title: A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein.
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.
  
  
  RESULTS
  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
  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.
authors: Department of Molecular Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors: Stefanie J J
authors: SJ
authors: Bartels
authors: Cornelia G
authors: CG
authors: Spruijt
authors: Arie B
authors: AB
authors: Brinkman
authors: Pascal W T C
authors: PW
authors: Jansen
authors: Michiel
authors: M
authors: Vermeulen
authors: Hendrik G
authors: HG
authors: Stunnenberg
mesh_terms: D001483:Base Sequence; D016384:Consensus Sequence / Q000235:genetics; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D059372:Nucleotide Motifs; D011485:Protein Binding; D020298:U937 Cells
chemical_list: D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C497570:RBPJ protein, human