation symmetrically methylated CpG (Lewis et al., 1992). in animals, occurring at the position ®ve of cytosine This protein is associated with chromosomes through- residues almost exclusively within the context of CpG out the cell cycle, colocalizes with methyl CpG rich dinucleotides. CpG methylation is non-random and the DNA (Lewis et al., 1992), and consists of two majority of potential sites in mammalian genomes are functional domains (Figure 1). The methyl CpG modi®ed (Cooper and Krawczak, 1989). Methylation binding domain or MBD is sucient to direct speci®c participates in the partitioning of genomes into active binding to methylated DNA (Nan et al., 1993). and inactive functional compartments (Cross and Bird, Regions outside the MBD contribute to overall binding 1995; Razin, 1998). The constitutents of the inactive energy through non-speci®c, presumably electrostatic compartment associated with DNA methylation in- interactions (Meehan et al., 1992). A second functional clude imprinted genes, the inactive female X chromo- domain, the transcriptional repression domain or some, and parasitic DNA elements (Bestor, 2000). The TRD, is required for transcriptional repression in vitro essential nature of DNA methylation in mammals has and in vivo (Nan et al., 1997; Jones et al., 1998; Kaludov and Wole, 2000). As expected for a chromosomal protein, MeCP2 is released from nuclei *Correspondence: PA Wade by treatment with nucleases or by extraction with salt, Methyl CpG binding proteins PA Wade 3167

Figure 1 Mammalian MBD family members. The ®gure depicts the mammalian MBD family in cartoon fashion. Notable sequence motifs are indicated in the ®gure and discussed in the text although it presents a biphasic extraction pro®le from containing DNA results in changes in the resonance of rat brain nuclei (Meehan et al., 1992). In addition, several residues in this loop, in b strands 2 ± 4 and in puri®ed recombinant Xenopus MeCP2 binds to methyl the alpha helix. These residues de®ne a surface of the CpG dinucleotides in a nucleosomal context. The wedge that likely interacts with DNA (Wake®eld et al., isolated MBD domain retains the capacity to bind 1999). This surface presents a set of basic residues (in methylated nucleosomes albeit with reduced anity strand b2 and the ¯exible loop) immediately ¯anking a compared to the intact protein (Chandler et al., 1999). hydrophobic patch in strand b3. Additional basic Several lines of evidence contributed to the notion residues line the opposite side of the hydrophobic that MeCP2 might function to repress transcription patch (Figure 2). The importance of structural within a chromatin infrastructure. Methylated DNA ¯exibility in the loop has been con®rmed by mutation injected into either mammalian cells or into Xenopus of a conserved glycine residue to proline, which results oocyte nuclei is transcriptionally repressed relative to in a minimally 25-fold reduction in binding anity unmethylated controls. This repression, however, re- (Free et al., 2000). Three solvent exposed hydrophobic quires sucient time to permit chromatin assembly residues (Tyrosine 123, Isoleucine 125, and Alanine (Buschausen et al., 1987; Kass et al., 1997). Further, 131) are postulated to interact with the methyl groups reactivation of genes silenced by aberrant promoter in the major groove, mutation of these residues methylation in cancer cell lines requires inhibition of individually results in reductions in binding anity both DNA methyltransferases and histone deacetylases (Free et al., 2000). (Cameron et al., 1999). The subsequent ®nding that MeCP2 is physically associated with the transcriptional corepressor Sin3 and histone deacetylases in both The MBD family of proteins mammalian cells and Xenopus oocytes (Nan et al., 1998; Jones et al., 1998) identi®ed candidate regulatory A survey of EST databases with the MBD domain of enzymes involved in the assembly of a specialized MeCP2 led to the identi®cation of an additional family chromatin state at methylated loci. The region of member initially termed PCM1 (Cross et al., 1997). interaction of Sin3 with mammalian MeCP2 was found Subsequent searches of the expanding EST databases to be largely coincident with the previously de®ned TRD identi®ed four proteins in mammals that all possessed (Nan et al., 1998). Importantly, in both mammalian cells the MBD sequence motif, MBD1 (identical to PCM1), and Xenopus oocytes, arti®cial recruitment of the MBD2, MBD3, and MBD4 (Hendrich and Bird, 1998). MeCP2 TRD to a promoter leads to transcriptional Outside of the MBD domain itself, these proteins bear repression that is partially relieved by inhibitors of little obvious resemblance to each other (Figure 1; histone deacetylase (Nan et al., 1998; Jones et al., 1998). Hendrich and Bird, 1998). The single exception to this The solution structure of the MBD domain from rat rule is the high degree of similarity between MBD2 and MeCP2 has recently been solved (Wake®eld et al., MBD3 which are approximately 70% identical in the 1999). The MBD is a wedge shaped structure (Figure region de®ned by MBD3 (Hendrich and Bird, 1998). 2) with one face of the wedge composed of a beta sheet The MBD proteins are ubiquitously expressed in and the other face consisting of an alpha helix and somatic tissues, while ES cells fail to express MBD1 hairpin loop (Wake®eld et al., 1999). The vertex of the and express very low levels of MBD2 (Hendrich and wedge is extended by a long loop between two of the Bird, 1998). Additionally, all four of these MBD beta strands that contains several basic residues proteins are alternatively spliced with some splice (Wake®eld et al., 1999). Addition of methyl CpG variants being tissue speci®c and others clearly

