title: In Vivo Mapping of FACT-Histone Interactions Identifies a Role of Pob3 C-terminus in H2A-H2B Binding.
abstract: Histone chaperones assist nucleosomal rearrangements to facilitate the passage of DNA and RNA polymerases through chromatin. The FACT (facilitates chromatin transcription) complex is a conserved histone chaperone involved in transcription, replication, and repair. The complex consists of two major subunits, Spt16 and SSRP1/Pob3 in mammals and yeast, which engage histones and DNA by multiple contacts. However, the precise mechanism of FACT function is largely unclear. Here, we used the genetically installed UV-activatable cross-linker amino acid p-benzoylphenylalanine (pBPA) to map the interaction network of FACT in living yeast. Unexpectedly, we found the acidic C-terminus of Pob3 forming cross-links to histone H2A and H2B most efficiently. This observation was independent of the performed cross-linking chemistry since similar histone cross-links were obtained using p-azidophenylalanine (pAzF). Further analyses identified a C-terminal nuclear localization sequence in Pob3. Its interaction with Importin-α interfered with H2A-H2B binding, which suggests a possible regulatory role in FACT recruitment to chromatin. Deletion of acidic residues from the Pob3 C-terminus creates a hydroxyurea-sensitive phenotype in budding yeast, suggesting a potential role for this domain in DNA replication.
authors.affiliation: Free Floater (Junior) Research Group "Applied Synthetic Biology", Georg-August University Göttingen , Institute for Microbiology and Genetics, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Hoffmann
authors.affiliation: Free Floater (Junior) Research Group "Applied Synthetic Biology", Georg-August University Göttingen , Institute for Microbiology and Genetics, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany.
authors.forename: Heinz
authors.initials: H
authors.lastname: Neumann
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D003432:Cross-Linking Reagents / Q000737:chemistry; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D056488:Histone Chaperones
chemical_list: D002352:Carrier Proteins; D002843:Chromatin; D003432:Cross-Linking Reagents; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C106768:POB3 protein, S cerevisiae; C581763:POB3 protein, S pombe; D056488:Histone Chaperones