BRCA1, a complex protein involved in the maintenance of genomic stability

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1 STATE-OF-THE-ART REVIEW BRCA1, a complex protein involved in the maintenance of genomic stability Kienan I. Savage and D. Paul Harkin Centre for Cancer Research and Cell Biology, Queen s University Belfast, UK Keywords BRCA1, breast cancer, DNA repair, DNA damage response, hereditary breast cancer, genomic instability Correspondence K. I. Savage, Centre for Cancer Research and Cell Biology, Queen s University Belfast, Belfast BT9 7BL, UK Fax: +44 (0) Tel: +44 (0) k.savage@qub.ac.uk (Received 18 September 2014, revised 10 November 2014, accepted 13 November 2014) BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mrna splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression. doi: /febs Introduction Approximately 10% of women diagnosed with breast cancer report a strong family history, prompting the search for breast cancer susceptibility genes. In 1994, the first breast and ovarian cancer susceptibility gene, BRCA1, was identified and cloned, followed closely by the discovery of BRCA2 in 1995 [1,2]. To date, over 800 distinct and clinically relevant mutations have been identified within the BRCA1 gene and, taken together, mutations within BRCA1 and BRCA2 may account for up to 20% of inherited breast cancer cases. Moreover, carriers of mutations within BRCA1 or BRCA2 have a life time risk of developing breast cancer of up to 60 80% and a lifetime risk of ovarian cancer of up to 20 40% [3]. Additionally, although BRCA1/2 mutations account for a relatively low proportion of breast cancer cases as a whole, it has been found that between 50% and 70% of sporadic breast tumours have lost at least one BRCA1 allele, suggesting that the loss of BRCA1 may also be important for sporadic breast cancer development [4]. In addition, hypermethylation of BRCA1 promoters and loss of BRCA1 transcript has also been found in a significant proportion of sporadic breast and ovarian tumours [5]. The most striking phenotype of BRCA1 deficient cells is severe genomic instability and sensitivity to Abbreviations AI, aromatase inhibitor; BARD1, BRCA1 associated RING domain protein 1; BRCT, BRCA1 C-terminal; DSB, double-strand break; ERa, estrogen receptor-a; HR, homologous recombination; ICL, interstrand cross-link; IR, ionizing radiation; MEF, mouse embryonic fibroblast; NER, nucleotide excision repair; NF-jB, nuclear factor-kappa B; NHEJ, nonhomologous end joining; PARP, poly ADP ribose polymerase; PARylated, poly(adp-ribosylated); RPA, replication protein A; SERM, selective estrogen receptor modulator; SSB, single-strand break; ssdna, single-stranded DNA; c-h2ax, phosphorylated H2AX. 630 FEBS Journal 282 (2015) ª 2014 FEBS

2 K. I. Savage and D. P. Harkin BRCA1 and maintenance of genomic stability genotoxic agents, with BRCA1 deficient cells often exhibiting chromosomal translocations incorporating large deletions and/or amplifications that involve multiple, nonhomologous chromosomes [6]. Subsequent to its identification, much attention has been focused on understanding the function of BRCA1, with roles identified in multiple cellular processes required for maintaining genomic integrity, including DNA damage signaling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodeling, transcriptional regulation, mrna splicing and apoptosis. Although the exact mechanistic role of BRCA1 in many of these processes remains to be defined, it is considered that BRCA1 carries out these diverse roles through its ability to interact with a wide range of different proteins. Indeed, to date, over 100 distinct BRCA1 interacting proteins have been reported and it is assumed that, through these interactions, the ability of BRCA1 to act as a scaffold for the formation of multiple different protein complexes with different cellular functions underlies its role in a diverse array of cellular processes. The remainder of this review will focus on the role of BRCA1 within these complexes and their functions, which contribute to the maintenance of genomic stability and carcinogenesis. Structural features of BRCA1 BRCA1 is located on chromosome 17q21.3, spans approximately 80 kb of genomic DNA, and is composed of 24 coding exons [1]. The BRCA1 gene encodes an 1863 amino acid protein, which shares limited homology with other known proteins but does contain two functional domains. At its N-terminus, BRCA1 contains a series of eight conserved Cys3-His-Cys4 motif repeats known as the RING domain, a catalytic domain involved in protein ubiquitination and protein protein interactions [7]. The most well characterized RING domain interaction occurs with the BRCA1 associated RING domain protein 1 (BARD1), forming the BRCA1/BARD1 heterodimer, a known E3 ubiquitin ligase complex [8]. It is now well accepted that BRCA1 and BARD1 are obligate heterodimers, with each protein required for the stability of the other [9]. The BRCA1/BARD1 heterodimer has been shown to ubiquitinate a plethora of substrates in vitro; however, the identification of bona fide in vivo substrates has remained challenging. Nevertheless, several promising candidate ubiquitination targets have been suggested, including the histone proteins H2A, H2B, H3 and H4, CtIP, NPM1, RPB8, TFIIE and BRCA1 itself [9]. Interestingly, BRCA1/BARD1 is considered to primarily mediate K6-linked ubiquitination as a posttranslational modification mechanism to regulate target protein function and/or recruitment, rather than signaling protein degradation. Although the ubiquitin ligase function if BRCA1 is well accepted, its biological relevance with respect to the tumour suppressor function of BRCA1 remains controversial. A recent report suggests that BRCA1/BARD1-mediated ubiquitination of histone H2A within heterochromatic regions leads to the repression of satellite DNA, which suppresses genomic instability [10]. It was demonstrated that expression of the E3 ligase dead I26A mutant of BRCA1 lead to a loss of H2A ubiquitination, de-repression of satellite DNA and genomic instability. Intriguingly, the I26A mutant is a synthetic mutant that does not occur naturally in high-risk breast/ovarian cancer families. In addition, the I26A mutation, although resulting in deficient E3 ligase function, does not