Identifying Specific Protein-DNA Interactions Using Quantitative Mass Spectrometry-Based Proteomics.
2013
A comprehensive and unbiased characterization of DNA-protein interactions is essential to increase our understanding of processes such as transcription and replication in the nucleus of a eukaryotic cell. In this regard, quantitative mass spectrometry-based proteomics has recently emerged as a powerful tool. Modern instrumentation and software enable the identification of hundreds of proteins in a sample in a few hours. Similar amounts of proteins can be identified in DNA affinity purifications from crude nuclear extracts. However, the majority of these proteins are highly abundant background proteins that bind non-specifically to the beads or DNA and only a small fraction represents sequence-specific interactors. This implies a need for a quantitative filter that can be used to discriminate specific interactors from non-specific background proteins. In recent years numerous methods have been developed that add a quantitative dimension to mass spectrometry measurements. We have established a single step DNA affinity purification protocol from crude nuclear extracts that makes use of an in vivo stable isotope labeling approach called SILAC (Stable Isotope Labeling by Amino acids in Cell culture). This generic method can be used to identify proteins binding to DNA sequences of interest, including transcription factor binding sites, single nucleotide polymorphisms and methylated CpG islands.
We have recently used this method to identify proteins binding to (hydroxy)methylatd DNA in (differentiated) mouse embryonic stem cells. These ‘readers’ for 5-methylcytosine and 5-hydroxymethylcytosine are only partially overlapping and some readers even display opposite binding patterns. Interactions are dynamic during differentiation, as evidenced by the 5-methylcytosine specific binding of Klf4 in ES cells, for example. Our data reveal that oxidized cytosine bases are intermediates of active DNA demethylation pathways but additionally point towards a DNA demethylation independent role for 5-hydroxymethylcytosine in certain cell types.
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