Structure of a BRCA1-BARD1 heterodimeric RING-RING complex

The RING domain of the breast and ovarian cancer tumor suppressor BRCA1 interacts with multiple cognate proteins, including the RING protein BARD1. Proper function of the BRCA1 RING domain is critical, as evidenced by the many cancer-predisposing mutations found within this domain. We present the solution structure of the heterodimer formed between the RING domains of BRCA1 and BARD1. Comparison with the RING homodimer of the V(D)J recombination-activating protein RAG1 reveals the structural diversity of complexes formed by interactions between different RING domains. The BRCA1-BARD1 structure provides a model for its ubiquitin ligase activity, illustrates how the BRCA1 RING domain can be involved in associations with multiple protein partners and provides a framework for understanding cancer-causing mutations at the molecular level.     

Identification of residues required for the interaction of BARD1 with BRCA1.

The breast and ovarian cancer predisposition gene product BRCA1, binds to BARD1 at its N terminus. In cells BRCA1 is found as a heterodimer with BARD1 and may represent the functionally active form of BRCA1. Using yeast two-hybrid and split-hybrid screens we have identified 16 independent missense mutations which prevent the ability of the BARD1 N terminus to heterodimerize with BRCA1. With reference to the recent structure of the BARD1center dotBRCA1 RING complex (Brzovic, P. S., Rajagopal, P., Hoyt, D. W., King, M-C., and Klevit, R. E. (2001) Nat. Struct. Biol. 8, 833--837) we note two classes of mutation; those that map to the hydrophobic core forming the BARD1:BRCA1 interface and are substitutions of leucine, and those that map to residues forming intramolecular contacts either in helical packing, or in the conserved zinc chelating cysteine residues of the RING itself. The directed mutation of charged residues predicted to play a role in the interaction could not alone prevent heterodimer formation suggesting that, while polar interactions may participate in the specificity of the interaction, they are not crucial. Together these data provide functional evidence for the requirement of a hydrophobic interface and illustrate that disruption of the tertiary structure by mutations away from the interface itself are able to prevent formation of the heterodimer.     

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms

The tumor‐suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N‐terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain‐containing proteins. RING domains bind and activate E2 ubiquitin‐conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer‐associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.     

BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export

BRCA1 is a tumor suppressor with several important nuclear functions. BRCA1 has no known cytoplasmic functions. We show here that the two previously identified nuclear localization signals (NLSs) are insufficient for nuclear localization of BRCA1 due to the opposing action of an NH2-terminal nuclear export signal. In transfected breast cancer cells, BRCA1 nuclear localization requires both the NLSs and NH2-terminal RING domain region; mutating either of these sequences shifts BRCA1 to the cytoplasm. The BRCA1 RING element mediates nuclear import via association with BARD1, and this is not affected by cancer-associated RING mutations. Moreover, BARD1 directly masks the BRCA1 nuclear export signal, and the resulting block to nuclear export is requisite for efficient import and nuclear localization of ectopic and endogenous BRCA1. Our results explain why BRCA1 exon 11 splice variants, which lack the NLSs but retain the RING domain, are frequently detected in the nucleus and in nuclear foci in vivo. In fact, co-expression of BARD1 promoted formation of DNA damage-induced nuclear foci comprising ectopic wild-type or NLS-deficient BRCA1, implicating BARD1 in nuclear targeting of BRCA1 for DNA repair. Our identification of BARD1 as a BRCA1 nuclear chaperone has regulatory implications for its reported effects on BRCA1 protein stability, ubiquitin ligase activity, and DNA repair.     

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

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin‐dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80‐ and RING‐dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding‐deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80–BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.     

Structural requirements for the BARD1 tumor suppressor in chromosomal stability and homology-directed DNA repair

The BRCA1 tumor suppressor exists as a heterodimeric complex with BARD1, and this complex is thought to mediate many of the functions ascribed to BRCA1, including its role in tumor suppression. The two proteins share a common structural organization that features an N-terminal RING domain and two C-terminal BRCT motifs, whereas BARD1 alone also contains three tandem ankyrin repeats. In normal cells, the BRCA1/BARD1 heterodimer is believed to enhance chromosome stability by promoting homology-directed repair (HDR) of double strand DNA breaks. Here we have investigated the structural requirements for BARD1 in this process by complementation of Bard1-null mouse mammary carcinoma cells. Our results demonstrate that the ankyrin and BRCT motifs of BARD1 are each essential for both chromosome stability and HDR. Tandem BRCT motifs, including those found at the C terminus of BARD1, are known to form a phosphoprotein recognition module. Nonetheless, the HDR function of BARD1 was not perturbed by synthetic mutations predicted to ablate the phospho-recognition activity of its BRCT sequences, suggesting that some functions of the BRCT domains are not dependent on their ability to bind phosphorylated ligands. Also, cancer-associated missense mutations in the BRCT domains of BARD1 (e.g. C557S, Q564H, V695L, and S761N) have been observed in patients with breast, ovarian, and endometrial tumors. However, none of these was found to affect the HDR activity of BARD1, suggesting that any increased cancer risk conferred by these mutations is not because of defects in this repair mechanism.     

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.     

HDAC Inhibitors Repress BARD1 Isoform Expression in Acute Myeloid Leukemia Cells via Activation of miR-19a and/or b

Over the past years BARD1 (BRCA1-associated RING domain 1) has been considered as both a BRCA1 (BReast Cancer susceptibility gene 1, early onset) interactor and tumor suppressor gene mutated in breast and ovarian cancers. Despite its role as a stable heterodimer with BRCA1, increasing evidence indicates that BARD1 also has BRCA1-independent oncogenic functions. Here, we investigate BARD1 expression and function in human acute myeloid leukemias and its modulation by epigenetic mechanism(s) and microRNAs. We show that the HDACi (histone deacetylase inhibitor) Vorinostat reduces BARD1 mRNA levels by increasing miR-19a and miR-19b expression levels. Moreover, we identify a specific BARD1 isoform, which might act as tumor diagnostic and prognostic markers.     

The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50

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.     

Negative feedback loop of BRCA1–BARD1 ubiquitin ligase on estrogen receptor alpha stability and activity antagonized by cancer-associated isoform of BARD1

Estrogen is involved in breast cancer risk, which is increased for BRCA1 mutation carriers, suggesting a role for BRCA1 in estrogen signaling. BRCA1 exerts its function through forming an E3 ubiquitin ligase with BARD1. We report that the estrogen receptor alpha is a target of the BRCA1–BARD1 ubiquitin ligase in vivo. BRCA1 and BARD1 are required for estrogen receptor alpha ubiquitination and degradation, and repression of either one leads to ERα accumulation, suggesting a feedback loop between BRCA1–BARD1 and estrogen receptor alpha, since BRCA1 and BARD1 are induced by estrogen receptor alpha. While the ubiquitin ligase activity maps to the N-terminal RING finger domains of BRCA1 and BARD1, we demonstrate that the BARD1 C-terminus is important for target recognition. Furthermore, a BARD1 isoform lacking the RING domain binds and stabilizes estrogen receptor alpha. Thus deficiencies of BRCA1 or BARD1 and/or upregulation of BARD1 isoforms lead to estrogen receptor alpha upregulation, providing a functional link between BRCA1 deficiency, estrogen signaling, and tumorigenesis.     

Ubiquitination and proteasome-mediated degradation of BRCA1 and BARD1 during steroidogenesis in human ovarian granulosa cells

Germ-line mutations in BRCA1 predispose women to early-onset, familial breast and ovarian cancers. However, BRCA1 expression is not restricted to breast and ovarian epithelial cells. For example, ovarian BRCA1 expression is enriched in ovarian granulosa cells, which are responsible for ovarian estrogen production in premenopausal women. Furthermore, recent tissue culture and animal studies suggest a functional role of BRCA1 in ovarian granulosa cells. Although levels of BRCA1 are known to fluctuate significantly during folliculogenesis and steroidogenesis, the mechanism by which BRCA1 expression is regulated in granulosa cells remains to be elucidated. Here we show that the ubiquitin-proteasome degradation pathway plays a significant role in the coordinated protein stability of BRCA1 and its partner BARD1 in ovarian granulosa cells. Our work identifies the amino-terminal RING domain-containing region of BRCA1 as the degron sequence that is both necessary and sufficient for polyubiquitination and proteasome-mediated protein degradation. Interestingly, mutations in the RING domain that abolish the ubiquitin E3 ligase activity of BRCA1 do not affect its own ubiquitination or degradation in ovarian granulosa cells. The proteasome-mediated degradation of BRCA1 and BARD1 also occurs during the cAMP-dependent steroidogenic process. Thus, the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells provide an excellent paradigm for investigating the regulation of this protein complex under physiological conditions.     

BARD1 translocation to mitochondria correlates with Bax oligomerization, loss of mitochondrial membrane potential, and apoptosis

The breast cancer regulatory protein-1 (BRCA1)-associated RING domain 1 (BARD1) gene is mutated in a subset of breast/ovarian cancers. BARD1 functions as a heterodimer with BRCA1 in nuclear DNA repair. BARD1 also has a BRCA1-independent apoptotic activity. Here we investigated the link between cytoplasmic localization and apoptotic function of BARD1. We used immunofluorescence microscopy and deconvolution analysis to resolve BARD1 cytoplasmic staining patterns and detected endogenous BARD1 at mitochondria. BARD1 was also detected in mitochondrial cell fractions by immunoblotting. The targeting of BARD1 to mitochondria was modestly stimulated by DNA damage and did not require BRCA1 as indicated by RNA interference and peptide-competition experiments. Transiently expressed yellow fluorescence protein-BARD1 localized to mitochondria, and the targeting sequences were mapped to both the N and C terminus of BARD1. Ectopic yellow fluorescence protein-BARD1 induced apoptosis and loss of mitochondrial membrane potential in MCF-7 breast tumor cells. BARD1 apoptotic function was associated with stimulation of Bax oligomerization at mitochondria. This distinguishes it from BRCA1, which is pro-apoptotic but did not induce Bax oligomerization. The cancer-associated BARD1 splice-variant DeltaRIN (lacks the BRCA1 binding domain and ankyrin repeats) was recruited to mitochondria but did not stimulate apoptosis or alter membrane permeability. We propose that BARD1 has two main sites of action in its cellular response to DNA damage, the nucleus, where it promotes cell survival through DNA repair, and the mitochondria, where BARD1 regulates apoptosis.     

53BP1 ablation rescues genomic instability in mice expressing ‘RING‐less’ BRCA1

BRCA1 mutations strongly predispose affected individuals to breast and ovarian cancer, but the mechanism by which BRCA1 acts as a tumor suppressor is not fully understood. Homozygous deletion of exon 2 of the mouse Brca1 gene normally causes embryonic lethality, but we show that exon 2‐deleted alleles of Brca1 are expressed as a mutant isoform that lacks the N‐terminal RING domain. This “RING‐less” BRCA1 protein is stable and efficiently recruited to the sites of DNA damage. Surprisingly, robust RAD51 foci form in cells expressing RING‐less BRCA1 in response to DNA damage, but the cells nonetheless display the substantial genomic instability. Genomic instability can be rescued by the deletion of Trp53bp1, which encodes the DNA damage response factor 53BP1, and mice expressing RING‐less BRCA1 do not show an increased susceptibility to tumors in the absence of 53BP1. Genomic instability in cells expressing RING‐less BRCA1 correlates with the loss of BARD1 and a defect in restart of replication forks after hydroxyurea treatment, suggesting a role of BRCA1–BARD1 in genomic integrity that is independent of RAD51 loading.     

Characterization of BRCA1 protein targeting, dynamics, and function at the centrosome: a role for the nuclear export signal, CRM1, and Aurora A kinase

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 γ-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 γ-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.     

The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation

BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1-304 of BRCA1 and residues 25-189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1(C61G), does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development.     

Autoubiquitination of the BRCA1*BARD1 RING ubiquitin ligase

The RING finger of BRCA1 confers ubiquitin ligase activity that is markedly enhanced when complexed with another RING-containing protein, BARD1, and is required for the function of this tumor suppressor protein in protecting genomic integrity. Here, we report that co-expression of BRCA1-(1-639) and BARD1 in bacteria can assemble a potent ubiquitin ligase activity. Purified BRCA1-(1-639)*BARD1 stimulated the Ubc5c-mediated monoubiquitination of histone H2A/H2AX in vitro, suggesting a possible role for BRCA1*BARD1 in modifying chromatin structure. Moreover, the truncated BRCA1*BARD1 complex exhibited efficient autoubiquitination activity in vitro capable of assembling non-lysine 48-linked polyubiquitin chains on both BRCA1-(1-639) and BARD1. When co-expressed in cells by transient transfection, the recombinant BRCA1-(1-300).BARD1 complex was found to be associated with polyubiquitin chains, suggesting that BRCA1-(1-300)*BARD1 was ubiquitinated in vivo as well. These results raise the possibility that BRCA1*BARD1 acts to assemble non-lysine 48-linked polyubiquitin chains that may serve as part of a signaling platform required for coordinating DNA repair-related events.