abrogate the interaction between BRCA1 and BARD1. Moreover, mice homozygous for the I26A BRCA1 mutation develop tumours at a similar frequency to BRCA1 wild-type mice and appear to have normal DNA repair capacity, suggesting that perhaps, at least in mice, the E3 ligase function of BRCA1 is dispensable for tumour suppression [11]. By contrast, mice harbouring the pathogenic C61G mutation, which has been suggested to disrupt both the E3 ligase function of BRCA1 and the interaction between BRCA1 and BARD1, display embryonic lethality, DNA repair deficiency and cancer predisposition, although their cells are less sensitive to DNA damaging agents [12]. Taken together, these two studies suggest that the interaction with BARD1 may be more important than the E3 ligase activity of BRCA1 in terms of tumour suppression and DNA repair activity. Nevertheless, studies investigating the role of BRCA1 in ubiquitination at sites of DNA damage in human cells have shown that decreased BRCA1 E3 ligase activity is associated with defective DNA repair. Indeed, after DNA damage BRCA1/BARD1 mediates the ubiquitination of histone H2A (and perhaps other substrates), with the E3-ligase activity of BRCA1/ BARD1 dramatically increased at double-strand breaks (DSBs) by PIAS1/4-dependent sumoylation of BRCA1. Intriguingly, depletion of PIAS1/4 results in defective DSB repair and sensitivity to DNA damaging agents, suggesting that BRCA1-dependent ubiquitination at DSB sites, which is dependent on PIAS1/4, is important for DSB repair and may play a role in tumour suppression. The C-terminus of BRCA1 contains two conserved BRCA1 C-terminal (BRCT) domains. These domains contain distinct hydrophobic/acidic patches and have been shown to have transactivation activity [13,14]. FEBS Journal 282 (2015) ª 2014 FEBS 631

3 BRCA1 and maintenance of genomic stability K. I. Savage and D. P. Harkin Subsequent to their discovery within the BRCA1 protein, BRCT domains have been found in many proteins involved in the DNA damage response, such as MDC1 and 53BP1, and have more recently been described as phosphospecific protein interaction motifs [15,16]. Not unexpectedly, a number of cancer predisposing mutations have also been found within the BRCT domains of BRCA1 that affect both the transactivation activity of BRCA1 (discussed below) and its phosphospecific interaction with other proteins [9]. BRCA1 also contains two nuclear localization signals at amino acids and , respectively, which target BRCA1 to the nucleus [17]. In addition, a nuclear export signal at amino acids is involved in nuclear cytoplasmic shuttling [18]. The central region of the BRCA1 protein is largely encoded by a single exon (exon 11) that is present in vertebrates but not in lower eukaryotes. This region, covering amino acids contains no known domain structures and is predicted to be intrinsically disordered by both in silico and experimental analysis [19,20]. This type of protein structure (i.e. a disordered region flanked by structural domains) is typical of scaffold or hub proteins. Disordered regions allow binding promiscuity as well as structural flexibility between ordered domains, thereby facilitating binding diversity and allowing the conformational flexibility to facilitate the formation of multiple different macromolecular complexes [20]. Indeed, this region of BRCA1 has been shown to interact with numerous different proteins and contains a number of DNA damage-induced phosphorylation sites that are considered to mediate the formation of various BRCA1 protein complexes [7]. BRCA1 complexes involved in DNA repair and cell cycle arrest DNA damage can arise as a consequence of different endogenous or exogenous events that are capable of causing many different types of damage, such as loss, alkylation or oxidation of bases; apurinic/apyrimidinic sites; DNA single-strand breaks (SSBs) and DNA DSBs. The most hazardous type of DNA damage is the DSB. Because broken DNA ends are able to dissociate, DSBs are not only more difficult to repair, but allow for the re-joining of unrelated ends, thus allowing for gross loss or amplification of genomic information, as well as chromosomal rearrangements, all of which are a common feature of BRCA1 loss/deficiency [21]. DSBs can be repaired through two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Both DSB repair pathways have been extensively reviewed elsewhere and therefore only a brief overview of the salient points of these pathways is provided here. NHEJ is initiated by binding of the Ku70/80 heterodimer directly to broken double-stranded DNA ends. Ku70/80 then recruits the catalytic subunit of the DNA-dependent protein kinase, thus creating the DNA-dependent protein kinase holoenzyme. Broken ends are then processed by DNA polynucleotide kinase and any latent gaps are filled by DNA polymerase-l. Processed ends are then ligated by the XRCC4 ligase IV complex. This process is relatively simple for the repair of blunt-ended DSBs. However, for complex or ragged DBSs, additional proteins are required. This process requires the ATM- and MRN-dependent activation of Artemis, a nuclease required for additional processing of complex breaks. After processing of ragged DSB ends by artemis, the core NHEJ process above is used to ligate DNA ends together. NHEJ is a rapid DSB repair mechanism and, in the case of localized simple breaks, can promote efficient DSB repair. However, as a result of the nature of this direct end ligation process, NHEJ can often result in small deletions (especially in the case of complex DSBs) and/ or chromosomal rearrangements, thereby contributing greatly to genomic instability. By contrast, HR utilizes an undamaged sister chromatid as a repair template to restore any damaged/lost sequence information at the DSB site. Firstly the 5 0 ends of the broken DSB ends are resected, generating a3 0 single-stranded region of DNA on either side of the DSB. This single-stranded DNA (ssdna) is rapidly bound by replication protein A (RPA), which prevents the formation of secondary DNA structures during resection. RPA is then displaced by Rad51, which is loaded onto RPA-coated ssdna in a Rad54, PALB2, BRCA2-dependent process. Rad51 loading then promotes sister strand invasion, generating a heteroduplex molecule known as a Holliday junction. Also required for this process are the structural maintenance of chromosomes proteins 1, 3, 5 and 6. These proteins, also known as cohesins, physically tether the sister chromatids together, stabilizing strand invasion and facilitating HR-mediated repair. DNA replication then ensues using the undamaged chromatid as a template. The Holliday junction is then resolved and the DNA ends ligated together, resulting in either crossover or noncross-over products depending on the direction of junction resolution [22]. Because HR requires a sister template for repair, this pathway is only available during S/G2 phases of the cell cycle. Consequently, NHEJ is the predominate repair pathway in G0/G1 cells. However, many DNA damaging agents cause replication-specific DNA dam- 632 FEBS Journal 282 (2015) ª 2014 FEBS

