Molecular Basis For Specific MCG And Non-mCG DNA Binding By The MBD Domain

DNA methylation is a fundamental epigenetic modification mark,which is essential for normal development and contributes to various biological processes,such as X-chromosome inactivation,genomic imprinting,and transcriptional repression.Cytosine methylation occurs prevalently at CG dinucleotide sites,with about 70%of CG sites being subject to methylation in the human genome.Nevertheless,cytosine methylation is also present at CH(H=A,T,or C)sites,which accounts for about 25%of the total cytosine methylation in both embryonic stem cells and neurons,contributing to transcriptional repression and imprinting,similar to CG methylation.Non-CG methylation occurs in virtually all human tissues and is associated with repression of development-related genes during differentiation of adult stem cells.The mCG-mediated transcriptional repression is through its binding to a family of proteins containing the MBD domain(Methyl-CpG-binding domain),a specific methyl-CpG binding domain of about 70 residues.11 MBD domains have been identified in human so far,including MeCP2(methyl-CpG-binding protein 2),MBD1-6,SETDB1/2(Histone-lysine N-methyltransferase SETDB1/2)and BAZ2A/B(Bromodomain adjacent to zinc finger domain protein 2A/B).In addition to methylated CG DNA,it has been reported that MeCP2 is able to recognize methylated CH(H=A,T or C)DNA and unmethylated Matrix/scaffold attachment regions(MARs/SARs)DNA.Therefore,this thesis systematically analyzed the structural characteristics and binding patterns of MeCP2 and MBD1-4 MBD domains to different DNA through sequence alignment,ITC binding experiments,mutagenesis and X-ray crystal structure analysis.We systematically compared the binding affinities of the MBD domains of human MeCP2 and MBD 1-4 with different DNAs containing mCG(different lengths and sequences),mCH and unmodified CH(H=A,C or T)motifs.Our ITC results showed that the MBD domains of MeCP2 and MBD1-4 bind to mCG sequences without sequence selectivity.To further clarify the molecular basis,we analyzed crystal structures of the MBD2 MBD domain in complex with two different mCG(mCGG and mCGT)DNAs.Our crystal structures indicate that base-specific protein-DNA interactions are primarily restricted by the mCG nucleotide motif.In addition to mCG DNA,we found that the MBD domain also recognizes mCA or CAC DNAs,but shows very weak affinity to mCH DNAs.The binding trend of the MBD domains with different DNA sequences is mCG>mCAC>mCAH-CAC.To further understand the molecular mechanism of MBD proteins binding to mCH and CA,we-determined the complex structures of MBD2-mCAT,MBD2-mCAC/GTG,MBD2-CAC/GTG and MeCP2-CAC/GTG.We found that MBD domains bind to mCA or CA DNA by recognizing the complementary TG dinucleotide,which explains why MeCP2 exhibits similar binding affinity to both mCA and hmCA dsDNAs.MeCP2 was firstly identified as a methyl-CpG DNA binding protein and involved in transcription repression.Interestingly,go back to 1991,even before MeCP2 was identified as a methyl-CpG binding protein,the chicken homolog of MeCP2,named as ARBP(Attachment region binding protein),has been found to bind to MARs/SARs without any CpG dinucleotide sequence,and MeCP2 specifically recognizes DNA sites containing a central sequence motif of 5'-GGTGT-3' on MARs/SARs.In this study,we investigated how MeCP2 recognizes unmethylated 5'-CAC/GTG-3'motif containing DNA by binding and structural studies.We found that MeCP2-MBD binds to MARs DNA with a comparable binding affinity to mCG DNA,and the MeCP2-CAC/GTG complex structure revealed that MeCP2 residues R111 and R133 form base-specific interactions with the GTG motif.For comparison,we also determined crystal structures of the MeCP2-MBD bound to mCG and mCAC/GTG DNA,respectively.Together,these crystal structures illustrate the adaptability of the MeCP2-MBD toward the GTG motif as well as the mCG DNA,and also provide structural basis of a biological role of MeCP2 as a transcription activator and its disease implications in Rett syndrome.The structures of MeCP2 and MBD1/2/4 MBD domain binding to mCG DNA have been extensive studies.MBD3 has been reported to be the only member of the MBD protein family does not bind to methylated DNA.However,recent reports show that MBD3 recognizes both mCG and hmCG DNA with a preference for hmCG,and our ITC result also showed that MBD3 is able to bind to mCG DNA.In order to reconcile the contradictory interpretations of MBD3 binding to mCG DNA,in this study,we carried out structural analysis coupled with isothermal titration calorimetry(ITC)binding assay and mutagenesis studies to address the structural basis for the mCG DNA binding ability of the MBD3 MBD domain.We found that the MBD3 MBD domain prefers binding mCG over hmCG through the conserved arginine fingers,and this MBD domain as well as other mCG binding MBD domains can recognize the mCG duplex without orientation selectivity.Furthermore,we found that the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for MBD3's weaker mCG DNA binding ability compared to other mCG binding MBD domains.In summary,our study demonstrates that the MBD3 MBD domain is a mCG binder,and also illustrates its binding mechanism to the methylated CG DNA.Our systematic study of MBD domians of MeCP2 and MBD 1-4 changes our traditional understanding that MBD protein is a mCG recognition protein,and reframes our understanding of genome-wide activity of MBD proteins in gene regulation.