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BARD1 Is Necessary for Ubiquitylation of Nucleosomal Histone H2A and for Transcriptional Regulation of Estrogen Metabolism Genes

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BARD1 Is Necessary for Ubiquitylation of Nucleosomal Histone H2A and for Transcriptional Regulation of Estrogen Metabolism Genes BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes Mikaela D. Stewarta,1, Elena Zelinb, Abhinav Dhallc, Tom Walshd,e, Esha Upadhyayc, Jacob E. Cornb,f, Champak Chatterjeec, Mary-Claire Kingd,e,2, and Rachel E. Klevita,2 aDepartment of Biochemistry, University of Washington, Seattle, WA 98195; bInnovative Genomics Institute, University of California, Berkeley, CA 94720; cDepartment of Chemistry, University of Washington, Seattle, WA 98195; dDepartment of Medicine, University of Washington, Seattle, WA 98195; eDepartment of Genome Sciences, University of Washington, Seattle, WA 98195; and fDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Contributed by Mary-Claire King, December 23, 2017 (sent for review September 4, 2017; reviewed by Vishva M. Dixit and Stanley Lipkowitz) Missense mutations that disrupt the RING domain of the tumor BRCA1/BARD1 heterodimer comprises the first ∼100 residues suppressor gene BRCA1 lead to increased risk of breast and ovar- of each protein, bringing their two RING domains into close ian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose proximity. As part of the heterodimer, the BRCA1 RING do- structure and function rely critically on forming a heterodimer main interacts with an E2 enzyme to activate transfer of ubiq- with BARD1, which also harbors a RING domain. The function of uitin onto a substrate, but the role of the BARD1 RING domain the BARD1 RING domain is unknown. In families severely affected has remained unknown. Mutations that result in truncation of with breast cancer, we identified inherited BARD1 missense muta- the BARD1 protein are associated with increased risk of breast tions Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zinc- and ovarian cancer (11–15), but the functional and clinical binding residues of the BARD1 RING domain. Each of these mutant consequences of missense mutations in the BARD1 RING BARD1 proteins retained the ability to form heterodimeric com- domain have not been characterized. To address these ques- plexes with BRCA1 to make an active ubiquitin ligase, but the GENETICS tions, we evaluated three missense mutations in the BARD1 mutant BRCA1/BARD1 complexes were deficient in binding to nu- RING domain, newly discovered in families severely affected cleosomes and in ubiquitylating histone H2A. The BARD1 muta- tions also caused loss of transcriptional repression of BRCA1- by breast cancer. CYP1A1 CYP3A4 regulated estrogen metabolism genes and ; breast Results epithelial cells edited to create heterozygous loss of BARD1 CYP1A1 CYP3A4 Germline Mutations in the BARD1 RING Domain in Breast Cancer showed significantly higher expression of and . BARD1 Reintroduction of wild-type BARD1 into these cells restored Families. Truncating mutations of are known to increase CYP1A1 and CYP3A4 transcription to normal levels, but introduc- risk of breast and ovarian cancer (16, 17), but much less is known tion of the cancer-predisposing BARD1 RING mutants failed to do of the functional and clinical consequences of missense mutations so. These results indicate that an intact BARD1 RING domain is crit- in the BARD1 RING domain. In the course of genomic analysis ical to BRCA1/BARD1 binding to nucleosomes and hence to ubiqui- of inherited predisposition to breast cancer, we identified three tylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism. Significance BARD1 | BRCA1 | breast cancer | ubiquitin | transcriptional repression Loss-of-function mutations in BRCA1 and its protein partner BARD1 lead to high risks of breast and ovarian cancer. Both mong inherited mutations of BRCA1 that increase risk of BRCA1 and BARD1 proteins harbor RING domains, and mis- Abreast and ovarian cancer are missense mutations that ab- sense mutations in the critical residues of the BRCA1 RING rogate the function of the BRCA1 RING domain. The BRCA1 domain are among those known to predispose to cancer. RING domain is a ubiquitin ligase (E3) enzyme, that catalyzes The BRCA1 RING domain is a ubiquitin ligase, but the func- the covalent attachment of the signaling protein ubiquitin (Ub) tion of the BARD1 RING domain and the consequences of onto protein substrates by binding to and activating E2 enzymes. mutations in it are unknown. Evaluation of missense muta- As is true for most Ub ligases, BRCA1 modifies numerous tions at evolutionarily conserved zinc-binding residues of proteins, including histone H2A, estrogen receptor α (ERα), and BARD1, each identified in a family severely affected with progesterone receptor (1–3). Although the biological conse- breast cancer, revealed that BARD1 is necessary for two core quences of protein ubiquitylation by BRCA1 are largely un- functions of the BRCA1/BARD1 complex: ubiquitylation of known, the BRCA1-dependent modification of histone H2A is histone 2A on nucleosomes and transcriptional regulation of critical for DNA damage repair and genome stability (4, 5). genes of estrogen metabolism. Cancer-predisposing missense mutations in the BRCA1 RING ’ Author contributions: M.D.S., T.W., J.E.C., C.C., M.