4 K. I. Savage and D. P. Harkin BRCA1 and maintenance of genomic stability age and/or replication fork stalling. HR plays a major role in the recovery of stalled replication forks where HR can be activated by accumulated ssdna at the stalled fork (and subsequent RPA accumulation) or a by one-sided DSB formed by a collapsed fork [21]. The role of BRCA1 in NHEJ, if any, remains unclear. BRCA1 has been reported to interact with Ku80 and has been reported to bind DSB sites in a Ku80-dependent manner, suggesting a role for BRCA1 in this pathway [23]. Additionally, NHEJ defects have been reported in BRCA1 null mouse embryonic fibroblasts (MEFs) [24,25]. However, contrasting results have been reported by other groups, where BRCA1 deficient embryonic stem cells exhibited decreased levels of homologous recombination and an increased frequency of NHEJ compared to BRCA1 wild-type parental lines [26,27]. Differences in these results might be explained by the use of distinct host cells (embryonic stem cells versus MEFs) and BRCA1 mutations (BRCA1 isoform missing exon 11 versus BRCA1 D5 6, which disrupts all BRCA1 isoforms). As a result, the exact role of BRCA1, if any, in NHEJ remains uncertain. By contrast, a number of roles for BRCA in HRmediated DSB repair have been well described, where BRCA1 exerts its effects through participation in at least four distinct protein complexes. The BRCA1 C complex consisting of BRCA1, Mre11:Rad50:Nbs1 (collectively known as the MRN complex) and CtIP plays a role in DSB end resection, a process that also involves EXO1 and DNA2 (Fig. 1) [28]. Initially, the BRCA1 C complex was considered to play a role in the initiation of end resection; however, a number of recent studies have shed further light on this. The interaction between BRCA1 and CtIP within this complex is mediated by CDK-dependent phosphorylation of CtIP-S327, whereas the ability of CtIP to promote end resection is mediated CtIP-T847 phosphorylation, which is also mediated by CDKs during the S/G2 phases of the cell cycle [29,30]. By generating different knock-in cell lines containing serine to alanine phosphorylation abrogation mutations at these sites, these studies have demonstrated that BRCA1 is dispensable for CtIP-mediated end resection. Specifically, Reczek et al. [31] showed that MEFs expressing CtIP S326A (equivalent to S327A in humans) displayed similar levels of Rad51 and RPA recruitment to DBSs as CtIP wild-type cells, indicating that the interaction between CtIP and BRCA1 is not required for DNA end resection. Accordingly, a loss of the CtIP-BRCA1 interaction did not affect HR or tumour development in mice [31]. Polato et al. [32] reported similar findings, also showing that, in contrast to the CtIP S327A mutant mice, mice harbouring the CtIP T847A mutation (which is essential for end resection but does not affect the BRCA1 interaction) had elevated levels of spontaneous chromosomal aberrations, as well as decreased levels of ionizing radiation (IR)-induced Rad51 foci, indicating that CtIP functions independently of BRCA1 to promote end resection. Nevertheless, further investigation of the role of the BRCA1 CtIP interaction in end resection using a high resolution technique (i.e. single molecule analysis of resection tracks) allowing visualization of the length of the resected DNA in a single molecule has shown that, although BRCA1 CtIP is expendable for the initiation of end resection, disruption of this interaction decreases the length and speed of resected DNA generated after DNA damage [33]. Therefore, although BRCA1 is not essential for CtIPmediated end resection, it appears as though interaction of BRCA1 with CtIP and within the BRCA1 C complex facilitates the efficiency of this process. The exact function of CtIP and BRCA1 within this complex and how they biochemically regulate end resection remains unclear. However, clarification was provided in a seminal study by Bunting et al. [34], which found that homologous recombination is completely restored in BRCA1 deficient cells by the codepletion/deletion of 53BP1. 53BP1, an NHEJ promoting protein, has subsequently been shown to block HR and promote the NHEJ-mediated repair of DSBs by recruiting a number of other pro-nhej proteins, including RIF1 and PTIP [34,35]. BRCA1 and CtIP have been shown to block DSB recruitment of RIF1, thereby promoting the initiation of DSB end resection, which is considered to help regulate the choice to repair DSBs via HR in S/G2 phase cells [36]. Moreover, BRCA1 has also been shown to reposition 53BP1 away from DSB ends, thereby further facilitating end resection (Fig. 1) [37 39]. Importantly, depletion of CtIP in BRCA1/53BP1 null cells results in a loss of DSB end resection, indicating that CtIP is responsible for end resection in the absence of BRCA1, although the role of BRCA1 within the BRCA1 C complex appears to comprises the repositioning of 53BP1 away from DSB ends and/or the blocking of RIF1 recruitment to DSB ends [32]. Additionally, by initiating DNA end resection and the subsequent generation of RPA-coated ssdna, the BRCA1 C complex assists in the activation of the ATR/Chk1 pathway responsible for intra-s phase arrest, thereby preventing the replication of damaged DNA and promoting the maintenance of genomic stability (Fig. 1). Additional to the role of the BRCA1 C complex in 53BP1 repositioning and initiation of resection, end FEBS Journal 282 (2015) ª 2014 FEBS 633

