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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.