-C.K., and R.E.K. designed research; M.D.S., domain cause loss of the domain s ligase activity, and hence a E.Z., A.D., T.W., E.U., M.-C.K., and R.E.K. performed research; T.W., J.E.C., C.C., and M.-C.K. general defect in ubiquitylation. The mutations also lead to ge- contributed new reagents/analytic tools; M.D.S., E.Z., T.W., M.-C.K., and R.E.K. analyzed nomic instability (2, 6–8). Nevertheless, there are conflicting data; and M.D.S., M.-C.K., and R.E.K. wrote the paper. hypotheses regarding the role of BRCA1 Ub ligase activity in Reviewers: V.M.D., Genentech; and S.L., National Cancer Institute. tumor suppression (4, 8–10). The alternative hypotheses have The authors declare no conflict of interest. been difficult to resolve because BRCA1-mutant RING domains Published under the PNAS license. are inactive toward all ubiquitylation substrates, so the specific 1Present address: Department of Biology, Texas Christian University, Fort Worth, TX 76129. target(s) involved in tumor suppression activity of BRCA1 has 2To whom correspondence may be addressed. Email: [email protected] or not been identified. [email protected]. Ub ligase activity of BRCA1 depends on BRCA1 forming a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex with BARD1, which also harbors a RING domain. The 1073/pnas.1715467115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1715467115 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 mutations in the RING domain of BARD1 in four families: BARD1 the BARD1 RING domain does not bind directly to E2 enzymes p.C53W (c.159T > G) at chr 2: 215,661,841 in families CF2947 and (22, 23), the consequences of comparable mutations in the BARD1 CF3031(whoarenotrelatedtoeachother);BARD1p.C71Y RING domain have remained undefined. (c.212G > A) at chr 2: 215,661,788 in family CF3490; and BARD1 To better understand the effects of mutations in zinc-binding p.C83R (c.247 A > G) at chr 2: 215,657,138 in family CF4638 (18) residues of the BARD1 RING domain, we evaluated BRCA1/ (Fig. 1A). Each of the three BARD1 mutations occurs at a residue BARD1 heterodimer complexes that contained each of the completely conserved across all sequenced species. None of the BARD1 missense mutations. For comparison, we similarly three mutations appear in the Genome Aggregation Database evaluated BARD1 p.C78S (c.233G > C) at chr 2: 215,657,152, (gnomAD) of 123,136 exome sequences (19) or among 10,000 which also lies in the BARD1 RING domain but alters a cysteine cancer-free older female participants of the Women’s Health residue that is not conserved and does not coordinate zinc. Initiative (whi.color.com/). For five of the 12 breast cancers in BARD1 p.C78S has not been observed in any family in our study, the four families, hormone receptor information was available: but was reported as a “variant of unknown significance” in one one tumor was triple negative and four were estrogen receptor individual of unknown phenotype in the ClinVar genetic data- and progesterone receptor positive. Most breast cancers from base (NIH clinical variation database) (24). patients with BRCA1 or truncating BARD1 mutations are triple negative (17). Whether triple negative breast cancer will also pre- Effect of BARD1 Mutations on Ubiquitylation of Nucleosomal Histone dominate among patients with missense mutations in the BARD1 H2A. The BARD1 RING domain binds to BRCA1 and stimulates RING domain awaits additional data. The observation in family BRCA1 ubiquitin ligase activity (6, 25, 26). To determine the CF3490 of two unaffected women, ages 67 and 68, who carry effect of BARD1 mutations, we carried out in vitro assays for BARD1 p.C71Y suggests that BARD1 RING domain mutations these activities using constructs of BRCA1 and BARD1 that have may not convey as high a risk as mutations in the comparable been shown to exhibit binding and ubiquitin ligase function similar residues of the BRCA1 RING domain (Fig. 1B)(20). to the full-length proteins (1, 2, 22). All wild-type and mutant RING domain structures are stabilized by two zinc atoms that BARD1 RING domains copurified with BRCA1, confirming that are bound by seven cysteine and one histidine residues. The all BARD1 variants retained their ability to form heterodimers three BARD1 RING domain mutations identified in breast with BRCA1 (SI Appendix,Fig.S1). cancer families alter cysteine residues that coordinate these zinc Ubiquitylation of lysine residues in the C-terminal tail of his- ions (Fig. 1 B and C). The amino acids of the resulting mutant tone H2A by the BRCA1/BARD1 heterodimer plays an im- proteins (tryptophan, tyrosine, and arginine) are not chemically portant role in DNA damage repair and gene silencing and is a capable of coordinating zinc in a manner similar to cysteine.
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