5 BRCA1 and maintenance of genomic stability K. I. Savage and D. P. Harkin Fig. 1. BRCA1 complexes involved in HRmediated DSB repair and cell cycle checkpoint control. During the S and G2 phases of the cell cycle, DSBs are faithfully repaired through HR. Initially, 53BP1 is displaced from DSB ends by the BRCA1 C complex, which then initiates DSB end resection. Both the BRCA1 C and B complexes, along with other proteins such as Exo1 and DNA2, are required to extend DNA end resection. Both the BRCA1 C and B complexes are also required for activation of the Intra-S checkpoint after DNA damage. Additionally, the BRCA1 A complex is recruited to flanking chromatin and is thought to prevent over resection of DSB ends. The BRCA1 A complex is also required for maintained DNA damage response signaling and activation of the G2/M checkpoint. Finally, the BRCA1 D complex facilitates the exchange of RPA bound to the single-stranded DNA overhangs with Rad51. Rad51 then catalyses the invasion of the sister chromatid and alignment with the complementary DNA sequence within, forming a Holiday junction. DNA polymerases restore the complementary DNA strand using the sister chromatid as a template. The Holiday junction in then resolved, resulting in noncross-over or cross-over products, depending on the direction of junction resolution. resection is extended to facilitate RPA loading and subsequent RPA-Rad51 exchange prior to sister strand invasion [34,36 39]. The repositioning of 53BP1 alone is not sufficient to facilitate resection with subsequent repositioning of RAP80, also bound to the chromatin surrounding DSBs, simultaneously required for continued/extended resection. The exact steps in this process remain unclear, although it is known that repositioning of RAP80 requires BRCA1 and POH1, a deubiquitinating enzyme and component of the proteasome, 634 FEBS Journal 282 (2015) ª 2014 FEBS

6 K. I. Savage and D. P. Harkin BRCA1 and maintenance of genomic stability which may function to locally degrade RAP80, facilitating the clearance of RAP80 and allowing end resection. Intriguingly, another BRCA1 complex, the BRCA1 A complex, which itself contains RAP80 along with MERIT40, BRCC36/45 and Abraxas, has been reported to inhibit DNA end resection, suggesting that, in some contexts, BRCA1 may function to limit and/or prevent over resection of DNA breaks [40,41]. This complex has also been shown to stabilize/ maintain DNA damage signaling from the break site and promote G2/M checkpoint arrest after DNA damage (Fig. 1). Another BRCA1 complex, the BRCA1 B complex containing BRCA1/TopBP1 and BACH1 (also known and BRIP1/FANCJ) has been reported to play a role in HR and S-phase cell cycle arrest. The exact role of this complex in HR remains unclear, although it is assumed that BACH1, a DNA helicase, contributes to end resection (possibly through its helicase activity) and RPA loading, whereas TopBP1 is required for ATR activation and subsequent S-phase checkpoint activation (Fig. 1) [42]. Finally, after resection, another BRCA1 complex, the BRCA1/BRCA2/PALB2/Rad51 complex (hereon referred to as the BRCA1 D complex), functions to promote the exchange of RPA for Rad51, and facilitate sister chromatid invasion. It is assuemd that the BRCA1 D complex facilitates the recruitment of PALB2, BRCA2 and Rad51 to DSB sites and that perhaps BRCA1 assists BRCA2 and Rad51 to promote strand invasion (Fig. 1) [43,44]. Exactly, how the BRCA1 A, B, C and D complexes are recruited to DSB sites remains unclear. Recruitment of the BRCA1 A complex to DSB sites has been well characterized and this occurs through a complex pathway involving recognition and binding of phosphorylated H2AX (c-h2ax) at break sites by MDC1 [45]. MDC1 itself is then phosphorylated, forming a binding site for RNF8, an E3 ubiquitin ligase that ubiquitinates histones H2A, H2B and H2AX surrounding the break site. These ubiquitinated histones are then recognized/bound by a second E3 ubiquitin ligase, RNF168, through its ubiquitin interaction motif, allowing amplification of the ubiquitin signal on histones flanking the DSB [46,47]. The BRCA1 A complex is then recruited to c-h2ax marked chromatin surrounding the DSB through the recognition and binding of ubiquitinated histones by RAP80, which contains two ubiquitin interaction motifs. Despite the well-understood process by which the BRCA1 A complex is recruited to DSBs, the recruitment of the other HR promoting BRCA1 complexes to these sites is less clear. Recruitment and binding of DSB repair factors can be sub-compartmentalized into two different regions surrounding the DSB: (a) factors that localize at DSB ends defined by regions of ssdna and (b) factors that localize to the chromatin flanking ssdna regions [48]. Intriguingly, BRCA1 has been identified in both micro-compartments and the temporal recruitment of BRCA1 to DNA break sites has been shown to be biphasic, with a rapid c-h2ax independent stage of recruitment followed by late c-h2ax-dependent recruitment. This suggests that BRCA1 may play distinct roles at different times and regions at DSB ends and the flanking chromatin [48 50]. Shedding light on this, a recent study by Li and Yu [51] found that the rapid c-h2ax independent recruitment of BRCA1 is mediated by binding of PARylated (poly(adp-ribosylated)) substrates surrounding the DSB through the BRCT domains of BARD1, which is obligately bound to BRCA1. Given that this rapid recruitment of BRCA1 to DSB ends occurs independently of c-h2ax and that recruitment of the BRCA1 A complex is dependent on c-h2ax, it is tempting to hypothesize that recruitment of the BRCA1 A complex may occur after recruitment of BRCA1 in other DSB-binding complexes. This may explain how the BRCA1 B and C complexes initiate and contribute to DNA end resection, whereas the BRCA1 A complex, which is likely recruited after this step, inhibits end resection. This hypothesis provides a model where BRCA1 within distinct macromolecular complexes, and recruited to the DSB ends and flanking chromatin at different times, is capable of initiating/promoting DSB end resection, as well as preventing over resection in flanking chromatin and assisting in the exchange of RPA/ssDNA for Rad51/ssDNA (Fig. 2). This model provides an overarching picture of how multiple different BRCA1 containing complexes are able to fine-tune the HR process to faithfully repair DSBs and help to safeguard the genome. BRCA1 has also been shown to form an S-phase specific complex with Rad51 and FANCD2, a protein mutated in a subgroup of Fanconi anaemia patients, a genomic instability syndrome characterized by cellular sensitivity to DNA cross-linking agents [52]. This complex appears to function exclusively to repair replication forks stalled at DNA interstrand cross-links (ICLs). Within this repair process, BRCA1 is considered to localize mono-ubiquitinated FANCD2 to ICL sites where it is required for ICL incision [53]. This function of BRCA1 appears to be distinct from its roles in HR because, although concomitant loss of BRCA1 and 53BP1 can restore HR and resistance to FEBS Journal 282 (2015) ª 2014 FEBS 635

