BRCA1-associated RING Domain Research Articles

Abstract 3816: DNA variation at BARD1 predicts response to aurora kinase inhibition in neuroblastoma.

Abstract Background: Neuroblastoma remains responsible for a disproportionately high number of pediatric cancer deaths, necessitating novel treatment approaches. Our lab has previously reported DNA variations in the BRCA1-associated RING domain 1 (BARD1) gene to be significantly associated with high-risk neuroblastoma in an ongoing genome-wide association study (Capasso Nat Gen 2009). The expression of an oncogenic isoform, BARD1β, correlated with this disease associated common variation, and isoform specific depletion showed differential growth inhibition in a risk haplotype dependent manner (Bosse Can Res 2012). Furthermore, this isoform was shown to bind to and stabilized aurora kinase A and B in neuroblastoma cells, suggesting a mechanism for its oncogenicity and a potential therapeutic strategy. Methods: Short interfering RNAs (siRNAs) targeting AURKA, AURKB and BARD1β were tested individually in a panel of neuroblastoma cell lines (n=8) stratified by risk haplotype. Cell growth was measured by real time cell electronic sensing (RT-CES), and mRNA knockdown was confirmed by quantitative PCR. The pharmacologic inhibitors MLN8237, a selective aurora kinase A inhibitor; AZD1152, a selective aurora kinase B inhibitor; and CCT137690, a pan-aurora kinase A and B inhibitor were tested in vitro in an expanded neuroblastoma cell line panel (n=10), and cell viability was measured with a CellTiter-Glo(R) Luminescent Cell Viability Assay. Results: AURKB knockdown resulted in a trend of greater growth inhibition in neuroblastoma cell lines harboring a BARD1 risk haplotype, directly correlating with growth inhibition following BARD1β knockdown (R2=0.57, p=0.03). AURKA depletion showed significant growth inhibition (mean inhibition=45%, p<0.0001), but no correlation with BARD1 genotype was observed. Aurora A kinase inhibition with MLN8237 resulted in significant cytotoxicity (IC50 30 -750nM), and further analysis of published studies using MLN8237 in vivo on murine xenograft models of neuroblastoma showed that the cell lines with the BARD1 risk haplotype had significantly greater tumor reduction with this single agent therapy. AZD1152 and CCT137690 were less potent inhibitors of growth (IC50 30nM-10mM and 250nM-3mM, respectively). Conclusion: Taken together, these data support a model of BARD1 acting in a pathway involving the aurora kinase proteins in neuroblastoma cells, suggesting that BARD1 genotypes have the potential to be a biomarker of aurora kinase inhibitor activity, but the associations are complex and unlikely to be binary predictors of sensitivity or insensitivity. These data support the continued investigation of MLN8237 in clinical trials for high-risk neuroblastoma and further exploration of the mechanisms by which DNA variation at BARD1 influences neuroblastoma oncogenicity and therapeutic vulnerabilities. Citation Format: Vanessa M. Pineros, Kris R. Bosse, Lori S. Hart, Sharon J. Diskin, JulieAnn Rader, John M. Maris. DNA variation at BARD1 predicts response to aurora kinase inhibition in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3816. doi:10.1158/1538-7445.AM2013-3816

Abstract 632: Characterization of BCCIP as a novel BARD1 interaction partner.