Oncogene Methyl CpG binding proteins PA Wade 3168

Figure 2 Three dimensional structure of the MBD domain from MeCP2. The structural coordinates for the rat MeCP2 MBD domain (Wake®eld et al., 1999) and the human MBD1 MBD domain were utilized in the Ribbons program to generate the cartoon. Open squares superimposed on the structure are basic residues making up the putative DNA interaction surface. The `hydrophobic patch' residues are indicated by open circles. The conserved tyrosine residue in strand b3 is indicated

aecting the MBD domain (Hendrich and Bird, 1998). not a component of the previously de®ned Mi-2/NuRD The genomic structures of the human and murine and Sin3 complexes (Ng et al., 2000). MBD1-MBD4 genes have been determined, all have an Recombinant MBD1 can repress transcription of intron within the MBD domain itself and the human methylated, but not unmethylated, templates in vitro and mouse genes are highly similar in their exon/intron much like MeCP2 (Cross et al., 1997; Fujita et al., organization (Hendrich et al., 1999a). 1999). The various isoforms of MBD1 repress transcription in both mammalian and insect cells (Fujita et al., 1999; Ng et al., 2000). The carboxyl terminus of the MBD1 protein contains a transcriptional repression domain with functions analogous to that of MeCP2 despite a MBD1 is the largest member of the family and lack of obvious sequence similarity (Ng et al., 2000). contains a sequence motif, the CXXC motif, shared Like MeCP2, transcriptional repression mediated by with DNA methyltransferase I (Cross et al., 1997). The MBD1 is sensitive to inhibitors of histone deacetylase mRNA for human MBD1 is found in at least ®ve although the precise HDAC associated with MBD1 alternatively spliced forms, aecting the carboxyl remains to be elucidated (Ng et al., 2000). The multiple terminus as well as the CXXC motifs (Fujita et al., splice variants of MBD1 in human cells also appear to 1999; Ng et al., 2000). Murine MBD1 apparently does dier substantially in their aects on transcription in not express the full complement of splice variants transient assays in both mammalian and insect cells found in the human mRNA (Hendrich et al., 1999a). (Fujita et al., 1999). Speci®c binding to methylated DNA substrates The solution structure of the MBD domain of MBD1 requires only the MBD domain and the CXXC motifs has also been solved (Ohki et al., 1999). The fold of this are unimportant for DNA binding in vitro (Cross et al., domain is essentially identical to that of MeCP2, 1997). In mammalian cells, MBD1 is localized indicating that all the MBD proteins are likely to adopt throughout euchromatin with additional concentra- a similar conformation (Figure 2). The placement and tions at a subset of pericentromeric regions on mitotic orientation of charged and hydrophobic residues in chromosomes (Fujita et al., 1999; Ng et al., 2000). strands b2 and b3 and the ¯exible loop are highly similar Initially, MBD1 was de®ned as a component of the to the MeCP2 structure (Ohki et al., 1999; Wake®eld et MeCP1 complex (Cross et al., 1997). A more recent re- al., 1999). In MeCP2, three solvent exposed hydro- examination of MBD1 using new reagents revealed phobic residues were predicted to directly contact the that it is not a component of MeCP1 and that its methyl groups in the major groove (Wake®eld et al., native molecular mass from HeLa nuclear extracts is 1999). In MBD1 however, only one of these positions 200 ± 400 kDa, inconsistent with the properties of contains a hydrophobic amino acid (tyrosine 34). In MeCP1 (Ng et al., 2000). While the identities of the fact, this residue is absolutely conserved in all members proteins associated with MBD1 in this complex remain of the MBD family that bind methylated DNA. elusive, MBD1 is not depleted by antisera speci®c for Mutation of this tyrosine to alanine in MeCP2 results MTA2, HDAC1 and SAP30, implying that it is also in a 10-fold reduction in binding anity (Free et al.,