7 BRCA1 and maintenance of genomic stability K. I. Savage and D. P. Harkin Fig. 2. Model for recruitment of BRCA1 complexes to DNA DSBs. Recruitment of BRCA1 to DSBs is biphasic. After DSB induction, histones around DSBs are rapidly PARylated by PARP1. Additionally, once activated, ATM phosphorylates histone H2AX, forming c-h2ax. (1) Rapid recruitment of BRCA1 B and C complexes to DSB ends, where they initiate and extend end resection, is mediated through the BARD1 BRCT domains, which recognize PARylated histones at DSB ends. (2) By contrast, the late recruitment of BRCA A complexes to DSB flanking chromatin, where it is hypothesized to prevent over resection of DNA ends, occurs through the recognition of c-h2ax by MDC1 and the subsequent activation of a ubiquitin signaling cascade, leading to recruitment of the BRCA1 A complex through binding of the BRCA1 BRCT domains to phosphorylated Abraxas. This model suggests that BRCA1, within distinct macromolecular complexes and recruited to the DSB ends and flanking chromatin at different times, is capable of initiating/promoting DSB end resection, as well as preventing over resection in flanking chromatin. Whether the BRCA1-D complex is recruited to DSBs in the rapid or late phase is unknown, although it is likely to be recruited to DSB ends, where it facilitates the exchange of RPA for Rad51. poly ADP ribose polymerase (PARP) inhibitors (which explicitly require HR-mediated repair), mice with a combined deletion of BRCA1 and 53BP1 remain sensitive to DNA interstrand cross-linking agents, such as cisplatin and mitomycin C [34,54]. This suggests the BRCA1/RAD51/FANCD2 complex may function as an independent BRCA1 protein complex that protects the genome against replication fork associated ICLs. In addition BRCA1 has been implicated in the nucleotide excision repair (NER) pathway as a result of its involvement in the BASC complex (BRCA1- associated genome surveillance complex), which includes MLH1, ATM, BLM, the MRN complex and the NER proteins Msh2 and Msh6 [55]. The NER pathway is utilized to repair DNA base modifications/ adducts, such as those caused by UV irradiation and DNA alkylating agents. The process involves the excision of a bp fragment of DNA surrounding the adducted base followed by re-synthesis and ligation using the complementary strand as a template [56]. NER is also utilized to repair actively transcribed DNA templates, known as transcription coupled repair, a process in which both Msh2 and Msh6 are involved [56]. In addition, BRCA1 has been shown to be required for transcription coupled repair of oxidative-8-oxoguanine lesions [57]. A mechanism for BRCA1 in this pathway has been suggested involving the BRCA1/BARD1 ubiquitin ligase-mediated degradation of RNA Pol II, thereby allowing access for the repair machinery [58,59]. Another mechanism through which BRCA1 may contribute to NER is via the transactivation of the xeroderma pigmentosum C and DDB2 (damaged DNA binding) proteins, both of which are involved in NER (the involvement of BRCA1 in transcriptional activation is discussed further below) [60]. BRCA1 forms multiple transcriptional complexes that contribute to genome stability As well as promoting DNA repair and genome stability directly, BRCA1 is known to form multiple complexes that regulate the transcription of genes involved in many cellular processes, including DNA repair (Fig. 3). A role for BRCA1 in transcription was first suggested by Miki et al. [1] in 1994 as a result of the high number of acidic amino acid residues in its C-terminus. BRCA1 transactivation activity was then demonstrated with experiments using the C-terminus of BRCA1 fused to the GAL4 DNA binding domain [14]. In addition, tumour derived mutations in the C-terminus of BRCA1 abolished GAL4-BRCA1- mediated transactivation and growth suppression, suggesting a physiological role for BRCA1 transcriptional regulation [14,61]. It is now widely accepted that BRCA1 affects transcription in a number of ways. First, BRCA1 is known to regulate signaling pathways that affect transcription factor activation. For example, BRCA1 phosphorylation in response to DNA damage by ATM and ATR at serines 1423 and 1524 is required for ATM-mediated p53 signaling and activation, thus regulating p53- dependent p21 transcription [62]. In addition, BRCA1 appears to modulate which p53 target genes are transactivated in response to DNA damage, with BRCA1 promoting the transcription of DNA repair and cell cycle arrest targets but not pro-apoptotic genes regulated by p53 [63,64]. By contrast to its role in promoting apoptosis, BRCA1 has also been shown to inhibit apoptosis through the formation a complex with the p65 subunit 636 FEBS Journal 282 (2015) ª 2014 FEBS

