Class I histone deacetylase inhibitors inhibit the retention of BRCA1 and 53BP1 at the site of DNA damage.
ABSTRACT: BRCA1 and 53BP1 antagonistically regulate homology-directed repair (HDR) and non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB). The histone deacetylase (HDAC) inhibitor trichostatin A directly inhibits the retention of 53BP1 at DSB sites by acetylating histone H4 (H4ac), which interferes with 53BP1 binding to dimethylated histone H4 Lys20 (H4K20me2). Conversely, we recently found that the retention of the BRCA1/BARD1 complex is also affected by another methylated histone residue, H3K9me2, which can be suppressed by the histone lysine methyltransferase (HKMT) inhibitor UNC0638. Here, we investigate the effects of the class I HDAC inhibitors MS-275 and FK228 compared to UNC0638 on histone modifications and the DNA damage response. In addition to H4ac, the HDAC inhibitors induce H3K9ac and inhibit H3K9me2 at doses that do not affect the expression levels of DNA repair genes. By contrast, UNC0638 selectively inhibits H3K9me2 without affecting the levels of H3K9ac, H3K56ac or H4ac. Reflecting their effects on histone modifications, the HDAC inhibitors inhibit ionizing radiation-induced foci (IRIF) formation of BRCA1 and BARD1 as well as 53BP1 and RIF1, whereas UNC0638 suppresses IRIF formation of BRCA1 and BARD1 but not 53BP1 and RIF1. Although HDAC inhibitors suppressed HDR, they did not cooperate with the poly(ADP-ribose) polymerase inhibitor olaparib to block cancer cell growth, possibly due to simultaneous suppression of NHEJ pathway components. Collectively, these results suggest the mechanism by that HDAC inhibitors inhibit both the HDR and NHEJ pathways, whereas HKMT inhibitor inhibits only the HDR pathway; this finding may affect the chemosensitizing effects of the inhibitors.
Project description:Stable retention of BRCA1/BARD1 complexes at sites of DNA damage is required for the proper response to DNA double-strand breaks (DSB). Here, we demonstrate that the BRCT domain of BARD1 is crucial for its retention through interaction with HP1. In response to DNA damage, BARD1 interacts with Lys9-dimethylated histone H3 (H3K9me2) in an ATM-dependent but RNF168-independent manner. This interaction is mediated primarily by HP1?. A conserved HP1-binding motif in the BARD1 BRCT domain directly interacted with the chromoshadow domain of HP1 in vitro. Mutations in this motif (or simultaneous depletion of all three HP1 isoforms) disrupted retention of BARD1, BRCA1, and CtIP at DSB sites and allowed ectopic accumulation of RIF1, an effector of nonhomologous end-joining, at damaged loci in S-phase. UNC0638, a small-molecule inhibitor of histone lysine methyltransferase (HKMT), abolished retention and cooperated with the PARP inhibitor olaparib to block cancer cell growth. Taken together, our findings show how BARD1 promotes retention of the BRCA1/BARD1 complex at damaged DNA sites and suggest the use of HKMT inhibitors to leverage the application of PARP inhibitors to treat breast cancer.
Project description:The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or nonhomologous end joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ, and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1; limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage; and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.
Project description:Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.
Project description:Caenorhabditis elegans has two histone H3 Lys9 methyltransferases, MET-2 (SETDB1 homolog) and SET-25 (G9a/SUV39H1 related). In worms, we found simple repeat sequences primarily marked by H3K9me2, while transposable elements and silent tissue-specific genes bear H3K9me3. RNA sequencing (RNA-seq) in histone methyltransferase (HMT) mutants shows that MET-2-mediated H3K9me2 is necessary for satellite repeat repression, while SET-25 silences a subset of transposable elements and tissue-specific genes through H3K9me3. A genome-wide synthetic lethality screen showed that RNA processing, nuclear RNA degradation, the BRCA1/BARD1 complex, and factors mediating replication stress survival are necessary for germline viability in worms lacking MET-2 but not SET-25. Unlike set-25 mutants, met-2-null worms accumulated satellite repeat transcripts, which form RNA:DNA hybrids on repetitive sequences, additively with the loss of BRCA1 or BARD1. BRCA1/BARD1-mediated H2A ubiquitination and MET-2 deposited H3K9me2 on satellite repeats are partially interdependent, suggesting both that the loss of silencing generates BRCA-recruiting DNA damage and that BRCA1 recruitment by damage helps silence repeats. The artificial induction of MSAT1 transcripts can itself trigger damage-induced germline lethality in a wild-type background, arguing that the synthetic sterility upon BRCA1/BARD1 and H3K9me2 loss is directly linked to the DNA damage provoked by unscheduled satellite repeat transcription.
Project description:The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here, we compare mice with mutations that ablate BRCT phospho-recognition by Bard1 (Bard1S563F and Bard1K607A) or Brca1 (Brca1S1598F). Brca1S1598F abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1 mutant tumors. Although not affecting HDR, the Bard1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, Bard1S563F/S563F and Bard1K607A/K607A mice, unlike Brca1S1598F/S1598F mice, are not tumor prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, because SFP, unlike HDR, is also impaired in heterozygous Brca1/Bard1 mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.
Project description:Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a "decision" determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (?H2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.
Project description: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 E2?ubiquitin 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.
Project description:Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.
Project description:BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.
Project description:Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.