Oncogene Methyl CpG binding proteins PA Wade 3169 2000), the same mutation in the context of MBD1 sequence of MBD3 is highly similar to that of MBD2 virtually abolishes binding (Ohki et al., 1999), high- throughout its length (Hendrich and Bird, 1998). The lighting its functional importance to interaction of mRNA is relatively abundant in most somatic tissues MBD proteins with methylated DNA. It seems likely and also in ES cells (Hendrich and Bird, 1998). MBD3 that the hydrophobic residues on strand beta three presents a relatively rich variety of splice variants contact the methyl groups in the major groove of DNA (Figure 3). The MBD is encoded by portions of exons and the charged residues in strand b2, b4, and the 1 and 2 (Figure 2; Hendrich et al., 1999a). A subset of ¯exible loop interact with the DNA backbone. mammalian MBD3 RNAs utilize an upstream splice donor in the ®rst exon resulting in deletion of major portions of the MBD domain (Figure 3), this form of MBD2 the protein has been termed MBD3D (Hendrich and Bird, 1998; Zhang et al., 1999). Xenopus laevis presents MBD2 is highly similar to MBD3 in a large region an additional alternatively spliced isoform (Figure 3), corresponding roughly to amino acids 140 ± 400 MBD3 Long Form, that inserts 20 amino acids (Hendrich and Bird, 1998). However, the MBD2 between exons 1 and 2 (Wade et al., 1999). Both the mRNA codes for *140 amino acids preceding this MBD3D and MBD3 Long Form splice variants fail to conserved region, a similar sequence is not found in bind methylated DNA substrates (Wade et al., 1999; MBD3. This region of MBD2 contains a repeat Zhang et al., 1999). While MBD2 maintains the same consisting of glycine and arginine residues (Figure 1, exon/intron organization as MBD3, there are no Hendrich and Bird, 1998). Compared to MBD3, known examples of MBD2 splice variants that aect MBD2 has a more restricted pattern of expression the integrity of the MBD domain (Hendrich et al., and an alternatively spliced mRNA is evident in testis 1999a). This suggests that MBD3 has assumed at least (Hendrich and Bird, 1998). MBD2 binds methylated some functions that are unrelated to DNA methylation DNA in a manner very similar to the isolated MBD since the gene duplication event resulting in these two domain of MeCP2 (Hendrich and Bird, 1998; Wade et very similar proteins. al., 1999). Surprisingly, MBD2b (a version lacking the Indeed, the binding properties of MBD3 appear to amino terminal 140 amino acids) has been reported to vary with species. The mammalian protein will only possess DNA demethylase activity (Bhattacharya et al., bind methylated DNA in vitro under certain condi- 1999), although this ®nding has been questioned (Wade tions, even then its selectivity is poor when compared et al., 1999; Ng et al., 1999). to MBD2 or MeCP2 (Hendrich and Bird, 1998; Wade Tethering of MBD2 near a promoter via a et al., 1999). In contrast, Xenopus MBD3 binds heterologous DNA binding domain results in moderate methylated DNA with an anity quite similar to the transcriptional repression that is sensitive to Trichos- isolated MBD domain from MeCP2 (Wade et al., tatin A (Ng et al., 1999). In cotransfection experiments 1999). Careful comparison of the sequences of the MBD2b increased transcriptional repression observed MBD domain in proteins experimentally proven to on methylated reporter constructs (Boeke et al., 2000). bind selectively to methylated DNA reveals two highly Surprisingly, mapping of the portion of MBD2b conserved residues which are altered in mammalian required for transcriptional repression in this assay MBD3 (Figure 4). First, an absolutely conserved identi®ed a small region that partially overlaps the tyrosine residue in beta strand 3 is changed to MBD domain (Boeke et al., 2000). phenylalanine. This residue is largely solvent exposed Immunoprecipitation studies demonstrate that in both the MeCP2 and MBD1 structures and MBD2 is physically associated with HDAC1 in mutations result in a dramatic decrease in binding mammalian cells and implicate MBD2 as the long- (Wake®eld et al., 1999; Ohki et al., 1999). Involvement sought methyl CpG binding component of the MeCP1 of the tyrosine hydroxyl in a hydrogen bond with a complex (Ng et al., 1999). Further, while MBD2 is DNA base could account for the absolute conservation associated with HDAC1 and with RbA p48/p46, by of this residue. The second highly conserved residue coimmunoprecipitation analysis it is not a component substituted in mammalian MBD3 is a conserved lysine of the previously de®ned Sin3 and Mi-2/NURD or arginine residue near the amino terminus of strand complexes (Ng et al., 2000). However, a direct b3 (Figure 4). If this residue makes an ionic contact interaction of Sin3A with MBD2b in the region with the DNA backbone, substitution with histidine sucient to direct transcriptional repression has also might suciently decrease binding energy to result in a been described (Boeke et al., 2000). It seems likely that qualitatively poor protein-DNA interaction. Indeed, the biochemical properties of MBD2 and its associa- mutation of the homologous residue in MBD1 to tions with other proteins, like the case of MBD1, are alanine virtually abolishes DNA binding (Ohki et al., not yet fully appreciated. 1999). The amino acid sequence of Zebra®sh MBD3 very closely resembles that of Xenopus MBD3. Speci®cally, MBD3 both the conserved tyrosine and lysine/arginine residues are present in the ®sh protein. It seems likely MBD3 is the smallest member of the MBD family, that this protein, like Xenopus MBD3, will bind coding for a protein of about 30 kDa. The coding methylated DNA selectively. Should this prediction