Abstract BARD1 (BRCA1-Associated Ring Domain 1) was originally identified in a yeast-two-hybrid (Y2H) screen as a binding partner of BRCA1. The functional heterodimer BRCA1/BARD1 is required for several of the cellular and tumor-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair pathway, transcription, cell cycle control, and ubiquitination. Although several BRCA1-interacting proteins were reported, few proteins were described to interact with BARD1. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a Y2H assay using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for BCCIP (BRCA2 and CDKN1A Interacting Protein) C-terminal region. BCCIP is known for its involvement in DNA damage repair and the cell cycle control. BCCIP interacts with BRCA2, and both proteins are important for RAD51 focus formation after ionizing radiation and homologous recombination (HR) repair of double-strand-breaks (DSBs). BCCIP enhances the p21 suppression activity towards CDK2, and BCCIP downregulation reduces p21 expression by abrogating p53 transcription activity. We confirmed BCCIP/BARD1 interaction by pull-down and co-immunoprecipitation (co-IP) analysis of ectopically expressed BARD1 and BCCIP. We also identified the constitutive BCCIP/BARD1 complex in human cells by co-IP assays. Interestingly the BCCIP observed in complex with BARD1 corresponds to a mono-ubiquitinated form, suggesting that BCCIP may be a putative natural target for BRCA1/BARD1 heterodimer E3 ubiquitin ligase. BRCA1 participation in a multicomponent complex with BCCIP is under investigation. In order to assess the biological significance of the interaction between BCCIP and BARD1 we are investigating its role in DNA damage repair, cell cycle control and apoptosis. We are also evaluating the BCCIP protein interaction network using a tandem affinity purification approach. Citation Format: Joao Séllos, Thales C. Nepomuceno, Renato S. Carvalho, Guilherme Suarez-Kurtz, Alvaro N.A. Monteiro, Marcelo A. Carvalho. Characterization of BCCIP as a novel BARD1 interaction partner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 632. doi:10.1158/1538-7445.AM2013-632

Clinical and Molecular Significance of Poly (ADP-Ribose) Polymerase-1 (PARP-1) in Breast Cancer of African Women and its Potential as a Targeted Therapy

Background: The therapeutic effects of Poly (ADP-ribose) polymerase-1 (PARP -1) inhibition are currently studied in a clinical trial that is recruiting African- American (A-A) women with breast cancer (BC). Although, A-A and West African women are likely to share the same ancestry, there are overwhelming evidences, that BC is undoubtedly heterogeneous which might influence results obtained in these Nationalities. Thus, this study aims to investigate PARP-1 expression in a large and annotated series of breast cancer from Nigerian women to determine its clinical and biological significance for the indigenous African women. Methods: PARP-1 protein expression was assessed immunohistochemically in 204 formalin fixed paraffin samples from Nigerian breast cancer women prepared as TMA. Results: PARP-1 was inversely associated with steroid hormone receptors (oestrogen (ER) and progesterone (PR) receptor), the Homologous Recombination marker BRCA1 associated ring domain 1 (BARD1) and p27. Conversely, a positive association was established between PARP-1 and high histologic grade, expression of basal markers (cytokeratins (CK) 5/6 and 14) and epidermal growth factor receptor (EGFR)), DNA damage-repair markers (protein inhibitor of activator signal transducer gamma (PIAS)), the BRCA1 inhibitor (metastasis tumour antigen-1 (MTA1), p53, the proliferation markers (KI-67, Phosphoinositide-3-kinases (PI3KCA)), the triple-negative and basal-like phenotypes. Outcome analysis indicated PARP-1 as a predictor of poor survival independent of tumour size, histological grade and lymph node involvement. Conclusion: These results provide evidence that PARP-1 plays an important role in Nigerian women with breast cancer. It is recommended that indigenous Black women from Africa are included in the ongoing clinical trial of PARP1 inhibitors that is aimed at determining the efficiency of the drug in black BC women outside United States.

Construction of a Peptide Microarray for Auto-anti- body Detection

Peptide and protein microarrays provide a multiplex approach to identification and quantification of protein-protein interactions (PPI), useful to study for instance antigen-antibody properties. Multivariate serology assays detecting multiple tumor auto-antibodies (TAA) is an emerging class of blood tests for cancer detection. Here we describe the efficient coupling of peptide baits derived from the BRCA1-associated RING domain protein 1 (BARD1) to a solid surface and detection of a commercially available anti-BARD1 antibody with this newly designed peptide microarray. Analytical sensitivity and specificity were shown to be comparable to a microtiter plate based enzyme-linked immunosorbent assay (ELISA).

Identification of functional SNPs in BARD1 gene and in silico analysis of damaging SNPs: based on data procured from dbSNP database.

