ABSTRACT
The opposing activities of 53BP1 and BRCA1 influence pathway choice in DNA double-strand-break repair. How BRCA1 counteracts the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2â¼ubiquitin and demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitination by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1-deficient cells. BRCA1-BARD1's function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin and optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning, and the need for SMARCAD1 in olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus, BRCA1-BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair.
Subject(s)
BRCA1 Protein/genetics , Chromatin/metabolism , DNA Helicases/genetics , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Recombinational DNA Repair , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , BRCA1 Protein/metabolism , Binding Sites , Camptothecin/pharmacology , Chromatin/chemistry , Chromatin/drug effects , Cloning, Molecular , DNA Breaks, Double-Stranded , DNA Cleavage/drug effects , DNA Helicases/metabolism , DNA, Neoplasm/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HeLa Cells , Histones/genetics , Histones/metabolism , Humans , Models, Molecular , Phthalazines/pharmacology , Piperazines/pharmacology , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effectsABSTRACT
BACKGROUND: Tumour metastasis to the brain is a common and deadly development in certain cancers; 18-30 % of breast tumours metastasise to the brain. The contribution that gene silencing through epigenetic mechanisms plays in these metastatic tumours is not well understood. RESULTS: We have carried out a bioinformatic screen of genome-wide breast tumour methylation data available at The Cancer Genome Atlas (TCGA) and a broad literature review to identify candidate genes that may contribute to breast to brain metastasis (BBM). This analysis identified 82 candidates. We investigated the methylation status of these genes using Combined Bisulfite and Restriction Analysis (CoBRA) and identified 21 genes frequently methylated in BBM. We have identified three genes, GALNT9, CCDC8 and BNC1, that were frequently methylated (55, 73 and 71 %, respectively) and silenced in BBM and infrequently methylated in primary breast tumours. CCDC8 was commonly methylated in brain metastases and their associated primary tumours whereas GALNT9 and BNC1 were methylated and silenced only in brain metastases, but not in the associated primary breast tumours from individual patients. This suggests differing roles for these genes in the evolution of metastatic tumours; CCDC8 methylation occurs at an early stage of metastatic evolution whereas methylation of GANLT9 and BNC1 occurs at a later stage of tumour evolution. Knockdown of these genes by RNAi resulted in a significant increase in the migratory and invasive potential of breast cancer cell lines. CONCLUSIONS: These findings indicate that GALNT9 (an initiator of O-glycosylation), CCDC8 (a regulator of microtubule dynamics) and BNC1 (a transcription factor with a broad range of targets) may play a role in the progression of primary breast tumours to brain metastases. These genes may be useful as prognostic markers and their products may provide novel therapeutic targets.
