title: The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6.
abstract: The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: S Branden
authors.initials: SB
authors.lastname: Van Oss
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Margaret K
authors.initials: MK
authors.lastname: Shirra
authors.affiliation: Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Alain R
authors.initials: AR
authors.lastname: Bataille
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Wier
authors.affiliation: Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, PA 16802, USA; Department of Developmental Biology, Southern Medical University, Guangzhou 510515, China.
authors.forename: Kuangyu
authors.initials: K
authors.lastname: Yen
authors.affiliation: Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Vinesh
authors.initials: V
authors.lastname: Vinayachandran
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: In-Ja L
authors.initials: IL
authors.lastname: Byeon
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Cucinotta
authors.affiliation: Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.
authors.forename: Annie
authors.initials: A
authors.lastname: Héroux
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea.
authors.forename: Jongcheol
authors.initials: J
authors.lastname: Jeon
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea.
authors.forename: Jaehoon
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: VanDemark
authors.affiliation: Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: B Franklin
authors.initials: BF
authors.lastname: Pugh
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address: arndt@pitt.edu.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Arndt
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D005557:Formaldehyde / Q000737:chemistry; D015966:Gene Expression Regulation, Fungal; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C472168:Bre1 protein, S cerevisiae; D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D006657:Histones; D009687:Nuclear Proteins; C107273:RTF1 protein, S cerevisiae; D011994:Recombinant Proteins; C080991:SPT4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor; D005557:Formaldehyde; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D012319:RNA Polymerase II
keywords: Bre1; H2B ubiquitylation; Paf1 complex; RNA Polymerase II; Rad6; Rtf1; chromatin; histone modifications; transcription elongation