Interaction of BARD1 and HP1 Is Required for BRCA1 Retention at Sites of DNA Damage.
ABSTRACT: 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 breast and ovarian cancer predisposition protein BRCA1 forms three mutually exclusive complexes with Fanconi anemia group J protein (FANCJ, also called BACH1 or BRIP1), CtIP, and Abraxas/RAP80 through its BRCA1 C terminus (BRCT) domains, while its RING domain binds to BRCA1-associated RING domain 1 (BARD1). We recently found that the interaction between heterochromatin protein 1 (HP1) and BARD1 is required for the accumulation of BRCA1 and CtIP at sites of DNA double-strand breaks. Here, we investigated the importance of HP1 and BARD1-HP1 interaction in the localization of FANCJ together with the other BRCA1-BRCT binding proteins to clarify the separate role of the HP1-mediated pathway from the RNF8/RNF168-induced ubiquitin-mediated pathway for BRCA1 function. FANCJ interacts with HP1? in a BARD1-dependent manner, and this interaction was enhanced by ionizing radiation or irinotecan hydrochloride treatment. Simultaneous depletion of all three HP1 isoforms with shRNAs disrupts the accumulation of FANCJ and CtIP, but not RAP80, at double-strand break sites. Replacement of endogenous BARD1 with a mutant BARD1 that is incapable of binding to HP1 also disrupts the accumulation of FANCJ and CtIP, but not RAP80. In contrast, RNF168 depletion disrupts the accumulation of only RAP80, but not FANCJ or CtIP. Consequently, the accumulation of conjugated ubiquitin was only inhibited by RNF168 depletion, whereas the accumulation of RAD51 and sister chromatid exchange were only inhibited by HP1 depletion or disruption of the BARD1-HP1 interaction. Taken together, the results suggest that the BRCA1-FANCJ and BRCA1-CtIP complexes are not downstream of the RNF8/RNF168/ubiquitin pathway, but are instead regulated by the HP1 pathway that precedes homologous recombination DNA repair.
Project description: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:Chromatin compaction represents a barrier for the repair of DNA double-strand breaks (DSBs). However, heterochromatin components are also required for DSB repair by homologous recombination. The BARD1/HP1 interaction, required for the retention of BRCA1, CTIP, and RAD51 at DSB sites, may play a critical role in the crosstalk between chromatin compaction and DSB repair.
Project description:Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.
Project description:BRCA1 is a DNA damage response protein and functions in the nucleus to stimulate DNA repair and at the centrosome to inhibit centrosome overduplication in response to DNA damage. The loss or mutation of BRCA1 causes centrosome amplification and abnormal mitotic spindle assembly in breast cancer cells. The BRCA1-BARD1 heterodimer binds and ubiquitinates ?-tubulin to inhibit centrosome amplification and promote microtubule nucleation; however regulation of BRCA1 targeting and function at the centrosome is poorly understood. Here we show that both N and C termini of BRCA1 are required for its centrosomal localization and that BRCA1 moves to the centrosome independently of BARD1 and ?-tubulin. Mutations in the C-terminal phosphoprotein-binding BRCT domain of BRCA1 prevented localization to centrosomes. Photobleaching experiments identified dynamic (60%) and immobilized (40%) pools of ectopic BRCA1 at the centrosome, and these are regulated by the nuclear export receptor CRM1 (chromosome region maintenance 1) and BARD1. CRM1 mediates nuclear export of BRCA1, and mutation of the export sequence blocked BRCA1 regulation of centrosome amplification in irradiated cells. CRM1 binds to undimerized BRCA1 and is displaced by BARD1. Photobleaching assays implicate CRM1 in driving undimerized BRCA1 to the centrosome and revealed that when BRCA1 subsequently binds to BARD1, it is less well retained at centrosomes, suggesting a mechanism to accelerate BRCA1 release after formation of the active heterodimer. Moreover, Aurora A binding and phosphorylation of BRCA1 enhanced its centrosomal retention and regulation of centrosome amplification. Thus, CRM1, BARD1 and Aurora A promote the targeting and function of BRCA1 at centrosomes.
Project description:Carriers of BRCA1 germline mutations are predisposed to breast and ovarian cancers. Accumulated evidence shows that BRCA1 is quickly recruited to DNA lesions and plays an important role in the DNA damage response. However, the mechanism by which BRCA1 is recruited to DNA damage sites remains elusive. BRCA1 forms a Ring-domain heterodimer with BARD1, a major partner of BRCA1 that contains tandem BRCA1 C-terminus (BRCT) motifs. Here, we identify the BRCTs of BARD1 as a poly(ADP-ribose) (PAR)-binding module. The binding of the BARD1 BRCTs to PAR targets the BRCA1/BARD1 heterodimer to DNA damage sites. Thus, our study uncovers a PAR-dependent mechanism of rapid recruitment of BRCA1/BARD1 to DNA damage sites.
Project description:The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.
Project description:Long non-coding RNAs (lncRNAs) are emerging regulators of genomic stability and human disease. However, the molecular mechanisms by which nuclear lncRNAs directly contribute to DNA damage responses remain largely unknown. Using RNA antisense purification coupled with quantitative mass spectrometry (RAP-qMS), we found that the lncRNA BGL3 binds to PARP1 and BARD1, exhibiting unexpected roles in homologous recombination. Mechanistically, BGL3 is recruited to DNA double-strand breaks (DSBs) by PARP1 at an early time point, which requires its interaction with the DNA-binding domain of PARP1. BGL3 also binds the C-terminal BRCT domain and an internal region (amino acids 127-424) of BARD1, which mediates interaction of the BRCA1/BARD1 complex with its binding partners such as HP1γ and RAD51, resulting in BRCA1/BARD1 retention at DSBs. Cells depleted for BGL3 displayed genomic instability and were sensitive to DNA-damaging reagents.
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:Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered γ-H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.