8 K. I. Savage and D. P. Harkin BRCA1 and maintenance of genomic stability Fig. 3. BRCA1 transcriptional complexes. BRCA1 forms multiple transcriptional complexes with the ability to both corepress or co-activate genes involved in different cellular processes. The proteins with which BRCA1 binds to form transcriptional activation complexes are coloured yellow, whereas co-repressor complex partners are coloured red. The functions of genes regulated by these different complexes are also highlighted. of the activation of nuclear factor-kappa B (NF-jB) transcriptional complex [65]. This complex constitutively binds to the promoters of a number anti-apoptotic NF-jB target genes, including BCL2 and XIAP. In response to DNA damage, BRCA1 recruits the p50 subunit of NF-jB to BRCA1/p60 bound promoters, resulting in transactivation of anti-apoptotic NF-jB targets, including BCL2 and XIAP, thereby inhibiting apoptosis and inducing cellular resistance to DNA damage [65]. The estrogen response signaling pathway is also known to be affected by BRCA1 at multiple different levels. Estrogens translocate into the nucleus where they bind to the estrogen receptor-a (ERa), stimulating receptor dimerization and activation. Activated ERs then bind estrogen response elements proximal to or within promoters and stimulate the transcription of target genes. Estrogen in general, promotes proliferation and drives the expression of a number of proliferative genes associated with cancer and subsequent genomic instability, such as cyclins E and D, the progesterone receptor, epithelial growth factor and vascular endothelial growth factor [66]. The link between estrogen signaling and the development of breast cancer is indicated by the potent efficacy of treatment of this disease with anti-estrogen drugs such as tamoxifen. BRCA1 is known to bind to ERa and is reported to repress both ligand-dependent and independent ERa signaling [67,68]. Concurrent with this, BRCA1 severely represses the expression of a number of endogenous ERa regulated genes, such as vascular endothelial growth factor, a protein implicated in tumour growth and angiogenesis [69]. In addition, wild-type BRCA1 inhibits proliferative signaling to the extracellular signal-related kinase by a membrane bound isoform of ERa. Another BRCA1 binding protein known as COBRA1 (i.e. a co-factor of BRCA1), a subunit of the negative elongation factor complex, also binds to ERa and inhibits ERa-mediated transcription by stalling RNA Pol II-mediated transcriptional elongation [70]. BRCA1 has also been associated directly with DNA promoters. BRCA1 does not contain any known DNA binding domains and does not bind DNA in a sequence specific manner. Instead, BRCA1 forms different transcription regulating complexes and is recruited to promoters via interaction with sequence specific transcription factors within these complexes. Consistent with this, we have shown that BRCA1 is recruited to the ERa promoter by the octamer binding protein Oct-1, where it is required for ERa expression [71]. Interestingly, we showed that RNA Pol II is present on the ERa promoter irrespective of BRCA1 promoter occupancy, suggesting, at least in the case of ERa, that BRCA1 does not recruit the core transcriptional machinery. Thus, exactly how BRCA1 stimulates transcription from the ERa promoter is unknown, although it is probably via recruitment of secondary factors and/or chromatin remodeling factors. Indeed, BRCA1 is known to bind to a number of chromatin remodeling factors such as the histone deacetylases HDAC1 and HDAC2, and the BRG1 and BRD7 components of the SWI/SNF chromatin remodeling complex [72 74]. Although SWI/SNF-mediated chromatin remodeling requires only the BRG1 BAF155 minimal complex, transcriptional activation requires an activation domain [75]. Thus, it is assumed that BRCA1 may provide this activation domain. In addition to regulating ERa expression, BRCA1 and Oct-1 are known to co-operate in the transactivation of the DNA damage-induced cell cycle inhibitor and pro-apoptotic gene GADD45, thereby preventing the propagation of damaged DNA to daughter cells and/ or promoting apoptosis, again contributing to the FEBS Journal 282 (2015) ª 2014 FEBS 637

9 BRCA1 and maintenance of genomic stability K. I. Savage and D. P. Harkin maintenance of genomic stability [76,77]. Furthermore, BRCA1/Oct-1 transcriptional activation complex has been shown to transactivate the expression of OGG1, NTH1 and REF1/APE1, which three key proteins within the base excision repair pathway, with BRCA1- dependent expression of these genes being required for resistance to H 2 O 2 -mediated oxidative stress [78]. In addition to Oct-1 and p53-mediated BRCA1 transactivation, BRCA1 functions as a transcriptional co-activator with a number of other transcription factors, such as the signal transducer and activator of transcription protein, Stat-1. BRCA1 co-activates a number of Stat-1 regulated genes such as the proapoptotic genes IRF7, MxA 2,5 OAS and ISG54 in response to interferon-c stimulation Although BRCA1 transactivates GADD45 in response to DNA damage, it has also been reported to repress GADD45 transcription in unperturbed cells, a mechanism that also requires the DNA damage response protein, CtIP [79 81]. The BRCA1/CtIP/ Zbrk1 complex has subsequently been shown to repress over a dozen genes including the DNA repair genes RFC1 and HMGA2 [82]. Zbrk1-mediated transcriptional repression requires the Kap1 co-repressor, which recruits the histone methyltransferase SETDB1 and the methylhistone binding protein HP1, resulting in chromatin compaction and transcriptional repression. Exactly how BRCA1 is involved in this repression mechanism remains unknown; however, Zbrk1- mediated GADD45 repression does not occur in the absence of BRCA1 [81]. BRCA1 also acts as a transcriptional co-repressor in a complex with c-myc and the c-myc interacting protein Nmi (n-myc-interacting protein), where it represses a number of cancer associated genes,such as htert and psoriasin (S100A7) [83,84]. The finding that BRCA1 forms part of, and is required for, both the Gadd45 pre-damage transcriptional repression complex (BRCA1/Zbrk1) and the post-damage transcriptional activation complex (BRCA1/Oct1) suggested that BRCA1 may receive and/or transduce some sort of switch signal to promoter binding proteins and/or the repression/activation machinery in response to DNA damage. Indeed, work from our own labotarory, which set out to define the BRCA1 transcriptome, found that BRCA1 is bound to a large subset of promoters throughout the genome, although it does not regulate the transcription of the majority of these genes in unperturbed cells [85]. However, in response to various stresses, such as DNA damage, the expression of many of these genes is regulated in a BRCA1-dependent manner. Given that BRCA1 is highly modified by ATM- and ATR-dependent phosphorylation events after DNA damage (as well as other phosphorylation and sumoylation events), it is conceivable that these phosphorylation events, or indeed DNA damage-induced modification of BRCA1 binding proteins, may regulate