Oncogene Methyl CpG binding proteins PA Wade 3170

Figure 3 Exon/Intron organization of the MBD domain of MBD3. The exons of various species' MBD3 are indicated in the ®gure. Exon 1 is solid black, Exon 2 is an open white rectangle. The alternative exon of MBD3 Long Form is indicated by a shaded rectangle with black outlining. Sequence present only in the Drosophila MBD homolog is indicated by shaded rectangles without outlining. Secondary structure features as extrapolated from the published structures of MeCP2 and MBD1 are depicted above each exon

Figure 4 Sequence alignment of MBD family members. The predicted protein sequences of several MBD family members are aligned in the ®gure. The upper set of proteins all bind methylated DNA speci®cally. Residues conserved in all these proteins that dier in mammalian MBD3 are in bold in the mammalian MBD3 sequence. MeCP2 residues mutated in Rett Syndrome are in bold in the human MeCP2 sequence. Secondary structures as predicted from the structures of MeCP2 and MBD1 are indicated at the top of the alignment

hold true, it suggests that at least some vertebrates MBD3 to bind methylated DNA and the prediction retain a requirement for a subset of MBD3 proteins to regarding the zebra®sh protein strongly suggest that interact with methylated DNA speci®cally. As EST recruitment of the Mi-2 complex to methylated loci sequences for MBD2 homologs exist in both these de®nes a crucial regulatory pathway during develop- organisms, this requirement is not likely to result from ment of these organisms. a lack of MBD2. However, both Zebra®sh (Macleod et al., 1999) and Xenopus (Stancheva and Meehan, 2000 and references therein) lack the global demethylation MBD4 event characteristic of early mammalian development. MBD3 is a component of a very abundant multi- MBD4 is the only known member of the MBD family protein complex containing a histone deacetylase and a not associated with histone deacetylase activity. While chromatin remodeling enzyme, the Mi-2 complex, in the MBD domain of MBD4 has the highest similarity Xenopus eggs (Wade et al., 1998). The developmental to MeCP2 in the MBD family, the carboxyl terminus transcription pattern of Xenopus and by extension of has homology to bacterial DNA repair enzymes zebra®sh undoubtedly utilizes DNA methylation in a (Hendrich and Bird, 1998). MBD4 is expressed in manner distinct from mammals. The ability of Xenopus most human tissues and splice variants are evident

