BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

BRCA1 is involved in maintaining genomic integrity and, as a regulator of the G2/M checkpoint, contributes to DNA repair and cell survival. The overexpression of BRCA1 elicits diverse cellular responses including apoptosis due to the stimulation of specific signaling pathways. BRCA1 is normally regulated by protein turnover, but is stabilized by BARD1 which can recruit BRCA1 to the nucleus to form a ubiquitin E3 ligase complex involved in DNA repair or cell survival. Here, we identify BARD1 as a regulator of BRCA1-dependent apoptosis. Using transfected MCF-7 breast cancer cells, we found that BRCA1-induced apoptosis was independent of p53 and was stimulated by BRCA1 nuclear export. Conversely, BARD1 reduced BRCA1-dependent apoptosis by a mechanism involving nuclear sequestration. Regulation of apoptosis by BARD1 was reduced by BRCA1 cancer mutations that disrupt Ub ligase function. Transfection of BRCA1 N-terminal peptides that disrupted the cellular BRCA1-BARD1 interaction caused a loss of nuclear BRCA1 that correlated with increased apoptosis in single cell assays, but did not alter localization or expression of endogenous BARD1. Reducing BARD1 levels by siRNA caused a small increase in apoptosis. Our findings identify a novel apoptosis inhibitory function of BARD1 and suggest that nuclear retention of BRCA1-BARD1 complexes contributes to both DNA repair and cell survival.     

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.     

Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains

Loss of the tumour suppressor BRCA1 results in profound chromosomal instability. The fundamental defect underlying this catastrophic phenotype is not yet known. In vivo, BRCA1 forms a heterodimeric complex with BARD1. Both proteins contain an N-terminal zinc RING-finger domain which confers E3 ubiquitin ligase activity. We have isolated full-length human BRCA1/BARD1 complex and have shown that it has a dual E3 ubiquitin ligase activity. First, it mediates the monoubiquitylation of nucleosome core histones in vitro, including the variant histone H2AX that co-localizes with BRCA1 at sites of DNA damage. Secondly, BRCA1/BARD1 catalyses the formation of multiple polyubiquitin chains on itself. Remarkably, this auto-polyubiquitylation potentiates the E3 ubiquitin ligase activity of the BRCA1/BARD1 complex >20-fold. Even though BRCA1 has been reported to associate with a C-terminal ubiquitin hydrolase, BAP1, this enzyme does not appear to function in the deubiquitylation of the BRCA1/BARD1 complex.     

Identification of BARD1 splice-isoforms involved in human trophoblast invasion

The tumor suppressor protein BARD1, originally discovered as BRCA1-binding protein, acts in conjunction with BRCA1 as ubiquitin ligase. BARD1 and BRCA1 form a stable heterodimer and dimerization, which is required for most tumor suppressor functions attributed to BRCA1. In addition, BARD1 has BRCA1-independent functions in apoptosis, and a role in control of tissue homeostasis was suggested. However, cancer-associated mutations of BARD1 are rare; on the contrary, overexpression of truncated BARD1 was found in breast and ovarian cancer and correlated with poor prognosis. Here we report that human cytotrophoblasts, which show a strong similarity with cancer cells in respect of their invasive behavior and capacity of matrix metalloprotease production, overexpress isoforms of BARD1 derived from differential splicing. We demonstrate that expression of BARD1 and its isoforms is temporally and spatially regulated by human chorionic gonadotropin and by hypoxia, both factors known to regulate the invasive phase and proliferation of cytotrophoblasts. Interestingly, we found a subset of BARD1 isoforms secreted by cytotrophoblasts. BARD1 repression by siRNAs, mitigates the interference of cytotrophoblasts with cell adhesion of collagen matrix-dependent epithelial cells, suggesting a role of BARD1 isoforms in extracellular matrix remodelling and in cytotrophoblasts invasion.     

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.     

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.     

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.     

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.     

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.     

In Vitro Repression of Brca1-associated RING Domain Gene,Bard1, Induces Phenotypic Changes in Mammary Epithelial Cells

BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1–BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.     

Nuclear targeting and cell cycle regulatory function of human BARD1

The BARD1 gene is mutated in a subset of breast and ovarian cancers, implicating BARD1 as a potential tumor suppressor. BARD1 gains a ubiquitin E3 ligase activity when heterodimerized with BRCA1, but the only known BRCA1-independent BARD1 function is a p53-dependent proapoptotic activity stimulated by nuclear export to the cytoplasm. We described previously the nuclear-cytoplasmic shuttling of BARD1, and in this study, we identify the transport sequences that target BARD1 to the nucleus and show that they are essential for BARD1 regulation of the cell cycle. We used deletion mapping and mutagenesis to define two active nuclear localization signals (NLSs) present in human BARD1 that are not conserved in rodent BARD1. Site-directed mutagenesis of the primary bipartite NLS abolished BARD1 nuclear import and caused its cytoplasmic accumulation. Using flow cytometry and 5-bromo-2-deoxyuridine incorporation assays, we discovered that transiently expressed BARD1 can elicit a p53-independent cell cycle arrest in G1 phase, and that this was abrogated by mutation of the BARD1 NLS but not by mutation of the nuclear export signal. Thus, BARD1 regulation of the cell cycle is a nuclear event and may be linked to its induced expression during mitosis and its possible involvement in the DNA damage checkpoint.     

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.     

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.     

BARD1 participates with BRCA1 in homology-directed repair of chromosome breaks

The BRCA1 tumor suppressor has been implicated in the maintenance of chromosomal stability through homology-directed repair of DNA double-strand breaks. Much of the BRCA1 in cells forms a heterodimeric complex with a structurally related protein BARD1. We report that expression of truncated mouse or human BARD1 peptides capable of interacting with Brca1 results in a homologous-repair deficiency. Repair is mildly reduced in Brca1 wild-type cells and severely reduced in cells that harbor a Brca1 splice product deleted for exon 11. Nuclear localization of the Brca1 or BARD1 peptides is not compromised, implying that the repair deficiency is caused by a more direct effect on repair. The tumor suppressor activity of BRCA1 may require the participation of BARD1 to maintain chromosome integrity through the homologous-repair pathway.     

New concepts on BARD1: Regulator of BRCA pathways and beyond

For nearly two decades most research on BARD1 was closely linked to research on BRCA1, the breast cancer predisposition gene. The co-expression of BARD1 and BRCA1 genes in most tissues, the nearly identical phenotype of Bard1 and Brca1 knock-out mice, and the fact that BRCA1 and BARD1 proteins form a stable complex, led to the general assumption that BARD1 acts as an accessory to BRCA1. More recent research on both proteins showed that BRCA1 and BARD1 might have common as well as separate functions. This review is an overview of how BARD1 functions and controls BRCA1. It highlights also experimental evidence for dominant negative, tumor promoting, functions of aberrant isoforms of BARD1 that are associated with and drivers of various types of cancer.