Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites

Background:

Pathogenic mutations in the BRCA1 gene are associated with an increased risk of developing breast and ovarian cancer (Easton, 1999; Gorodetska 2019). BRCA1 tumor suppressor activity is mainly linked to its function in the repair of DNA double-strand breaks (DSBs), toxic DNA lesions that activate the DNA damage response (DDR), and whose inaccurate repair leads to genome instability and tumorigenesis (Jackson and Bartek, 2009).

The N-terminal RING domain of BRCA1 mediates the interaction with BARD1 and E2-ubiquitin-conjugating enzymes, thus allowing ubiquitylation of target proteins (Figure 1) (Huen, 2010).

Figure 1. BRCA1 domains architecture. From Huen, M., Sy, S. & Chen, J. Nat Rev Mol Cell Biol, 2010

The C-terminal BRCA1 BRCT domains recognize various phosphorylated DDR proteins, forming distinct BRCA1-containing complexes. Interaction of the BRCT domains with phosphorylated ABRAXAS mediates the formation of the BRCA1-A complex. This complex is thought to recruit BRCA1 to DSBs via RAP80-mediated recognition of the poly-ubiquitin chains deposited by RNF8 and RNF168 (Figure 2).

Figure 2. A model for RNF8-/RNF168-mediated ubiquitylation in mediating BRCA1 recruitment to DSBs. Adapted from Huen, M., Sy, S. & Chen, J. Nat Rev Mol Cell Biol, 2010

Key findings: 

• BRCA1 is efficiently recruited to DSBs in the absence of RAP80. To investigate the role of chromatin ubiquitylation at DSBs in BRCA1 recruitment, the authors generated knockout (KO) cells for RAP80 by CRISPR/Cas9-mediated genome editing. As expected, these cells fail to form ABRAXAS foci at DSBs induced by ionizing radiation (IR). In contrast, BRCA1 recruitment to DSBs is not affected, while its maintenance is, which suggests that RAP80 (and the BRCA1-A complex) is not necessary for the initial recruitment of BRCA1 to DSBs.
• BRCA1 recruitment to DSBs relies on RAP80 in the absence of RNF168. At DSBs, RAP80 recognizes the K63-ubiquitin chains deposited by RNF8 and RNF168. By using siRNA and KO approaches, the authors show that RNF8 depletion prevents BRCA1 foci formation. However, BRCA1 foci are only reduced by RNF168 depletion and completely lost in the absence of both RNF168 and RAP80. These findings indicate that RAP80-mediated recruitment of BRCA1 becomes essential in the absence of RNF168.
• RAP80-independent BRCA1 recruitment to DSBs occurs via the RING domain. To understand the dependence of RAP80 on BRCA1 recruitment to DSBs, the authors expressed the truncated BRCT domain of BRCA1, whose localization to DSB showed RAP80 dependency. This implies that RAP80-independent recruitment of BRCA1 to DNA lesions must be carried out by a region other than the tandem BRCT domains. To identify this region, the authors expressed various GFP-tagged siRNA-resistant BRCA1 mutants in BRCA1 depleted cells. They observed that two mutants unable to interact with the BRCA1-A complex (via deletion of the BRCT domain or mutation of the ABRAXAS phosphopeptide-binding site) and one mutant consisting only of the RING finger domain are still able to localize to DSBs independently of RAP80. From these observations, they conclude that the RING domain may be important for BRCA1 recruitment redundantly with RAP80.
• RAP80-independent BRCA1 recruitment to DSBs requires interaction with the nucleosome acidic patch or with the E2-ubiquitin ligases via the RING domain. Since mutation or deletion of the RING domain can impair BRCA1 stability, the authors generated BRCA1 constructs harbouring two distinct mutations within the RING domain that abolish the interaction with either the E2-ubiquitin-conjugating enzymes or the nucleosome acidic patch, whilst retaining protein stability. Both mutants accumulate at DSBs in wild-type cells but not in RAP80 KO, implying that both the recognition of the ubiquitylated chromatin via RAP80/BRCA1-A complex and the RING domain are required for BRCA1 recruitment to DSBs. In these mutants, DSB localization of the HR-marker RAD51 also relies on RAP80.
• Inactivation of the RING- and the RAP80-dependent BRCA1 recruitment to DSBs hypersensitizes cells to PARP inhibition. To investigate the clinical relevance of their findings, the authors reconstituted the BRCA1 deficient breast cancer cell line MDA-MB-436 cells with BRCA1 constructs lacking either the BRCT domain or lacking the BRCT domain and harbouring the E2-ubiquitin ligase binding mutation within the RING domain. They found that the double mutant fails to form BRCA1 and RAD51 foci. In line with this observation, cells expressing the double mutant are hyper-sensitive to PARP inhibitor, a drug that kills selectively HR-deficient cancer cells.

