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DNA methylation is required for chromatin binding by Mbd2 and Mbd3 in ES cells (mRNA)

ABSTRACT: Cytosine methylation on DNA is an important epigenetic and regulatory mark. Chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins modulate chromatin structure and transcription at methylated loci. Two MBD proteins, Mbd2 and Mbd3, are mutually exclusive members of the NuRD chromatin remodeling complex, and have been shown to bind methylated or hydroxymethylated DNA, respectively. However, a recent study called both results into question, showing that chromatin binding by these proteins is partially (Mbd2) or completely (Mbd3) independent of DNA methylation/hydroxymethylation. Here we re-analyze these data and observe discrepancies with both conclusions. Furthermore, we describe multiple new datasets that demonstrate the dependence of endogenous Mbd2 and Mbd3 on DNA methylation. Interestingly, we find that Mbd2 and Mbd3 are also dependent on one another for binding, likely due to the fact that both are required for normal levels of DNA methylation/hydroxymethylation. These findings describe a regulatory loop controlling the DNA methylation machinery and its readers.

ORGANISM(S): Mus musculus

PROVIDER: GSE79769 | GEO | 2016/11/15

SECONDARY ACCESSION(S): PRJNA316978

REPOSITORIES: GEO

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Project description:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in â??rewritingâ?? the cancer methylome at specific regulatory regions. LNCaP prostate cancer cell line clones with reduced MBD2 expression were establised by using shRNA to MBD2 and scrambled control clones were established with scrambled control shRNA. To interrogate methylation changes induced by MBD2 knock-down we profiled three stably transfected scrambled control clones and three MBD2 knockdown clones on Illumina HumanMethylation450K arrays. Differential methylation analysis was carried out to identified CpG sites hypo-/hyper-methylated as a result of MBD2 knockdown.

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DNA methylation is required for chromatin binding by Mbd2 and Mbd3 in ES cells (mRNA)

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