BackgroundThe BARD1 gene encodes for the BRCA1-associated RING domain (BARD1) protein. Germ line and somatic mutations in BARD1 are found in sporadic breast, ovarian and uterine cancers. There is a plethora of single nucleotide polymorphisms (SNPs) which may or may not be involved in the onset of female cancers. Hence, before planning a larger population study, it is advisable to sort out the possible functional SNPs. To accomplish this goal, data available in the dbSNP database and different computer programs can be used. To the best of our knowledge, until now there has been no such study on record for the BARD1 gene. Therefore, this study was undertaken to find the functional nsSNPs in BARD1.Result2.85% of all SNPs in the dbSNP database were present in the coding regions. SIFT predicted 11 out of 50 nsSNPs as not tolerable and PolyPhen assessed 27 out of 50 nsSNPs as damaging. FastSNP revealed that the rs58253676 SNP in the 3′ UTR may have splicing regulator and enhancer functions. In the 5′ UTR, rs17489363 and rs17426219 may alter the transcriptional binding site. The intronic region SNP rs67822872 may have a medium-high risk level. The protein structures 1JM7, 3C5R and 2NTE were predicted by PDBSum and shared 100% similarity with the BARD1 amino acid sequence. Among the predicted nsSNPs, rs4986841, rs111367604, rs13389423 and rs139785364 were identified as deleterious and damaging by the SIFT and PolyPhen programs. Additionally, I-Mutant showed a decrease in stability for these nsSNPs upon mutation. Finally, the ExPASy-PROSIT program revealed that the predicted deleterious mutations are contained in the ankyrin ring and BRCT domains.ConclusionUsing the available bioinformatics tools and the data present in the dbSNP database, the four nsSNPs, rs4986841, rs111367604, rs13389423 and rs139785364, were identified as deleterious, reducing the protein stability of BARD1. Hence, these SNPs can be used for the larger population-based studies of female cancers.

The BARD1 Cys557Ser variant and risk of familial breast cancer in a South-American population

Since the discovery of the BRCA1 and BRCA2 genes, much work has been carried out to identify further breast cancer (BC) susceptibility genes. BARD1 (BRCA1-associated ring domain) was originally identified as a BRCA1-interacting protein but has also been described in tumor-suppressive functions independent of BRCA1. Some association studies have suggested that the BARD1 Cys557Ser variant might be associated with increased risk of BC, but others have failed to confirm this finding. To date, this variant has not been analyzed in Spanish or South-American populations. In this study, using a case-control design, we analyzed the C-terminal Cys557Ser change in 322 Chilean BC cases with no mutations in BRCA1 or BRCA2 and in 570 controls in order to evaluate its possible association with BC susceptibility. BARD1 Cys557Ser was associated with an increased BC risk (P = 0.04, OR = 3.4 [95 % CI 1.2-10.2]) among cases belonging to families with a strong family history of BC. No difference between single cases affected with age <50 years at diagnosis (n = 117) and controls was observed for carriers of Cys/Ser genotype. It is likely that this variant is not involved in BC risk in this group of women. We also analyzed a possible interaction between BARD1 557Ser/XRCC3 241Met variants considering the role of both genes in the maintenance of genome integrity. The combined genotype Cys/Ser-carrier with the XRCC3 241Met allele was associated with an increased BC risk (P = 0.02, OR = 5.01 [95 % CI 1.36-18.5]) among women belonging to families with at least three BC and/or ovarian cancer cases. Our results suggest that BARD1 557Ser and XRCC3 241Met may play roles in BC risk in women with a strong family history of BC. Nevertheless there is no evidence of an interaction between the two SNPs. These findings should be confirmed by other studies and in other populations.

Abstract 2132: Characterization of galectin 3 as a novel BARD1 interaction partner

