Defining the budding yeast chromatin‐associated interactome

We previously reported a novel affinity purification (AP) method termed m odified ch romatin i mmuno p urification (mChIP), which permits selective enrichment of DNA‐bound proteins along with their associated protein network. In this study, we report a large‐scale study of the protein network of 102 chromatin‐related proteins from budding yeast that were analyzed by mChIP coupled to mass spectrometry. This effort resulted in the detection of 2966 high confidence protein associations with 724 distinct preys. mChIP resulted in significantly improved interaction coverage as compared with classical AP methodology for ∼75% of the baits tested. Furthermore, mChIP successfully identified novel binding partners for many lower abundance transcription factors that previously failed using conventional AP methodologies. mChIP was also used to perform targeted studies, particularly of Asf1 and its associated proteins, to allow for a understanding of the physical interplay between Asf1 and two other histone chaperones, Rtt106 and the HIR complex, to be gained. Synopsis The maintenance of cellular fitness requires living organisms to integrate multiple signals into coordinated outputs. Central to this process is the regulation of the expression of the genetic information encoded into DNA. As a result, there are numerous constraints imposed on gene expression. The access to DNA is restricted by the formation of nucleosomes, in which DNA is wrapped around histone octamers to form chromatin wherein the volume of DNA is considerably reduced. As such, nucleosome positioning is critical and must be defined precisely, particularly during transcription (Workman, 2006 ). Furthermore, nucleosomes can be actively assembled/disassembled by histone chaperones and can be made to ‘slide’ along DNA by the actions of chromatin remodelers. Moreover, the histone proteins are heavily regulated at the expression level and by extensive post‐translational modifications (PTMs) (Campos and Reinberg, 2009 ). Histone PTMs have also been shown to help recruit numerous chromatin‐associated factors in accordance with the histone code (Strahl and Allis, 2000 ). Although our understanding of chromatin and its roles has improved, we still have limited knowledge of the chromatin‐associated protein complexes and their interactions. The characterization of biological systems and of specific subdomain within them, such as chromatin, remains a difficult task. An efficient approach to gain insight in the func