Oncogene Methyl CpG binding proteins PA Wade 3171 (Hendrich and Bird, 1998). In keeping with its mutations exhibit markedly dierent symptoms similarity to MeCP2, an MBD4-GFP fusion is (Huppke et al., 2000). Several recent reports have localized at densely methylated sequences in mouse con®rmed MeCP2 mutations in males with mental cells (Hendrich and Bird, 1998). defects (Orrico et al., 2000; Meloni et al., 2000). Several lines of evidence implicate a role for MBD4 Rett syndrome mutations in MeCP2 tend to cluster in DNA repair. It interacts with the DNA repair in three distinct locations in the coding region: the protein MLH1in a two-hybrid screen (Bellacosa et al., MBD, the TRD, and the C terminus. The properties of 1999). Recombinant MBD4 induces nicks and linear- a subset of these mutations have been examined. In the ization of supercoiled plasmids (Bellacosa et al., 1999) context of Xenopus MeCP2, missense mutations in the although the protein lacks endonuclease activity on MBD domain (R106W, R133C, F155S, and T158M) oligonucleotide substrates (Hendrich et al., 1999b). reduced the ability of the protein to bind methylated MBD4 clearly has speci®c DNA N-glycosylase activity DNA (Ballestar et al., 2000). Selective binding was with a strong preference for G : T mismatches (Hen- essentially eliminated in all the mutants examined with drich et al., 1999b; Petronzelli et al., 2000). Surpris- the exception of T158M, which reduced binding anity ingly, the MBD domain does not appear to in¯uence by a factor of two (Ballestar et al., 2000). An analysis the speci®city of the glycosylase activity for G : T of Rett mutants in the context of the MBD domain of mismatches (Hendrich et al., 1999b; Petronzelli et al., rat MeCP2 revealed that the missense mutants R106W, 2000). However, while the MBD domain binds to R133C and F155S have a signi®cant eect on DNA symmetrically methylated CpG dinucleotides, it prefers binding anity (Free et al., 2000). Of these mutations, 5-methyl CpG paired with TpG (Hendrich et al., both R106W and F155S appear to reduce binding 1999b). As this particular G : T mismatch is the anity through structural perturbations in the MBD expected product of deamination of a single 5-methyl fold (Free et al., 2000). Mutation of arginine 133 to C in a symmetrically methylated CpG dinucleotide, cysteine reduces binding anity and also perturbs MBD4 has been designated a repair enzyme speci®c for tyrosine 123 ± a residue postulated to interact with the methylated DNA (Hendrich et al., 1999b; Petronzelli et methyl groups on the DNA substrate (Free et al., 2000; al., 2000). Interestingly, mutations in the MBD4 gene Wake®eld et al., 1999). Finally, the T158M mutation have been isolated in carcinomas with microsatellite had near wild-type anity for methylated DNA (Free instability, where DNA mismatch repair activity is et al., 2000). In the context of full length human defective (Bader et al., 1999; Riccio et al., 1999). MeCP2, none of the missense mutations in the MBD domain, including T158M, retained the capacity to bind methylated DNA, emphasizing the importance of Rett syndrome and MeCP2 context for functional analysis of Rett mutants (Yusufzai and Wole, 2000). As expected, truncation In October 1999, the surprising ®nding that several mutations in the TRD have no discernible eect on patients with the neurological disorder Rett Syndrome selectivity for methylated DNA, although overall had mutations in the MeCP2 gene was announced binding anity is aected (Yusufzai and Wole, (Amir et al., 1999). Rett Syndrome is a neurological 2000). A set of TRD nonsense mutations were unable disorder occurring predominantly in females character- to repress transcription in a tethering assay in Xenopus ized by normal early development followed by a period oocytes, although no defect was observed for the of regression. Patients lose speech and purposeful hand R306C missense mutation (Yusufzai and Wole, movements while acquiring a variety of neurological 2000). Interestingly, truncation mutations lacking the symptoms. Stabilization generally occurs and most intact carboxyl terminus were seen to be signi®cantly patients survive into adulthood (Rett, 1966; Hagberg et less stable in oocytes, suggesting involvement of this al., 1983; Hagberg, 1985). In the past year, several region in global folding and stability of MeCP2 groups have reported similar ®ndings, expanding the (Yusufzai and Wole, 2000). The defects observed in number of MeCP2 mutations reported to in excess of MeCP2 function as a result of these mutations lead to 90 mutations in more than 400 individuals (Amir et al., the inevitable conclusion that the neurological defects 2000; Buyse et al., 2000; Bienvenu et al., 2000; Cheadle in Rett Syndrome result from loss of MeCP2 function. et al., 2000; Hampson et al., 2000; Huppke et al., 2000; Further experiments are clearly required to de®ne Kim and Cook, 2000; Obata et al., 2000; Wan et al., precise molecular mechanisms resulting in pathology. 1999; Xiang et al., 2000). Most groups report that However, it also seems that most Rett mutations do more than 80% of classic Rett cases examined have not result in subtle changes in the properties of mutations in the X-linked MeCP2 gene. Additionally, a MeCP2, but rather catastrophically impact DNA large number of non-disease associated polymorphisms binding, protein stability, or both. in MeCP2 have been reported (see for example, Wan et al., 1999; Buyse et al., 2000). In some cases, skewed X- inactivation pro®les result in asymptomatic carrier Perspectives females (Wan et al., 1999; Amir et al., 2000; Dragich et al., 2000). There appears to be little correlation The past decade has seen considerable progress in the between phenotype of Rett patients and speci®c understanding of how DNA methylation is translated mutations in MeCP2, in fact, patients with identical into functional states in the genome. A family of