Abstract BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid (Y2H) screen as a binding partner of BRCA1. The functional heterodimer BRCA1/BARD1 is required for several of the cellular and tumor-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair pathway, transcription, cell cycle control, and ubiquitination. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a Y2H screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for galectin 3 C-terminal region, residues 170-250 (OMIM 153619). Galectin 3 is a galactoside binding protein involved in several cellular processes including apoptosis control and RNA splicing. Moreover there are evidences that galectin 3 variants are correlated with tumor development and progression in breast and thyroid cancer. We confirmed galectin 3/BARD1 interaction conducting co-immunoprecipitation analysis of ectopically expressed BARD1 and galectin-3 in HEK293T cells. The interaction between endogenous galectin 3 and BARD1 were validated in HeLa nuclear extracts, and by confocal microscopy. Using ectopically expressed galectin 3 fragments we determined the interaction region (aa 100-215). Moreover, galectin 3 fragment comprising aminoacids 100-165 is able to disrupt the endogenous interaction with BARD1. Co-immunoprecipitation assays suggested the presence of BRCA1, BARD1 and galectin-3 in the same complex. Interestingly the galectin 3 observed in this complex corresponds to a mono-ubiquitinated form. This ubiquitination reaction occurs mainly in the nucleus but is not BRCA1 dependent. The role of galectin 3 in DNA damage pathway was investigated using galectin 3 silenced HeLa cells exposed to increasing doses of ionizing radiation. These cells exhibited a slightly increase in radioresistance, but no effect was observed in cell cycle control. In addition the phosphorylation of histone H2AX seems to be delayed after damage in cells lacking galectin 3. Other DNA damage agents, such as carboplatin and etoposide did not affected differently the cell viability of these cells. We are now evaluating the protein interaction network of galectin 3 using a tandem affinity purification approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2132. doi:1538-7445.AM2012-2132

Lack of association between BARD1 Cys557Ser variant and breast cancer risk: a meta-analysis of 11,870 cases and 7,687 controls

The BRCA1-associated RING domain (BARD1) gene has been identified as a high-penetrance gene for breast cancer, whose germline and somatic mutations were reported in both non-BRCA1/2 hereditary site-specific and sporadic breast cancer cases. Some association studies suggested that the BRAD1 Cys557Ser variant might be associated with increased risk of breast cancer, but the results remain conflicting rather than conclusive. In order to derive a more precise estimation of the relationship, this meta-analysis was performed. Eligible studies were identified by searching several databases for relevant reports published before March 2011. In total, 14 studies (11,870 cases and 7,687 controls) were included in the present meta-analysis. The pooled odds ratio (OR) with 95% confidence interval (CI) for breast cancer risk associated with Cys557Ser carrier was estimated. The carrier frequency of the Cys557Ser mutation was 3.85% (457/11,870) in patients with breast cancer and 3.29% (253/7,687) in healthy controls. When all studies were pooled into the meta-analysis, there was no evidence for significant association between Cys557Ser mutation and breast cancer risk (OR 1.14, 95% CI 0.94-1.34). In the subgroup analyses by design of experiment and family history with BRCA1/2 status (unselected cases, family history with non-BRCA1/2 cases, and family history with BRCA1/2-positive cases), no significant associations were found in any subgroup of population. This meta-analysis strongly suggests that BARD1 Cys557Ser mutation is not associated with increased breast cancer risk.

Abstract 3927: Characterization of galectin 3 as a putative BRCA1/BARD1 ubiquitin E3 ligase substrate

Abstract BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid screen as a binding partner of BRCA1. The functional heterodimer BRCA1/BARD1 is required for several of the cellular and tumor-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair pathway, transcription, cell cycle control, and ubiquitination. BARD1 also contains two tandem BRCT repeats at its C-terminus, a region shown to bind serine-phosphorylated peptides in vitro. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a yeast-two-hybrid screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for galectin 3 C-terminal region, residues 170-250 (OMIM 153619). Galectin 3 is a galactoside binding protein involved in several cellular processes including apoptosis control and RNA splicing. Moreover there are evidences that galectin 3 variants are correlated with tumor development and progression in breast and thyroid cancer. We confirmed galectin 3/BARD1 interaction by conducting co-immuneprecipitation analysis of ectopically expressed BARD1 and galectin-3 in HEK293T cells. The interaction between endogenous galectin 3 and BARD1 were validated in HeLa nuclear extracts. Using the same approach we co-immuneprecipitated BRCA1 and galectin-3, suggesting the presence of BRCA1, BARD1 and galectin-3 in the same complex. Interestingly the galectin 3 observed in this complex corresponds to a mono-ubiquitinated form, corroborating the idea that galectin 3 is a putative natural target of BRCA1-BARD1 heterodimer E3 ubiquitin ligase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3927. doi:10.1158/1538-7445.AM2011-3927

Cancer predisposing BARD1 mutations in breast–ovarian cancer families

The breast cancer susceptibility gene BARD1 (BRCA1-associated RING domain protein, MIM# 601593) acts with BRCA1 in DNA double-strand break (DSB) repair and also in apoptosis initiation. We screened 109 BRCA1/2 negative high-risk breast and/or ovarian cancer patients from North-Eastern Poland for BARD1 germline mutations using a combination of denaturing high-performance liquid chromatography and direct sequencing. We identified 16 different BARD1 sequence variants, five of which are novel. Three of them were suspected to be pathogenic, including a protein truncating nonsense mutation (c.1690C>T, p.Gln564X), a splice mutation (c.1315-2A>G) resulting in exon 5 skipping, and a silent change (c.1977A>G) which alters several exonic splicing enhancer motifs in exon 10 and results in a transcript lacking exons 2-9. Our findings suggest that BARD1 mutations may be regarded as cancer risk alleles and warrant further investigation to determine their actual contribution to non-BRCA1/2 breast and ovarian cancer families.

BARD1 (BRCA1 associated RING domain 1)

Review on BARD1 (BRCA1 associated RING domain 1), with data on DNA, on the protein encoded, and where the gene is implicated.

BARD1: a New Target In Leukemia

AbstractAbstract 4642 BackgroundBARD1 (BRCA1-associated RING domain protein 1) was first described as a BRCA1 partner and tumour suppressor protein, muted in most cases of breast and ovarian cancer. BARD1 functions are BRCA1-dependent, requiring formation of a stable heterodimer through interaction of the RING finger domains. BRCA1-independent functions for BARD1 have recently been described, making it an interesting and important molecule for study.BARD1 is expressed in almost all human tissues, including haematological cells, testis and breast tissue, but it is over-expressed in leukaemias, sarcomas and testis cancer, implicating BARD1 in development of cancer. Different BARD1 isoforms are up-regulated in breast, ovarian and uterine cancers but markedly down-regulated or absent in healthy tissues. Therefore, the presence of these isoforms might be a risk factor or causal event in the cancer pathogenesis.We investigated the role of BARD1 isoforms in leukaemia, through the study of the epigenetic mechanisms involved in regulation of its expression and function. As such, we determined the expression of a specific BARD1 isoform in different human leukaemia cell lines the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid, Vorinostat) of expression of BARD1. Results.Four human leukaemia cell lines (U937, NB4, K562 and HL60) express the BARD1 isoform of interest. Treatment of these cell lines with SAHA at 5 μ M resulted in decreased expression of this isoform (Fig. 1).Decreased expression of BARD1 by SAHA was also observed in in human breast cancer MCF7 cells, the usual model for BARD1 experiments and in the human neuroblastoma cell line Kelly (Fig. 2), but not in HeLa cells or an human epithelial carcinoma cell line (Fig. 2). The reduced expression of BARD1 was attributed to the action of 2 specific micro-RNAs (miRNAs), that directly bind its 3′untranslated region (UTR). SAHA-induced expression of miR-19a and miR-19b in primary human leukemia cells as demonstrated using miRNA microarray expression analysis and confirmed by Real-Time PCR (Fig. 3). These 2 miRNA were up-regulated after SAHA stimulation in human leukaemia cells. BARD1 is the predicted target for miR-19a and miR-19b based on miRBase database analyses. Transient transfection in NB4 cells with mimic miR-19a and mimic miR-19b confirmed expression of these miRNAs was associated with BARD1 down-regulation (Fig. 4 5.). The BARD1 3′UTR was cloned into a pGL3 vector with a downstream the luciferase gene reporting gene (Fig. 6). The plasmid was co-transfected in HeLa cells with each mimic miRNA and a luciferase assay was performed. We demonstrate that expression of miR-19a and miR-19b can directly bind BARD1 3′UTR, reducing luciferase activity in this system (Fig. 7), thereby confirming that BARD1 is a target of these miRNAs.These findings support our hypothesis that BARD1 is a promising target in leukemia and should be further investigated for its diagnostic and prognostic features. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

BRCA1 16 years later: nuclear import and export processes

Over the past several years, the importance of regulated nuclear transport processes for tumor suppressors has become evident. Proteins with a molecular mass greater than 40 kDa can enter the nucleus only by active transport across the nuclear membrane. The most common pathway by which this occurs is via the importin alpha/beta pathway, whereby the cargo protein binds importin alpha. This heterodimer binds importin beta and the heterotrimer passes through nuclear pores at the expense of GTP. Breast cancer susceptibility gene 1 (BRCA1) is one such protein. As a mediator of transcription and DNA repair, two exclusively nuclear functions, BRCA1, at 220 kDa, can enter the nucleus only via active transport mechanisms. In addition to the classical importin alpha/beta pathway, BRCA1 can also enter the nucleus in a piggyback mechanism with BRCA1-associated RING domain protein 1 (BARD1). The interaction between BRCA1 and BARD1 is also important in the retention of BRCA1 in the nucleus. This is important because BRCA1 also undergoes active nuclear export. BRCA1 is also involved in apoptotic processes. Whether this occurs within the nucleus or cytoplasm is still unclear; thus, the consequences of BRCA1 nuclear export have not been clearly elucidated. This review will discuss the literature regarding the subcellular localization of BRCA1, with particular emphasis on its nuclear import and export processes.

Thermodynamic study of the BRCT domain of BARD1 and its interaction with the -pSER-X-X-Phe- motif-containing BRIP1 peptide

The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRCA1/BARD1 complex demonstrates ubiquitin ligase activity and has been implicated in genomic stability and tumor suppression. Both proteins possess a structurally conserved C-terminal domain (BRCT). While BRCA1–BRCT has been shown to mediate BRCA1 interactions with phosphoproteins such as BRIP1 by recognizing the pSer-X-X-Phe motif, attempts to demonstrate analogous interactions of its dimeric counterpart BARD1–BRCT, have so far been unsuccessful. In this study, chemical-denaturation experiments of BARD1–BRCT domain suggest that its low thermodynamic stability (Δ G = 2.5 kcal/mol) at room temperature, may affect some of its biochemical properties, such as its interaction with phosphopeptides. The stability of BARD1–BRCT domain at 10 °C, increases to 7.5 kcal/mol and isothermal titration calorimetry (ITC) experiments at this lower temperature showed binding to the BRIP1 phosphopeptide via an enthalpy-driven interaction, which appears to be specific to the pSer-X-X-Phe peptide-binding motif. Substitution of either pSer at position 0 with Ser (non-phosphorylated peptide) or Phe with Val at position + 3, leads to no-binding ITC results. While these findings are indicative that BRIP1 is a potential BARD1 binding partner, it becomes evident that in vitro binding assays involving the entire BARD1 protein and in vivo experiments are also needed to establish its binding partners and its potential role in tumor suppression pathways.

Abstract 1117: Characterization of BAT1 (UAP56) interaction with BARD1

Abstract BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid screen as a binding partner of BRCA1. The functional heterodimer BRCA1-BARD1 is required for several of the cellular and tumour-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair (DDR) pathway, transcription, cell cycle control, and ubiquitination. BARD1 also contains two tandem BRCT repeats at its C-terminus, a region shown to bind serine-phosphorylated peptides in vitro. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a yeast two hybrid (Y2H) screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for residues 308-418 of BAT1 (UAP56, C-terminal region [OMIM 142560]). UAP56 is a member of DEAD box family of ATP-dependent RNA helicases. Several studies indicate that UAP56 protein is not only essential in pre-mRNA splicing but is also important in mRNA nuclear export and cytoplasmic mRNA localization. We confirmed BAT1-BARD1 interaction by conducting co-IP analysis of ectopic expressed BARD1 and BAT1 in HEK293T cells. We are using a Y2H system approach to determine the minimal region of interaction between BARD1 and BAT1; in parallel, we are also checking for the presence of a multicomponent complex, investigating the presence of BRCA1. In order to assess the biological significance of the interaction between BARD1 and BAT1 we will determine whether this interaction is important for the activation of, or recovery from, the G2/M cell cycle checkpoint after IR and also investigate its putative involvement in p53-dependent apoptosis pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1117.

Abstract 3867: Understanding the neuroblastoma predisposition signal at chromosome 2q35: Identification of the β-BARD1 isoform as an oncogenic driver

Abstract Genome-wide association studies have proven to be a powerful tool to identify susceptibility loci in cancer, however the molecular basis of a majority of these association signals remain undefined. We have previously reported nine common single nucleotide polymorphism (SNP) alleles at chromosome 2q35, within and 5′ upstream of the BRCA1-associated RING domain (BARD1) gene, to be significantly associated with high-risk neuroblastoma (Capasso, M et al., Nature Genetics 2009). Here, we focus on understanding the biological underpinnings of this genetic association by investigating how common variation at this locus correlates with changes in BARD1 expression in both the germline and in neuroblastoma samples, and further how these BARD1 alterations are involved in driving neuroblastoma tumorigenesis. We first showed that the neuroblastoma associated SNP variation at 2q35 is in linkage disequilibrium (LD) with multiple genomic areas dense with regulatory domains, including many putative binding sites for the neuronal splicing regulator, FOX2. We next demonstrated that in peripheral blood mononuclear cells, these SNPs correlate both with a trend towards increased overall BARD1 expression and with differential BARD1 exon expression. Specifically, a majority of the neuroblastoma associated SNPs were significantly associated with decreased expression of the RING domain forming BARD1 exons 2 and 3 (P = 0.0008-0.034). We have identified 16 unique BARD1 isoforms in the neuroblastoma tissue of origin, sympathetic fetal ganglia, with the β-BARD1 isoform specifically splicing out exons 2 and 3. β-BARD1 was found to be ubiquitously expressed in a panel of fetal sympathetic ganglia, neuroblastoma cell lines, and high-risk primary tumors, with its expression being significantly increased in cell lines harboring a homozygous BARD1 risk allele genotype at the most significantly associated 2q35 SNP, rs6435862 (N = 22; P = 0.04). Transient transfection of a specific siRNA targeting the alternatively spliced boundary of β-BARD1 in a panel of neuroblastoma cell lines (N = 12) showed that neuroblastoma cells harboring at least one risk allele at SNP rs6435862 and the highest β-BARD1 expression showed a significant inhibition of cell proliferation upon β-BARD1 knockdown (% growth inhibition = 45-81%, P = 0.0004-0.034), while six of seven cell lines with a homozygous major allele genotype at rs6435862 and lower β-BARD1 expression did not show significant growth inhibition. These data are consistent with β-BARD1 acting as an oncogene in neuroblastoma and we hypothesize that common genomic variation drives formation of this isoform, underlying neuroblastoma predisposition on 2q35. Ongoing work will focus on further defining the role of common variation in BARD1's RNA splicing patterns and identifying the molecular basis of β-BARD1's oncogenicity in neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3867.

Distinct Roles of BARD1 Isoforms in Mitosis: Full-Length BARD1 Mediates Aurora B Degradation, Cancer-Associated BARD1β Scaffolds Aurora B and BRCA2

The BRCA1-associated ring domain protein 1 (BARD1) interacts with BRCA1 via its RING finger domain. The BARD1-BRCA1 complex participates in DNA repair, cell cycle control, genomic stability, and mitotic spindle formation through its E3 ubiquitin ligase activity. Cancer cells express several BARD1 protein isoforms, including the RING finger-deficient variant BARD1beta. Here, we show that BARD1 has BRCA1-dependent and BRCA1-independent functions in mitosis. BARD1, but not BRCA1, localizes to the midbody at telophase and cytokinesis, where it colocalizes with Aurora B. The 97-kDa full-length (FL) BARD1 coimmunoprecipates with BRCA1, but the 82-kDa BARD1beta coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1beta. Depletion of FL BARD1 had only minor effects on cell growth and did not abolish midbody localization of BARD1 staining, but resulted in massive up-regulation of Aurora B. In contrast, suppression of FL BARD1 and BARD1beta led to growth arrest and correlated with various mitotic defects and disappearance of midbody localization of BARD1 staining. Our data suggest a novel function of FL BARD1 in Aurora B ubiquitination and degradation, opposing a proproliferative function of BARD1beta in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 and up-regulation of Aurora B, as observed in cancer cells, can be explained by an imbalance of FL BARD1 and BARD1beta.

BARD1 may be renamed ROW1 because it functions mainly as a REPRESSOR OF WUSCHEL1

Human BRCA1 (BRreast CAncer susceptible gene1) is known to involve in cell-cycle control, transcriptional regulation, DNA recombination, DNA repair and many other processes. hBARD1 (BRCA1-Associated Ring Domain 1) forms heterodimer via its N-terminal conserved RING domain with BRCA1. In Arabidopsis, two genes, At4g21070 and At1g04020, that share N-terminal RING domain and C-terminal BRCT (for BRCA1 C-Terminal) domains with no substantial similarities for other motifs, have been identified. AtBRCA1 was induced by γ-ray while AtBARD1 was required for DNA repair. Recently, we find that AtBARD1 may function to confine WUS transcription in the shoot apical meristem organization center, together with the ATPase-dependent chromatin remodeling factor, SYD. In bard1-3 Arabidopsis knock-out mutant, WUS was released to the outer layers and expressed at extremely high level comparing to wild-type. Our data suggest that BARD1 mainly function as a REPRESSOR OF WUSCHEL1 (ROW1). Extensive motif analyses carried out here showed that ROW1 possesses substantial sequence identity with a reported transcription repressor, MLL and also a potential PHD domain which recognizes histone tail codes, in its uncharacterized middle region. We suggest that ROW1 represses transcription in a chromatin-related mechanism.

Crystal Structure of the BARD1 Ankyrin Repeat Domain and Its Functional Consequences

BARD1 is the constitutive nuclear partner to the breast and ovarian cancer-specific tumor suppressor BRCA1. Together, they form a heterodimeric complex responsible for maintaining genomic stability through nuclear functions involving DNA damage signaling and repair, transcriptional regulation, and cell cycle control. We report the 2.0A structure of the BARD1 ankyrin repeat domain. The structure includes four ankyrin repeats with a non-canonical C-terminal capping ankyrin repeat and a well ordered extended loop preceding the first repeat. Conserved surface features show an acidic patch and an acidic pocket along the surface typically used by ankyrin repeat domains for binding cognate proteins. We also demonstrate that two reported mutations, N470S and V507M, in the ankyrin repeat domain do not result in observable structural defects. These results provide a structural basis for exploring the biological function of the ankyrin repeat domain and for modeling BARD1 isoforms.

Mutation ofArabidopsis BARD1Causes Meristem Defects by Failing to ConfineWUSCHELExpression to the Organizing Center

Stem cell fate in the Arabidopsis thaliana shoot apical meristem (SAM) is controlled by WUSCHEL (WUS) and CLAVATA. Here, we examine BARD1 (for BRCA1-associated RING domain 1), which had previously been implicated in DNA repair functions; we find that it also regulates WUS expression. We observed severe SAM defects in the knockout mutant bard1-3. WUS transcripts accumulated >238-fold in bard1-3 compared with the wild type and were located mainly in the outermost cell layers instead of the usual organizing center. A specific WUS promoter region was recognized by nuclear protein extracts obtained from wild-type plants, and this protein-DNA complex was recognized by antibodies against BARD1. The double mutant (wus-1 bard1-3) showed prematurely terminated SAM structures identical to those of wus-1, indicating that BARD1 functions through regulation of WUS. BARD1 overexpression resulted in reduced WUS transcript levels, giving a wus-1-like phenotype. Either full-length BARD1 or a clone that encoded the C-terminal domain (BARD1:C-ter;bard1-3) was sufficient to complement the bard1-3 phenotype, indicating that BARD1 functions through its C-terminal domain. Our data suggest that BARD1 regulates SAM organization and maintenance by limiting WUS expression to the organizing center.