The Histone Variant MacroH2A1 Is a BRCA1 Ubiquitin Ligase Substrate

Cell Rep. 2017 May 30;19(9):1758-1766. doi: 10.1016/j.celrep.2017.05.027.

Abstract

The breast- and ovarian-cancer-specific tumor suppressor BRCA1 and its heterodimeric partner BARD1 contain RING domains that implicate them as E3 ubiquitin ligases. Despite extensive efforts, the bona fide substrates of BRCA1/BARD1 remain elusive. Here, we used recombinant GST fused to four UBA domains to enrich ubiquitinated proteins followed by a Lys-ε-Gly-Gly (diGly) antibody to enrich ubiquitinated tryptic peptides. This tandem affinity purification method coupled with mass spectrometry identified 101 putative BRCA1/BARD1 E3 substrates. We identified the histone variant macroH2A1 from the screen and showed that BRCA1/BARD1 ubiquitinates macroH2A1 at lysine 123 in vitro and in vivo. Primary human fibroblasts stably expressing a ubiquitination-deficient macroH2A1 mutant were defective in cellular senescence compared to their wild-type counterpart. Our study demonstrates that BRCA1/BARD1 is a macroH2A1 E3 ligase and implicates a role for macroH2A1 K123 ubiquitination in cellular senescence.

Keywords: BRCA1; E3 ligase substrate; macroH2A1.1; senescence; ubiquitination.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • BRCA1 Protein / metabolism*
  • Cell Line
  • Cellular Senescence
  • Chromatography, Affinity
  • Histones / chemistry
  • Histones / metabolism*
  • Humans
  • Lysine / metabolism
  • Substrate Specificity
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination

Substances

  • BRCA1 Protein
  • Histones
  • Ubiquitin
  • macroH2A histone
  • Ubiquitin-Protein Ligases
  • Lysine