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PLoS One. 2011;6(10):e25884. doi: 10.1371/journal.pone.0025884. Epub 2011 Oct 3.

A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein.

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  • 1Department of Molecular Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.

Abstract

BACKGROUND:

DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs). Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD) family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA.

METHODOLOGY/PRINCIPAL FINDINGS:

For the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC). In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs) showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif.

CONCLUSIONS/SIGNIFICANCE:

The here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

PMID:
21991380
[PubMed - indexed for MEDLINE]
PMCID:
PMC3185043
Free PMC Article

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