the activity and/or composition of proteins within various BRCA1 promoter bound complexes, thereby allowing the fine tuning of BRCA1-regulated transcription in response to different types of genotoxic stress. A BRCA1-mRNA splicing complex that promotes genomic stability BRCA1 has been reported to form a large DNA damage-induced protein complex containing RNA and hnrnp proteins, suggesting it may form a complex that regulates RNA processing after DNA damage [86]. Accordingly, a new BRCA1 complex involved in mrna splicing has been identified. This complex includes BRCA1, BCLAF1, U2AF35/65, Prp8 and SF3B1 and forms in response to DNA damage. BRCA1 is recruited to this complex in a BCLAF1-dependent manner after the phosphorylation of BRCA1 on serine-1423 in response to DNA damage [87]. Through this assembly mechanism, BRCA1 recruits the core mrna splicing machinery to a large subgroup of BRCA1 bound promoters, and promotes the efficient mrna splicing of these genes. Upregulated splicing of these genes, many of which are involved in DNA repair and maintenance of genomic stability, appears to maintain the expression of proteins that are turned over more rapidly after DNA damage, such as ATRIP, BACH1 and EXO1 (all of which are proteins involved in HR-mediated DSB repair). As a result, depletion of core components of this complex, such as BCLAF1 or U2AF65, results in defective DNA DSB repair and subsequent genomic instability. It should be noted that, when carrying out these studies, we found that BCLAF1 depletion was unable to further sensitize BRCA1 mutant cells to IR using clonogenic survival assays, suggesting that BRCA1 and BCLAF1 function in an epistatic manner within the same pathway. However, clonogenic survival assays only demonstrate long-term cellular survival after a specific genotoxic insult and do not allow the functional separation of different pathways that contribute to cell death. Therefore, although demonstrating that the pathway in which BRCA1 and BCLAF1 cooperate is required for cellular survival after IR, it is unlikely that all of the tumor-suppressive function of BRCA1 is mediated through its role in mrna splicing with BCLAF1 alone. 638 FEBS Journal 282 (2015) ª 2014 FEBS

10 K. I. Savage and D. P. Harkin BRCA1 and maintenance of genomic stability Intriguingly, BRCA1 has been previously reported to inhibit 3 0 mrna poly-adenylation, and thereby mrna stability, through BRCA1/BARD1 ubiquitindependent degradation of RNA-Pol II [58]. This suggests a general role for the BRCA1/BARD1 complex in blocking active transcription on a genome wide level after DNA damage, presumably to prevent the transcription of damaged genes. Despite this, it is accepted that specific genes, for example ATRIP, are actively transcribed in response to DNA damage, suggesting an additional mechanism that facilitates the transcription/expression of a subset of genes in the context of a more genome wide shutdown of transcription. We propose that, as part of this mechanism, BRCA1 recruits the mrna splicing machinery to a subset of promoters of genes required for an efficient DNA damage response (such as ATRIP, BACH1 and EXO1), thereby promoting the co-transcriptional splicing of these genes, positively regulating the stability of their transcripts and subsequent protein expression. BRCA1 tissue-specific tumourigenesis It is clear that BRCA1 plays a role in multiple different protein complexes, many of which contribute either directly or indirectly to the maintenance of genomic stability. However, given that BRCA1 plays this role in many, if not all, cells within the body, one of the most perplexing features of BRCA1 mutation is that it predominantly predisposes carriers to cancers of the breast and ovaries: both representing estrogen-regulated tissues. Several observations suggest estrogen has an important role in the development of BRCA1- dependent breast cancer. Oophorectomy in BRCA1 mutation carriers significantly reduces the risk of breast cancer onset and recurrence [88 90]. Furthermore, pregnancy increases the risk of early-onset breast cancer in BRCA1 mutation carriers, in contrast to noncarriers for whom pregnancy is protective [91]. Estrogen is postulated to promote tumourigenesis directly through stimulation of ERa and the downstream activation of pro-mitogenic transcriptional programs. However, this is confounded by observations that approximately 70 80% of BRCA1 mutated breast tumours are ERa negative [92,93]. Furthermore, BRCA1 drives ERa expression, suggesting the role of estrogen in BRCA1-dependent tumour development may be independent of ERa [71]. However, an ERa independent mechanism for estrogen-mediated tumourigenesis has been postulated, through the endogenous conversion of estrogen to genotoxic metabolites, which could then trigger genomic instability. Indeed, recent work from our own group has demonstrated that estrogen and its metabolites are capable of causing S-phase specific DNA DSBs, which appear to be induced through the collapse of stalled replication forks during DNA replication [94]. The same study also found that BRCA1 was required for the repair of estrogen and estrogen metabolite-mediated DSBs and that depletion of BRCA1, or indeed heterozygous mutation of BRCA1, as is the case in BRCA1 mutation carriers, lead to defective repair of estrogen metabolite-induced DSBs and subsequent genomic instability. Importantly, estrogeninduced DNA damage and genomic instability occurs independently of ERa. Intriguingly, the study also found that BRCA1 directly transcriptionally represses the expression of the estrogen metabolizing enzyme CYP1A1, resulting in reduced estrogen metabolism and estrogen-mediated DNA damage in human breast cells. It is well accepted that estrogen levels in human breast cells are up to six- to seven-fold higher than that of other tissues. Therefore, it is likely that the increased estrogenic burden on these tissues requires the co-ordinated effort of multiple BRCA1 functional complexes, including DNA repair and transcriptional complexes functioning together, to protect breast and ovarian cells from estrogen-induced genomic instability and subsequent tumourigenesis. In addition, studies have demonstrated that a BRCA1 transcriptional complex, involving the NRF2 transcription factor, drives antioxidant