Conclusions and future perspectives

In summary, Sherker and colleagues show that chromatin ubiquitylation controls BRCA1 recruitment to DSBs in two ways: one via the BRCT domain-mediated interaction with the BRCA1-A complex (including RAP80) that recognizes K63-ubiquitin chains, the other through the RING domain and dependent on RNF168-mediated ubiquitylation of H2A K13 and K15. Although BARD1 recognizes the H2AK13/K15 ubiquitylation marks (Becker et al., 2021; Dai et al., 2021), the BRCA1 RING mutants used in this preprint (unable to interact with the E2-ubiquitin ligases or with the nucleosomes acidic patch) are still able to interact with BARD1, thus leading the authors to hypothesise alternative mechanisms to explain the observed phenotype.

They propose that BRCA1-mediated H2A ubiquitylation (H2A K125/K127/K129) could promote its own recruitment by favoring SMARCAD1 localization to DSBs and 53BP1 displacement from nucleosomes (Densham et al., 2016). This, in turn, would allow BRCA1-BARD1 to access the H2AK13/K15 ubiquitylated nucleosomes. Alternatively, the interaction with the E2 could be important for positioning BARD1 for the recognition of the H2AK13/15 ubiquitylation.

Since various pathogenic mutations affect the BRCA1 RING domain (Bouwman et al., 2020; Findlay et al., 2018) and tumors expressing RING-less BRCA1 isoforms acquire resistance to therapy, these findings suggest that targeting RAP80, or its interaction with BRCA1, could represent a novel strategy to overcome the resistance of those tumors to DNA damaging agents.

What I liked about this preprint and questions for the authors

I love the idea that BRCA1-mediated H2A ubiquitylation mediates its own recruitment to DSB and it would be very interesting to see what happens after mutating different H2A lysines. Do you believe that other ubiquitylation targets rather than the histones could mediate the observed phenotype?

References:

• Becker, J.R., Clifford, G., Bonnet, C. et al. BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination. Nature (2021).
• Bouwman, P., van der Heijden, I., van der Gulden, H., de Bruijn, R., Braspenning, M.E., Moghadasi, S., Wessels, L.F.A., Dutch-Belgian, V.U.S.w., Vreeswijk, M.P.G., and Jonkers, J. Functional Categorization of BRCA1 Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays. Clin Cancer Res (2020).
• Coleman, K.A., and Greenberg, R.A. The BRCA1-RAP80 complex regulates DNA repair mechanism utilization by restricting end resection. The Journal of biological chemistry (2011).
• Dai, L., Dai, Y., Han, J., Huang, Y., Wang, L., Huang, J., and Zhou, Z. Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin. Mol Cell (2021).
• Dever, S.M., Golding, S.E., Rosenberg, E., Adams, B.R., Idowu, M.O., Quillin, J.M., Valerie,N., Xu, B., Povirk, L.F., and Valerie, K. Mutations in the BRCT binding site of BRCA1result in hyper-recombination. Aging (2011).
• Densham, R.M., Garvin, A.J., Stone, H.R., Strachan, J., Baldock, R.A., Daza-Martin, M., Fletcher, A., Blair-Reid, S., Beesley, J., Johal, B., et al. Human BRCA1-BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection. Nat Struct Mol Biol (2016).
• Easton, D. F., How many more breast cancer predisposition genes are there? Breast Cancer Research (1999).
• Findlay, G.M., Daza, R.M., Martin, B., Zhang, M.D., Leith, A.P., Gasperini, M., Janizek, J.D.,Huang, X., Starita, L.M., and Shendure, J. Accurate classification of BRCA1 variants with saturation genome editing. Nature (2018).
• Gorodetska, I., Kozeretska, I., Dubrovska, A., BRCA genes: The role in genome stability, cancer stemness and therapy resistance, Journal of Cancer (2019).
• Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature (2009).
• Huen, M., Sy, S. & Chen, J. BRCA1 and its toolbox for the maintenance of genome integrity. Nat Rev Mol Cell Biol (2010).

Tags: brca1, dsb

doi: https://doi.org/10.1242/prelights.30276

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