title: Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes.
abstract: Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex. We explored the background signal produced by the BioID approach and found that using distinct types of controls increased the stringency of our statistical analysis with SAINTexpress. A direct comparison of BioID with our AP-MS protocol optimized for chromatin-associated protein complexes revealed that the approaches identified few shared interaction partners and enriched for distinct biological processes; yet, both approaches permitted the recovery of biologically meaningful interactions. While no clear bias could be observed for either technique toward protein complexes of particular functions, BioID allowed for the purification of proteins of lower cellular abundance. Finally, we were able to identify a strong association of MED4 with the centrosome by BioID and validated this finding by immunofluorescence. In summary, BioID complements AP-MS for the study of chromatin-associated protein complexes.
  
  
  CONCLUSIONS
  This manuscript describes the application of BioID, a proximity biotinylation approach, to chromatin-associated proteins, namely core histones and members of the mediator complex. We observed that BioID was successful at identifying known interaction partners for the baits tested, but also allowed novel putative interaction partners to be identified. By performing a detailed comparison of BioID versus a standard method for interactome mapping (affinity purification coupled to mass spectrometry, AP-MS), we show that the approaches were complementary, allowing for purification of different interaction partners. These interaction partners were different in the biological processes they are associated with, but also in their abundance. BioID represents a significant technical development in the field of chromatin research by expanding the search space for interactome mapping beyond what is possible with AP-MS. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital 600 University Avenue, Toronto, M5G 1X5, Canada.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Lambert
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital 600 University Avenue, Toronto, M5G 1X5, Canada.
authors.forename: Monika
authors.initials: M
authors.lastname: Tucholska
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital 600 University Avenue, Toronto, M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada.
authors.forename: Christopher
authors.initials: C
authors.lastname: Go
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital 600 University Avenue, Toronto, M5G 1X5, Canada.
authors.forename: James D R
authors.initials: JD
authors.lastname: Knight
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital 600 University Avenue, Toronto, M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada. Electronic address: gingras@lunenfeld.ca.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
mesh_terms: D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D056892:Mediator Complex / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002843:Chromatin; D029968:Escherichia coli Proteins; C491863:MED4 protein, human; D056892:Mediator Complex; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
keywords: Affinity purification coupled to mass spectrometry; BioID; Chromatin; Protein–protein interactions; Proximity biotinylation; Systems biology