The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties.
The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD-DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16(INK4a) genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.
Fraga
Mario F
MF
Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Center (CNIO), C/ Melchor Fernandez Almagro, no. 3, E-28029, Madrid, Spain.
Ballestar
Esteban
E
Montoya
Guillermo
G
Taysavang
Panya
P
Wade
Paul A
PA
Esteller
Manel
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002868:Chromosomal Proteins, Non-Histone; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007700:Kinetics; D051783:Methyl-CpG-Binding Protein 2; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014981:Xenopus
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C115369:MBD3 protein, human; C497392:MECP2 protein, human; C492013:Mbd3 protein, mouse; C497393:Mecp2 protein, mouse; D051783:Methyl-CpG-Binding Protein 2; D009841:Oligonucleotides; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA