Abstract
DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.
Highlights
In vertebrates, DNA methylation at transcriptional start sites (TSSs) is an epigenetic modification associated with the downregulation of gene transcription [1]
We identified methylated DNA regions by Methylated-DNA precipitation sequencing (MeDPseq) experiments performed in both cell lines using the MethylMiner kit (Invitrogen) for selecting methylated DNA regions
Methylated regions identified by MeDPseq were enriched in Methylated-DNA precipitations (MeDP)-chip experiments performed with recombinant proteins containing four methyl-CpG binding domain (MBD) domains from MBD1 [22,39] (Supplementary Figure S1A and B)
Summary
DNA methylation at transcriptional start sites (TSSs) is an epigenetic modification associated with the downregulation of gene transcription [1]. DNA methylation at specific sites can impair the direct binding of transcription factors to their targets and, in turn, may lead to transcriptional downregulation [5,6,7,8], these epigenetic signals are interpreted by specific proteins [9] These proteins have been classified into three families [10,11,12] according to their methyl-DNA binding domain: the methyl-CpG binding domain (MBD) proteins; the UHRF proteins that bind methylated DNA through there SRA domain proteins; and a subclass of zinc finger proteins that preferentially bind methylated DNA sequences (ZBTB33, ZBTB4, ZBTB38, ZFP57, KLF4). In human cells and Xenopus oocytes these proteins are found associated with chromatin remodeling complexes
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