Oncogene Methyl CpG binding proteins PA Wade 3172 proteins has been identi®ed that bind to methylated The answers to these and other unresolved issues will DNA, these proteins are found in association with be crucial in understanding the functions of these key enzymes that alter the fundamental properties of regulatory molecules. chromatin. At a super®cial level, one can attribute the functional properties of methylated DNA to these MBD associated proteins. However, a number of interesting questions remain unanswered. Only one member of the family, MBD3, has been described in Acknowledgments biochemical detail. Proteins associated with MBD1 and I thank Timur Yusufzai and Alan Wole for communica- MBD2, in particular, remain to be identi®ed. A second tion of results prior to publication. I thank Dr Paula major unanswered question is the distribution of MBD Vertino and Dr Xiaodong Cheng for critical reading of this manuscript. Research in my laboratory is supported by proteins within the genome. While these ®ve proteins grants from the National Institute of Child Health and share a common DNA binding surface and recognize Human Development (1 K22 HD01238-01) and from the the same binding site, they are clearly dierentially Rett Syndrome Research Foundation. I gratefully ac- partitioned. Finally, what are the consequences of knowledge The Massachusetts Rett Syndrome Association disruption of this family, as occurs in Rett Syndrome? for ®nancial support.

References

Amir RE, Van den Veyver IB, Schultz R, Malicki DM, Tran Fujita N, Takebayashi S, Okumura K, Kudo S, Chiba T, CQ, Dahle EJ, Philippi A, Timar L, Percy AK, Motil KJ, Saya H and Nakao M. (1999). Mol. Cell. Biol., 19, 6415 ± Lichtarge O, Smith EO, Glaze DG and Zoghbi HY. 6426. (2000). Ann. Neurol., 47, 670 ± 679. Hagberg B. (1985). Acta Paediatr. Scand., 74, 405 ± 408. AmirRE,VandenVeyverIB,WanM,TranCQ,FranckeU Hagberg B, Aicardi J, Dias K and Ramos O. (1983). Ann. and Zoghbi HY. (1999). Nat. Genet., 23, 185 ± 188. Neurol., 14, 471 ± 479. Bader S, Walker M, Hendrich B, Bird A, Bird C, Hooper M Hampson K, Woods CG, Latif F and Webb T. (2000). J. and Wyllie A. (1999). Oncogene, 18, 8044 ± 8047. Med. Genet., 37, 610 ± 612. Ballestar E, Yusufzai TM and Wole AP. (2000). Biochem- Hendrich B and Bird A. (1998). Mol. Cell. Biol., 18, 6538 ± istry, 39, 7100 ± 7106. 