title: Defining the budding yeast chromatin-associated interactome.
abstract: We previously reported a novel affinity purification (AP) method termed modified chromatin immunopurification (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.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Lambert
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Fillingham
authors.forename: Mojgan
authors.initials: M
authors.lastname: Siahbazi
authors.forename: Jack
authors.initials: J
authors.lastname: Greenblatt
authors.forename: Kristin
authors.initials: K
authors.lastname: Baetz
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D040081:Protein Array Analysis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors
chemical_list: C109731:ASF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C078808:HIR1 protein, S cerevisiae; D006657:Histones; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D012097:Repressor Proteins; C505157:Rtt106 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors