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Supplementary Figures S1-S13. Interactions of exogenously overexpressed proteins in vivo (S1); Purified recombinant proteins used in the experiments shown in Figure 2 (S2); SPR analyses of the HP1α, β, or HP1γ− chromoshadow domain binding to BARD1-BRCT (S3); SPR analyses of the BRCA1 fragments binding to HP1α, β, or γ (S4); Inhibition of HP1γ only mildly decreases the stable retention of BRCA1/BARD1 at sites of DNA damage (S5); Inhibition of HP1γ increases the interaction of BARD1 with HP1α (S6); BARD1 is dispensable for recruitment of HP1γ at DSB sites (S7); ATM-dependent interaction of BARD1 with HP1γ/H3K9me2 (S8); Ectopic accumulation of RIF1 and inhibition of RAD51 retention at DSB sites by depletion of all three isoforms of HP1 or BARD1 mutant that does not bind HP1 (S9); Dissolution kinetics of γH2AX after DSB damage in G2 cells (S10); Cell cycle progression of cells treated with UNC0638 (S11); An H3K9-specific HKMT inhibitor UNC0638 disrupts BRCA1/BARD1 retention at sites of DNA damage (S12); A model for the role of BRCA1/BARD1/HP1 complex in the DSB repair pathways (S13).

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 HP1γ. 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. Cancer Res; 75(7); 1311–21. ©2015 AACR.

Legends for Supplementary Figures S1-S13.

Supplemenatary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR (“Critical sequence”) contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

The tumor-suppressor proteins BRCA1 and BARD1 function as an E3 ubiquitin ligase to facilitate transcriptional repression and DNA damage repair. This is mediated in-part through its ability to mono-ubiquitylate histone H2A in nucleosomes. Studies in Caenorhabditis elegans have been used to elucidate numerous functions of BRCA1 and BARD1; however, it has not been established that the C. elegans orthologs, BRC-1 and BRD-1, retain all the functions of their human counterparts. Here we explore the conservation of enzymatic activity toward nucleosomes which leads to repression of estrogen-metabolizing cytochrome P450 (cyp) genes in humans. Biochemical assays establish that BRC-1 and BRD-1 contribute to ubiquitylation of histone H2A in the nucleosome. Mutational analysis shows that while BRC-1 likely binds the nucleosome using a conserved interface, BRD-1 and BARD1 have evolved different modes of binding, resulting in a difference in the placement of ubiquitin on H2A. Gene expression analysis reveals that in spite of this difference, BRC-1 and BRD-1 also contribute to cyp gene repression in C. elegans. Establishing conservation of these functions in C. elegans allows for use of this powerful model organism to address remaining questions regarding regulation of gene expression by BRCA1 and BARD1.

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1- nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.

Background: Breast cancer type 1 susceptibility protein (BRCA1) is a tumor suppressor gene involved in DNA double strand break repair with well-known cancer implications. BRCA1 heterodimerizes with BRCA1 associated Ring domain 1 (BARD1) to form a complex with DNA binding and ubiquitin E3 ligase function capable of interacting with proteins of diverse biological processes, most notably homology-directed DNA repair. During DNA repair, BRCA1-BARD1 directly interfaces with nucleosomes and transfers mono ubiquitin (Ub) to lysine residues on the C-terminal tail of histone H2A. Although truncation of the enzymatic BRCA1-BARD1 RING-RING domain retains H2A ubiquitylating activity, full-length BRCA1-BARD1 binds more tightly with nucleosomes and displays higher H2A-Ub activity. However, the molecular basis and biological significance for this enhanced nucleosome binding and H2A-Ub activity is uncharacterized. Methods: Full length BRCA1-BARD1 or truncated mutants and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. To determine the biological significance, mammalian cell lines that stably express wild type or mutant forms of BARD1 were established for cellular fractionation, foci analysis, and clonogenic survival studies alongside various DNA damage agents. Results: Our results show multiple interaction sites exist between BRCA1-BARD1 and nucleosomes which allow high-affinity chromatin binding and promote increased histone H2A ubiquitylation activity. Multivalent BARD1-nucleosome interactions, namely those using strong binding motifs located in the intrinsically disordered region (IDR) of BARD1, and the weak “kiss” interaction mediated by the RING domains of both BRCA1 and BARD1, are essential for H2A ubiquitylation by BRCA1-BARD1. Further, we isolated two types of specific histone binding and/or ubiquitylation-defective mutants of BARD1: a BARD1-IDR mutant with disrupted nucleosome binding withe retained H2A ubiquitylation ability, and a RING mutant that solely impairs H2A ubiquitylation. In both cases, we demonstrate that these mutants are hypersensitive to DNA damage agents, including polyADP-ribose polymerase (PARP) inhibitors, and demonstrate reduced capacity of BARD1 to associate with chromatin and foci formation owing to attenuated repair capacity. Conclusion: Our studies provide convincing evidence BRCA1-BARD1 interacts with nucleosomes and ubiquitylates histones via its E3 ligase activity. Further, it plays a critical role in DNA damage response and repair that contributes to genome stability, which when disrupted sensitizes them to DNA damage agents. Our results open new avenues towards understanding whether and how these mutations in BRCA1-BARD1 affect its tumor suppression functions and their implications clinically, ultimately with the goal to translate these findings for the benefit of cancer patients. Citation Format: Wenjing Li, Samuel R. Witus, Meiling Wang, Peter S. Brzovic, Rachel E. Klevit, Weixing Zhao. BRCA1-BARD1 ubiquitylates histones for genome maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6096.

Small heat shock proteins (sHSPs) are chaperones whose importance in protein homeostasis is exemplified by dozens of missense mutations associated with tissue-specific disease states. Despite decades of studies, the structure, dynamics, and mechanism of chaperone activity remain unclear. Here we show that the human sHSP HSPB5 distinguishes native lens protein γD-crystallin from damaged γD-crystallin even though the mutant/damaged client is folded. The disordered N-terminal region of HSPB5 (NTR) is essential for its chaperone activity, whereas the structured domain (ACD) has no intrinsic activity. Nevertheless, two sHSP mutational hotspots associated with disease, D109 and R120, are located in the ACD. Our studies on wild-type HSPB5 oligomers reveal that distinct regions within the NTR interact with specific grooves presented on the ACD dimer and/or with other NTR sub-regions and that the number of binding partners is greater than the number of binding sites, leading to a large, but finite number of potential combinations of interactions at any given time. The ACD mutations result in increased dynamics and accessibility of the disordered NTR and enhanced chaperone activity in vitro. Our findings reveal that HSPB5 quasi-order is delicately balanced and that perturbations arising from mutations within the structured core cause alterations that contribute to misbalance in eye lens protein homeostasis that lead to cataract formation.

RING-between-RING (RBR) E3 ligases mediate ubiquitin transfer through an obligate E3-ubiquitin thioester intermediate prior to substrate ubiquitination. Although RBRs share a conserved catalytic module, substrate recruitment mechanisms remain enigmatic, and the relevant domains have yet to be identified for any member of the class. Here we characterize the interaction between the auto-inhibited RBR, HHARI (AriH1), and its target protein, 4EHP, using a combination of XL-MS, HDX-MS, NMR, and biochemical studies. The results show that (1) a di-aromatic surface on the catalytic HHARI Rcat domain forms a binding platform for substrates and (2) a phosphomimetic mutation on the auto-inhibitory Ariadne domain of HHARI promotes release and reorientation of Rcat for transthiolation and substrate modification. The findings identify a direct binding interaction between a RING-between-RING ligase and its substrate and suggest a general model for RBR substrate recognition.