6547. Bellacosa A, Cicchillitti L, Schepis F, Riccio A, Yeung AT, Hendrich B and Bird A. (2000). Curr. Top. Microbiol. MatsumotoY,GolemisEA,GenuardiMandNeriG. Immunol., 249, 55 ± 74. (1999). Proc. Nat. Acad. Sci. USA, 96, 3969 ± 3974. Hendrich B, Abbott C, McQueen H, Chambers D, Cross S Bestor TH. (2000). Hum. Mol. Genet., 9, 2395 ± 2402. and Bird A. (1999a). Mamm. Genome, 10, 906 ± 912. Bhattacharya SK, Ramchandani S, Cervoni N and Szyf M. HendrichB,HardelandU,NgHH,JiricnyJandBirdA. (1999). Nature, 397, 579 ± 583. (1999b). Nature, 401, 301 ± 304. BienvenuT,CarrieA,deRouxN,VinetMC,JonveauxP, Huppke P, Laccone F, Kramer N, Engel W and Hanefeld F. Couvert P, Villard L, Arzimanoglou A, Beldiord C, (2000). Hum. Mol. Genet., 9, 1369 ± 1375. Fontes M, Tardieu M and Chelly J. (2000). Hum. Mol. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Genet., 9, 1377 ± 1384. Landsberger N, Strouboulis J and Wole AP. (1998). Nat. Bird AP and Wole AP. (1999). Cell, 99, 451 ± 454. Genet., 19, 187 ± 191. Boeke J, Ammerpohl O, Kegel S, Moehren U and Renkawitz Kaludov NK and Wole AP. (2000). Nucl. Acids Res., 28, R. (2000). J. Biol. Chem., 275, 34963 ± 34967. 1921 ± 1928. Buschhausen G, Wittig B, Graessmann M and Graessmann Kass SU, Landsberger N and Wole AP. (1997). Curr. Biol., A. (1987). Proc.Natl.Acad.Sci.USA,84, 1177 ± 1181. 7, 157 ± 165. Buyse IM, Fang P, Hoon KT, Amir RE, Zoghbi HY and Roa Kim SJ and Cook Jr EH. (2000). Hum. Mutat. (Online), 15, BB. (2000). Am.J.Hum.Genet.,67, 1428 ± 1436. 382 ± 383. CameronEE,BachmanKE,MyohanenS,HermanJGand Lewis JD, Meehan RR, Henzel WJ, Maurer-Fogy I, Baylin SB. (1999). Nat. Genet., 21, 103 ± 107. Jeppesen P, Klein F and Bird A. (1992). Cell, 69, 905 ± 914. Chandler SP, Guschin D, Landsberger N and Wole AP. Li E, Bestor TH and Jaenisch R. (1992). Cell, 69, 915 ± 926. (1999). Biochemistry, 38, 7008 ± 7018. Macleod D, Clark VH and Bird A. (1999). Nat. Genet., 23, Cheadle JP, Gill H, Fleming N, Maynard J, Kerr A, Leonard 139 ± 140. H, Krawczak M, Cooper DN, Lynch S, Thomas N, Meehan RR, Lewis JD and Bird AP. (1992). Nucl. Acids Hughes H, Hulten M, Ravine D, Sampson JR and Clarke Res., 20, 5085 ± 5092. A. (2000). Hum. Mol. Genet., 9, 1119 ± 1129. Meloni I, Bruttini M, Longo I, Mari F, Rizzolio F, D'Adamo Cooper DN and Krawczak M. (1989). Hum. Genet., 83, 181 ± P, Denvriendt K, Fryns JP, Toniolo D and Renieri A. 188. (2000). Am.J.Hum.Genet.,67, 982 ± 985. Cross SH and Bird AP. (1995). Curr. Opin. Genet. Dev., 5, Nan X, Campoy FJ and Bird A. (1997). Cell, 88, 471 ± 481. 309 ± 314. Nan X, Meehan RR and Bird A. (1993). Nucl. Acids Res., 21, Cross SH, Meehan RR, Nan X and Bird A. (1997). Nat. 4886 ± 4892. Genet., 16, 256 ± 259. Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Dragich J, Houwink-Manville I and Schanen C. (2000). Eisenman RN and Bird A. (1998). Nature, 393, 386 ± 389. Hum. Mol. Genet., 9, 2365 ± 2375. Ng HH, Jeppesen P and Bird A. (2000). Mol. Cell. Biol., 20, Free A, Wake®eld RI, Smith BO, Dryden DT, Barlow PN 1394 ± 1406. and Bird AP. (2000). J. Biol. Chem., 276, 3353 ± 3360.

