Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae

Abstract

DNA–protein interactions play critical roles in the control of genome expression and other fundamental processes. An essential element in understanding how these systems function is to identify their molecular components. We present here a novel strategy, Hybridization Capture of Chromatin Associated Proteins for Proteomics (HyCCAPP), to identify proteins that are interacting with any given region of the genome. This technology identifies and quantifies the proteins that are specifically interacting with a genomic region of interest by sequence-specific hybridization capture of the target region from in vivo cross-linked chromatin, followed by mass spectrometric identification and quantification of associated proteins. We demonstrate the utility of HyCCAPP by identifying proteins associated with three multicopy and one single-copy loci in yeast. In each case, a locus-specific pattern of target-associated proteins was revealed. The binding of previously unknown proteins was confirmed by ChIP in 11 of 17 cases. The identification of many previously known proteins at each locus provides strong support for the ability of HyCCAPP to correctly identify DNA-associated proteins in a sequence-specific manner, while the discovery of previously unknown proteins provides new biological insights into transcriptional and regulatory processes at the target locus.

Multiplexed Programmable Release of Captured DNA

“…We show here that toehold-mediated branch migration permits the selective release of target sequences from solid supports, making it possible to multiplex strategies that involve the sequence-specific capture of nucleic acids on solid supports such as PICh 11 , GENECAPP 12 , HyCCAPP 13 and DNA enrichment for sequencing 22 . The strategy is characterized and feasibility is demonstrated on a model oligonucleotide system, the locus-specific capture of crosslinked yeast chromatin and on DNA microarrays.…”

“…This is useful for the mass spectrometric identification of locus-specific DNA-associated proteins 11–13 , a powerful emerging technology. Current methodologies to sequence-specifically capture crosslinked chromatin regions utilize desthiobiotinylated oligonucleotides and soluble biotin for subsequent release 11, 13 . This strategy is not amenable to multiplexing, however, as all capture oligonucleotides will be released similarly upon addition of biotin, without discrimination as to target sequence.…”

“…This strategy is not amenable to multiplexing, however, as all capture oligonucleotides will be released similarly upon addition of biotin, without discrimination as to target sequence. We first designed experiments to compare the rates of release of 25s rDNA 13 chromatin by toehold-mediated exchange and desthiobiotin/biotin exchange, using qPCR to measure release of the target DNA.…”

“…Cell lysate containing fragmented chromatin was prepared from 3% formaldehyde crosslinked cells 13 (see SI and ref. 13 for sample preparation details).…”

“…13 for sample preparation details). Hybridization using either toehold-mediated exchange or desthiobiotin-modified oligonucleotides was carried out at 37°C, followed by capture using streptavidin-coated magnetic particles and washing steps to remove non-specifically bound material.…”