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Updated: May 28, 2026
11:19
Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets
Published on: July 7, 2010
A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific
Stefanie J J Bartels1, Cornelia G Spruijt, Arie B Brinkman
1Department of Molecular Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
Plos One
|October 13, 2011
Summary
Researchers developed a new method to find proteins that bind to methylated DNA. This technique identified known DNA methylation readers and a novel protein, RBP-J, advancing the study of the methyl-DNA interactome.
Area of Science:
- Epigenetics
- Molecular Biology
- Genomics
Background:
- DNA methylation is a key epigenetic mark regulating biological processes.
- Methyl-CpG Binding Proteins (MBPs) recognize and bind methylated DNA.
- Existing MBP identification methods rely on homology, potentially missing novel binders.
Purpose of the Study:
- To identify novel proteins that bind to methylated DNA (DNAme readers).
- To establish a new experimental approach for discovering methyl-DNA interacting proteins.
Main Methods:
- Utilized a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC).
- Performed pull-down assays and electrophoretic mobility shift assays (EMSAs) for validation.
Main Results:
- Successfully identified known MBPs and MBD2/NuRD complex subunits, validating the method.
- Discovered RBP-J, a transcription factor, binds methylated DNA in a methylation-dependent manner.
- Confirmed RBP-J binding to methylated DNA even with a mutated consensus motif.
Conclusions:
- The SILAC/methyl-CpG pull-down assay is an effective new approach for identifying novel DNAme readers.
- This method will significantly advance the understanding of the complete methyl-DNA interactome.
- The discovery of RBP-J as a methyl-DNA binder opens new avenues for research.
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