The histone variant macroH2A1 is a BRCA1 ubiquitin ligase substrate
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Jun Qin
PROVIDER: MSV000081054 | MassIVE | Sun Apr 30 17:31:00 BST 2017
SECONDARY ACCESSION(S): PXD006411
REPOSITORIES: MassIVE
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Project description: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.
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Project description:The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3) ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.
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Project description:This SuperSeries is composed of the following subset Series: GSE16882: Histone H1 binding is restricted by histone variant H3.3 (Nucleosome) GSE16883: Histone H1 binding is restricted by histone variant H3.3 (DamID) GSE16884: Histone H1 binding is restricted by histone variant H3.3 (Expression) GSE19764: Histone H1 binding is restricted by histone variant H3.3 (FAIRE) Refer to individual Series
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Project description:Although PARP inhibitors (PARPi) now form part of the standard-of-care for the treatment of homologous recombination defective cancers, de novo and acquired resistance limits their overall effectiveness. Previously, overexpression of the BRCA1-∆11q splice variant has been shown to cause PARPi resistance. How cancer cells achieve increased BRCA1-∆11q expression has remained unclear. Using isogenic cells with different BRCA1 mutations, we show that reduction in HUWE1 leads to increased levels of BRCA1-∆11q and PARPi resistance. This effect is specific to cells able to express BRCA1-∆11q (e.g. BRCA1 exon 11 mutant cells) and is not seen in BRCA1 mutants that cannot express BRCA1-∆11q, nor in BRCA2 mutant cells. As well as increasing levels of BRCA1-∆11q protein in exon 11 mutant cells, HUWE1 silencing also restores RAD51 nuclear foci and platinum salt resistance. HUWE1 catalytic domain mutations were also seen in a case of PARPi resistant, BRCA1 exon 11 mutant, high grade serous ovarian cancer. These results suggest how elevated levels of BRCA1-∆11q and PARPi resistance can be achieved, identify HUWE1 as a candidate biomarker of PARPi resistance for assessment in future clinical trials and illustrate how some PARPi resistance mechanisms may only operate in patients with particular BRCA1 mutations.
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Project description:The breast cancer suppressor protein, BRCA1, is a ubiquitin ligase expressed in a wide range of tissues. However, inheritance of a single BRCA1 mutation significantly increases a woman's lifetime chance of developing tissue-specific cancers in the breast and ovaries. Recently, studies have suggested this tissue specificity may be linked to inhibition of estrogen receptor alpha (ERalpha) transcriptional activation by BRCA1. Here, we show that ERalpha is a putative substrate for the BRCA1/BARD1 ubiquitin ligase, suggesting a possible mechanism for regulation of ERalpha activity by BRCA1. Our results show ERalpha is predominantly monoubiquitinated in a reaction that involves interactions with both BRCA1 and BARD1. The regions of BRCA1/BARD1 necessary for ERalpha ubiquitination include the RING domains and at least 241 and 170 residues of BRCA1 and BARD1, respectively. Cancer-predisposing mutations in BRCA1 are observed to abrogate ERalpha ubiquitination. The identification of ERalpha as a putative BRCA1/BARD1 ubiquitination substrate reveals a potential link between the loss of BRCA1/BARD1 ligase activity and tissue-specific carcinoma.
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Project description:It has been recognized that BRCA1, in the form of the BRCA1/BARD1 heterodimer, acting as an ubiquitin E3 ligase offered a possible mechanism to explain its pleiotrophic nature of BRCA1 activity. Our observation that mice lacking BRCA1 enzymatic activity are viable apart from male sterility was unexpected. Our results suggest that the E3 ligase activity of BRCA1 is largely dispensable for normal development and is not essential for all BRCA1 functions. Thus, many of the known and unknown functions of BRCA1 are likely to be mediated independent of its ability to catalyze ubiquitination. The genome copy number patterns were studied on the mice tumors that lacks E3 ubiqitin ligase activity of BRCA1 and were compared to copy number profile of mice lacking p53 and both brca1 and p53. Array CGH was performed using Agilent mouse CGH microarray 244K kit. Genomic DNA isolated from tumor tissue and its corresponding mouse tail were labelled with two different dyes and hybridized simultaneously on to microarray slides to perform comparitive genomic hybridization.
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Project description:It has been recognized that BRCA1, in the form of the BRCA1/BARD1 heterodimer, acting as an ubiquitin E3 ligase offered a possible mechanism to explain its pleiotrophic nature of BRCA1 activity. Our observation that mice lacking BRCA1 enzymatic activity are viable apart from male sterility was unexpected. Our results suggest that the E3 ligase activity of BRCA1 is largely dispensable for normal development and is not essential for all BRCA1 functions. Thus, many of the known and unknown functions of BRCA1 are likely to be mediated independent of its ability to catalyze ubiquitination. The genome copy number patterns were studied on the mice tumors that lacks E3 ubiqitin ligase activity of BRCA1 and were compared to copy number profile of mice lacking p53 and both brca1 and p53.
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