PloS one, 2011-10, Vol.6 (10), p.e25884-e25884
2011
Volltextzugriff (PDF)
Details
- Autor(en) / Beteiligte
- Titel
- A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein
- Ist Teil von
- PloS one, 2011-10, Vol.6 (10), p.e25884-e25884
- Ort / Verlag
- United States: Public Library of Science
- Erscheinungsjahr
- 2011
- Quelle
- Free E-Journal (出版社公開部分のみ）
- Beschreibungen/Notizen
- 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. 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. 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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1932-6203eISSN: 1932-6203DOI: 10.1371/journal.pone.0025884Titel-ID: cdi_plos_journals_1309098576
- Format
- –
- Schlagworte
- Amino acid sequence, Amino acids, Base Sequence, Binders, Binding proteins, Biological activity, Biology, Cancer, Cell culture, Chromatin, Consensus Sequence - genetics, CpG islands, CpG Islands - genetics, Cytomegalovirus, Deoxyribonucleic acid, DNA, DNA damage, DNA methylation, DNA Methylation - genetics, Electrophoretic mobility, Epigenetic inheritance, Hepatitis, Histone deacetylase, Homology, Humans, Identification, Immunoglobulin J Recombination Signal Sequence-Binding Protein - metabolism, Isotope Labeling - methods, Life sciences, Mammals, Mass spectrometry, MeCP2 protein, Medical research, Methyl-CpG binding protein, Methylation, Molecular biology, Molecular Sequence Data, Mutation - genetics, Notch protein, Nucleotide Motifs, Nucleotide sequence, NuRD protein, Peptides, Protein Binding, Proteins, Proteomics, Readers, Receptors, Scientific imaging, Search engines, Transcription (Genetics), U937 Cells, Zinc, Zinc compounds