Abstract
BackgroundDNA 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 FindingsFor 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/SignificanceThe 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.
Highlights
DNA methylation is an epigenetic modification that is essential for a variety of biological processes
In a forward experiment, fully methylated synthetic DNA is used as a bait for Methyl-CpG Binding Proteins (MBPs) in a heavy-labeled nuclear extract, whereas unmethylated DNA is used in a light extract
Using a DNA sequence that is based on the GSTP1 CpG-island and U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders
Summary
DNA methylation is an epigenetic modification that is essential for a variety of biological processes. DNA methylation primarily occurs at cytosines in a CpG dinucleotide context. CpG-island methylation is generally associated with transcriptional repression, and more recently gene-body DNA methylation has been associated with transcriptional activity [2]. DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is 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
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