The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6

Mol Cell. 2016 Nov 17;64(4):815-825. doi: 10.1016/j.molcel.2016.10.008. Epub 2016 Nov 10.

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.

Keywords: Bre1; H2B ubiquitylation; Paf1 complex; RNA Polymerase II; Rad6; Rtf1; chromatin; histone modifications; transcription elongation.

MeSH terms

  • Amino Acid Motifs
  • Binding Sites
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • Cross-Linking Reagents / chemistry
  • Crystallography, X-Ray
  • Formaldehyde / chemistry
  • Gene Expression Regulation, Fungal*
  • Histones / chemistry
  • Histones / genetics
  • Histones / metabolism*
  • Models, Molecular
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Interaction Domains and Motifs
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • TATA-Box Binding Protein / chemistry
  • TATA-Box Binding Protein / genetics
  • TATA-Box Binding Protein / metabolism*
  • Transcription, Genetic
  • Transcriptional Elongation Factors / genetics
  • Transcriptional Elongation Factors / metabolism
  • Ubiquitin-Conjugating Enzymes / chemistry
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitination

Substances

  • Bre1 protein, S cerevisiae
  • Chromosomal Proteins, Non-Histone
  • Cross-Linking Reagents
  • Histones
  • Nuclear Proteins
  • RTF1 protein, S cerevisiae
  • Recombinant Proteins
  • SPT4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • TATA-Box Binding Protein
  • Transcriptional Elongation Factors
  • SPT5 transcriptional elongation factor
  • Formaldehyde
  • RAD6 protein, S cerevisiae
  • Ubiquitin-Conjugating Enzymes
  • RNA Polymerase II