Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10553-8. doi: 10.1073/pnas.1606863113. Epub 2016 Sep 6.

Abstract

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.

Keywords: Bre1–Rad6; RING E3 ligase; cross-linking mass spectrometry; nucleosome; ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation, Enzymologic
  • Histones / chemistry*
  • Histones / genetics
  • Humans
  • Molecular Docking Simulation
  • Nucleosomes / chemistry
  • Nucleosomes / genetics
  • Protein Conformation
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Ubiquitin / chemistry
  • Ubiquitin / genetics
  • Ubiquitin-Conjugating Enzymes / chemistry*
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitination / genetics*

Substances

  • Bre1 protein, S cerevisiae
  • Histones
  • Nucleosomes
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • RAD6 protein, S cerevisiae
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases