It has long been appreciated that chromatin-associated proteins and epigenetic factors play central roles in cell-fate reprogramming of genotypically identical stem cells thorough lineage-specific transcription or repression of precise genes and large chromosomal regions (Martin, 1981
; Ho and Crabtree, 2010
; Rossant, 2008
). However, the hierarchy of chromatin-templated events orchestrating the formation and inheritance of different epigenetic states remains poorly understood at a molecular level. Since mis-regulation of chromatin structure and post-translational modification of histones (PTMs) is linked to cancer and other epigenetic diseases (Jones and Baylin, 2007
; Chi et al., 2010
), it is imperative to establish new methodologies that will allow comprehensive studies and unbiased screens for participants in epigenetic mechanisms. Unfortunately, defining how chromatin regulators collectively assemble and operate on a precise region of the genome is difficult to elucidate; there are no current methodologies that allow for determination of all proteins present at a defined, small region of chromatin.
Technical challenges have precluded the ability to determine positioning of chromatin factors along the chromosome. Chromatin immunoprecipitation (ChIP) assays have been used to better understand genome-wide distribution of proteins and histone modifications within a genome at the nucleosome level (Dedon et al., 1991
; Ren et al., 2000
; Pokholok et al., 2005
; Robertson et al., 2007
; Johnson et al., 2007
; Barski et al., 2007
; Mikkelsen et al., 2007
). However, major drawbacks of ChIP-based chromatin enrichment methods are that experiments are largely confined to examining singular histone PTMs or proteins rather than simultaneous profiling of multiple targets, the inability to determine the co-occupancy of particular histone PTMs, and that ChIP is reliant on the previous identification of the molecular target. Affinity purification approaches have been devised for the isolation of a chromatin region using an engineered recombinase excision method (Griesenbeck et al., 2003
); however, these approaches were not done at a level for proteomic analysis and they do not provide a mechanism for determining the specificity of protein interactions. More recently, groups biochemically enriching for intact chromatin have reported characterization of proteins associated with large chromatin structures such as telomeres (Dejardin and Kingston, 2009
) and engineered plasmids (Akiyoshi et al., 2009
; Unnikrishnan et al., 2010
); however, these approaches do not enrich for a small integrated genomic locus, and do not employ specialized mass spectrometric techniques to detect protein contamination in purified material.
We sought to compare differences in chromatin between the transcriptionally active and silent states of a single genomic locus, and developed a technology, called Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS). ChAP-MS provides for the site-specific enrichment of a given ~1,000 base-pair section of a chromosome followed by unambiguous identification of both proteins and histone PTMs associated with this chromosome section using highly selective mass spectrometry. Using ChAP-MS, we were able to purify chromatin at the S. cerevisiae GAL1 locus in transcriptionally silent and active states. We identified proteins and combinatorial histone PTMs unique to each of these functional states, and validated these findings with ChIP. The ChAP-MS technique will greatly improve the field of epigenomics as an unbiased approach to study regulatory mechanisms on chromatin.