MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density.. Mus musculus

Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C>T transitions resulting from spontaneous deamination of 5’-methylcytosine. Despite its significance, this DNA repair pathway is still poorly understood. Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. The described data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and illustrates how MBD4 functions in normal and pathological conditions. Overall design: Immunoprecipitation of 5mC- and 5hmC-modified DNA isolated from WT (Mbd4+/+) and KO (Mbd4-/-) mouse embryonic fibroblasts. 10 µg of DNA was used as input, and 2 µl of 5mC antibody (Active Motif, 39649) or 4 µl of 5hmC antibody (Active Motif, 39791) was used to immunoprecipitate modified DNA. Four independent DNA immunoprecipitations were pooled for each condition before sequencing analysis.