Systematic measurement of transcription factor-DNA interactions by targeted mass spectrometry identifies candidate gene regulatory proteins

Institute for Systems Biology, Seattle, WA 98109.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2013; 110(9). DOI: 10.1073/pnas.1216918110
Source: PubMed


Regulation of gene expression involves the orchestrated interaction of a large number of proteins with transcriptional regulatory elements in the context of chromatin. Our understanding of gene regulation is limited by the lack of a protein measurement technology that can systematically detect and quantify the ensemble of proteins associated with the transcriptional regulatory elements of specific genes. Here, we introduce a set of selected reaction monitoring (SRM) assays for the systematic measurement of 464 proteins with known or suspected roles in transcriptional regulation at RNA polymerase II transcribed promoters in Saccharomyces cerevisiae. Measurement of these proteins in nuclear extracts by SRM permitted the reproducible quantification of 42% of the proteins over a wide range of abundances. By deploying the assay to systematically identify DNA binding transcriptional regulators that interact with the environmentally regulated FLO11 promoter in cell extracts, we identified 15 regulators that bound specifically to distinct regions along ∼600 bp of the regulatory sequence. Importantly, the dataset includes a number of regulators that have been shown to either control FLO11 expression or localize to these regulatory regions in vivo. We further validated the utility of the approach by demonstrating that two of the SRM-identified factors, Mot3 and Azf1, are required for proper FLO11 expression. These results demonstrate the utility of SRM-based targeted proteomics to guide the identification of gene-specific transcriptional regulators.

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Available from: Theo A Knijnenburg, Nov 18, 2015
    • "Another recent study using a similar enrichment strategy identified four Mediator subunits bound to their promoter sequences (Foulds et al, 2013). Recent advances in mass spectrometry-based protein identification technologies with improved sensitivity and reproducibility, such as targeted (Mirzaei et al, 2013) and dataindependent peptide identification approaches (Gillet et al, 2012; Egertson et al, 2013; Lambert et al, 2013), hold great promise for overcoming these limitations and are currently being explored. Whether aberrant targeting of NCOA5 to the Abca1 promoter to repress its expression exacerbates disease progression remains an important question with therapeutic relevance. "
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    • "This could be achieved by quantitatively comparing the protein abundance across pulldown experiments from different genomic regions. This concept was recently used in a targeted approach to identify proteins binding to the FLO11 promoter region (Mirzaei et al. 2013). An alternative could be a discovery-based mass spectrometric acquisition method combined with a label-free quantification algorithm. "
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