gene expression, resulting in a reduction in toxic reactive oxygen species [95]. Subsequently, it was shown that loss of the BRCA1/NRF2-mediated antioxidant response, upon BRCA1 depletion, generally leads to apoptosis. However, in breast cells, estrogen is able to promote the NRF2-dependent antioxidant response in a BRCA1 independent manner, through a PI3K/mTORdependent pathway [96]. Finally, estrogen was shown to stimulate the survival of BRCA1 deficient breast cells, which would normally undergo apoptosis, and it was suggested that this may allow cells exposed to higher levels of estrogen, such as those in the breast and ovaries, to accumulate further tumourigenic mutations, such as the TP53 or PTEN mutations usually found in BRCA1 mutated breast and ovarian tumours. Therapeutic implications of BRCA1 function Cancer therapy Given that BRCA1 plays such a determinate role in the DNA damage response, including the promotion FEBS Journal 282 (2015) ª 2014 FEBS 639

11 BRCA1 and maintenance of genomic stability K. I. Savage and D. P. Harkin of DNA DSB repair and cell cycle checkpoint arrest, it should not be unexpected that BRCA1 plays an important role in modulating the response to genotoxic chemotherapeutic agents. A number of preclinical studies have shown that BRCA1 drives resistance to a number of commonly used DNA damaging chemotherapeutics, such as etoposide, bleomycin and the platinum compounds cisplatin and carboplatin [97]. Consistent with this, a number of retrospective clinical studies have shown that patients whose tumours express low levels of BRCA1 respond better to platinum-based therapy compared to those whose tumours express moderate or high levels of BRCA1 [97,98]. By contrast, BRCA1 appears to mediate sensitivity to antimicrotubule agents; however, the mechanism behind this remains unknown [97]. More recently, the DNA double-strand break repair defect in BRCA1 mutated cells has been exploited to identify a synthetically lethal relationship with loss or inhibition or PARP1 [99]. An initial report by Farmer et al. [99] demonstrated that BRCA1 and 2 deficient cells were exquisitely sensitive to inhibition of PARP1 using small molecule inhibitors of this protein. PARP1 is a core component of the base excision repair pathway, within which it functions to signal SSBs through the poly-adp-ribosylation of histones around the SSB. It is assumed that inhibition of PARP1 leads to an accumulation of SSBs which, when met by the advancing replication fork during DNA synthesis, results in replication fork stalling. Rescue of stalled replication forks requires a functional HR pathway involving BRCA1. Therefore, in BRCA1/HR deficient cells, PARP1 inhibition leads to replication fork collapse, resulting in an accumulation of unrepaired DSBs and cell death. A number of different PARP inhibitors are currently being investigated in both breast and ovarian cancer clinical trials where BRCA1 is known to be mutated. In addition, further clinical trials are being conducted in other cancer types such as prostate and pancreatic cancers with an underlying defect in HR-mediated DNA repair pathways [100]. One of the outstanding issues in the appropriate use of PARP inhibitors across multiple cancer types relates to the need for appropriate biomarker to select patients that are likely to respond to this type of targeted therapy. The direct sequencing of BRCA1 is one method already being used to select patients for enrolment in PARP inhibitor trials. However, this approach is not without its issues because not all BRCA1 mutations will result in a repair defect. Furthermore, this approach will not identify those patients who are likely to respond to PARP inhibitors through epigenetic inactivation of BRCA1 or indeed through mutation or loss of other components of the HR repair pathway. Based on these issues, we recently developed and independently validated a gene expression signature, termed DDRD, that identifies breast tumours with an underlying DNA repair defect [101]. More importantly, we see the same biology in multiple different cancers, suggesting that the assay can be translated across different tumour types for the purpose of patient selection. Chemoprevention in BRCA1 carriers Currently, women in the UK who carry a BRCA1 mutation are offered more intensive screening and risk reducing mastectomy and/or oophorectomy. The only other risk-reduction strategy available to these women is chemoprevention. The selective ER modulators (SERMs) tamoxifen and raloxifene are recommended for use in high-risk women in the UK. However, evidence for the benefit of tamoxifen as a chemopreventive agent in BRCA1 mutation carriers is conflicting. One small study [102] showed no benefit from tamoxifen in reducing breast cancer risk in BRCA1 mutation carriers, although it has been suggested that tamoxifen use in BRCA1 mutation carriers with breast cancer may reduce the incidence of contralateral tumours [103,104]. Additionally, given that the majority of tumours that develop in BRCA1 mutation carriers are ER negative, it is unlikely that SERMs will reduce breast cancer risk. Moreover, our own findings demonstrating that estrogen can promote genomic instability (i.e. an early hallmark of BRCA1 mutant tumours) independently of the estrogen receptor cast some mechanistic doubt on the efficacy of SERMS as chemopreventive agents in BRCA1 carriers. A phase III trial termed the Prevention of Breast Cancer by Letrozole in Postmenopausal Women carrying a BRCA1/2 Mutation (LIBER) (ClinicalTrials.gov number NCT ) is currently enrolling postmenopausal women for treatment with letrozole, an aromatase inhibitor (AI) that leads to reduced estrogen synthesis and circulating estrogen levels in postmenopausal women, aiming to evaluate its ability to prevent the development of breast cancer in patients with a BRCA1/2 mutation. However, AIs may have little preventative effect in premenopausal women, in whom the majority of BRCA1-linked tumours develop, and in whom estrogen production occurs predominantly in the ovaries through an aromatase independent biosynthesis pathway. In these women, oophorectomy has been shown to reduce the risk of breast cancer by up to 60% [105]. Taking our findings into account, it may also be worth 640 FEBS Journal 282 (2015) ª 2014 FEBS