Oncogene Methyl CpG binding proteins PA Wade 3173 NgHH,ZhangY,HendrichB,JohnsonCA,TurnerBM, Robertson KD and Wole AP. (2000). Nat. Rev. Genet., 1, Erdjument-Bromage H, Tempst P, Reinberg D and Bird 11 ± 19. A. (1999). Nat. Genet., 23, 58 ± 61. Stancheva I and Meehan RR. (2000). Genes Dev., 14, 313 ± Obata K, Matsuishi T, Yamashita Y, Fukuda T, Kuwajima 327. K, Horiuchi I, Nagamitsu S, Iwanaga R, Kimura A, WadePA,GegonneA,JonesPL,BallestarE,AubryFand Omori I, Endo S, Mori K and Kondo I. (2000). J. Med. Wole AP. (1999). Nat. Genet., 23, 62 ± 66. Genet., 37, 608 ± 610. Wade PA, Jones PL, Vermaak D and Wole AP. (1998). Ohki I, Shimotake N, Fujita N, Nakao M and Shirakawa M. Curr. Biol., 8, 843 ± 846. (1999). EMBO J., 18, 6653 ± 6661. Wake®eld RI, Smith BO, Nan X, Free A, Soteriou A, Uhrin Okano M, Bell DW, Haber DA and Li E. (1999). Cell, 99, D, Bird AP and Barlow PN. (1999). J. Mol. Biol., 291, 247 ± 257. 1055 ± 1065. Orrico A, Lam C, Galli L, Dotti MT, Hayek G, Tong SF, Wan M, Lee SS, Zhang X, Houwink-Manville I, Song HR, Poon PM, Zappella M, Federico A and Sorrentino V. Amir RE, Budden S, Naidu S, Pereira JL, Lo IF, Zoghbi (2000). FEBS Lett., 481, 285 ± 288. HY, Schanen NC and Francke U. (1999). Am. J. Hum. Petronzelli F, Riccio A, Markham GD, Seeholzer SH, Genet., 65, 1520 ± 1529. Stoerker J, Genuardi M, Yeung AT, Matsumoto Y and Xiang F, Buervenich S, Nicolao P, Bailey ME, Zhang Z and Bellacosa A. (2000). J. Biol. Chem., 275, 32422 ± 32429. Anvret M. (2000). J. Med. Genet., 37, 250 ± 255. Razin A. (1998). EMBO J., 17, 4905 ± 4908. Yusufzai TM and Wole AP. (2000). Nucl. Acids Res., 28, Rett A. (1966). Wien. Med. Wochenschr., 116, 723 ± 726. 4172 ± 4179. Riccio A, Aaltonen LA, Godwin AK, Loukola A, Percesepe Zhang Y, Ng HH, Erdjument-Bromage H, Tempst P, Bird A A, Salovaara R, Masciullo V, Genuardi M, Paravatou- and Reinberg D. (1999). Genes Dev., 13, 1924 ± 1935. Petsotas M, Bassi DE, Ruggeri BA, Klein-Szanto AJ, Testa JR, Neri G and Bellacosa A. (1999). Nat. Genet., 23, 266 ± 268.

Oncogene