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Irmgard Irmingerfinger - One of the best experts on this subject based on the ideXlab platform.
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BARD1 splice variants display mislocalization in breast cancer cells and can alter the apoptotic response to cisplatin
Cancer Letters, 2016Co-Authors: Kamila A Marzec, Irmgard Irmingerfinger, Estefania Martinoecharri, Beric R HendersonAbstract:Abstract We previously showed that BARD1 is a shuttling protein with pro-apoptotic activity in MCF-7 breast cancer cells. BARD1 is expressed as splice variant isoforms in breast cancer. Here we characterized YFP-tagged BARD1 splice variants (beta, omega, phi, ΔRIN, epsilon) for subcellular localization and apoptotic efficacy. We found that loss of nuclear localization (NLS) or export (NES) sequences influenced cellular distribution. The beta and omega variants (+NLS/−NES) shifted exclusively to the nucleus. In contrast, BARD1-epsilon (−NLS/+NES) was mostly cytoplasmic. Variants that lacked both NLS and NES were evenly distributed. Interestingly, the more nuclear isoforms (omega and beta) were least apoptotic in MCF-7 cells as measured by FACS. The cytoplasmic localization of BARD1 isoforms correlated with increased apoptosis. This relationship held in cells exposed to low dose (5 µM) of cisplatin. At 20 µM cisplatin, the main observation was a protective effect by the omega isoform. Similar analyses of HCC1937 cells revealed less pronounced changes but a significant protective influence by BARD1-epsilon. Thus BARD1 variants differ in localization and apoptotic ability, and their expression profile may aid prediction of drug efficacy in breast cancer.
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new concepts on BARD1 regulator of brca pathways and beyond
The International Journal of Biochemistry & Cell Biology, 2016Co-Authors: Irmgard Irmingerfinger, Magda Ratajska, Maxim PilyuginAbstract: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.
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long non coding rna and micrornas might act in regulating the expression of BARD1 mrnas
The International Journal of Biochemistry & Cell Biology, 2014Co-Authors: Maxim Pilyugin, Irmgard IrmingerfingerAbstract:Abstract Long non-coding RNAs (lncRNAs) are ubiquitously expressed RNA molecules of more than 200 nucleotides without substantial ORFs. LncRNAs could act as epigenetic regulators of gene expression affecting transcription, mRNA stability and transport, and translation, although, precise functions have been attributed to only few of them. Competing endogenous RNAs (ceRNAs) represent one recently emerged type of functional lncRNAs that share microRNA recognition sequences with mRNAs and may compete for microRNA binding and thus affect regulation and function of target mRNAs. We studied the epigenetic regulation of the BARD1 gene. The BARD1 protein acts as tumor suppressor with BRCA1. In cancer, mRNAs encoding the tumor suppressor full length BARD1 are often down-regulated while the expression of oncogenic truncated isoforms is boosted. We found that the BARD1 3′UTR is almost 3000 nt long and harbors a large number of microRNA binding elements. In addition we discovered a novel lncRNA, BARD1 9′L, which is transcribed from an alternative promoter in intron 9 of the BARD1 gene and shares part of the 3′UTR with the protein coding BARD1 mRNAs. We demonstrate with the example of two microRNAs, miR-203 and miR-101, that they down-regulate the expression of FL BARD1 and cancer-associated BARD1 mRNAs, and that BARD1 9′L counteracts the effect of miR-203 and miR-101, As BARD1 9′L is abnormally over-expressed in human cancers, we suggest it might be a tumor promoting factor and treatment target. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.
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abstract b44 development of a serum test for the detection of lung cancer based on oncogenic BARD1 isoform expression
Clinical Cancer Research, 2012Co-Authors: Irmgard Irmingerfinger, Maxim Pilyugin, Pierre-alain André, Balazs Hegedus, Geoffrey J Laurent, Andrea BiancoAbstract:The BARD1 protein was originally identified as binding partner of the breast cancer gene product, BRCA1. Highly upregulated expression of aberrant isoforms of BARD1, derived from differential splicing, was correlated with poor prognostic factors in breast and ovarian cancer (Wu et al. Int J Can, 2006; Li et al. Can Res, 2007) and decreased patient survival in lung cancer (Zhang et al. Int J Can, 2011). Previous and ongoing studies have shown that BARD1 isoforms act antagonistically to the functions of BARD1 and BRCA1 as ubiquitin ligase. In particular, BARD1beta is promoting cell proliferation by stabilizing the Aurora kinases (Ryser et al. Can Res 2009; Bosse et al. submitted). Isoforms BARD1-beta and BARD1-pi are specifically upregulated in lung cancer and correlated with poor prognosis (Zhang et al., Int J Can, 2011). Thus, BARD1 isoforms might be drivers of tumorigenesis and potential markers of lung cancer progression, if detectable in the patients9 sera. To this goal, we performed ELISA tests with antibodies against different regions of BARD1 for the detection of BARD1 isoforms in the blood of lung cancer patients. We also generated a peptide library representing 40 epitopes mimicking BARD1 isoforms, for the detection of autoimmune antibodies recognizing epitopes expressed by BARD1 isoforms. BARD1 protein isoforms could be detected by ELISA in various serum samples, however this detection was always reproducible due to the BARD1 protein instability. We then performed inverse ELISA assays, using peptides for capturing autoimmune antibodies directed against BARD1 isoforms. Using serum samples from 60 non-small cell lung cancer (NSCLC) from time of diagnosis, and 40 control sera from phenotypically healthy volunteers, we could distinguish NSCLC cancer patients and controls. When applying a combination of seven peptides lung cancer was detected with 87 percent sensitivity and 68 percent specificity. Thus, our data show convincingly that antibodies against BARD1 isoforms are telltales of lung cancer and their detection can be further developed towards a blood test for the early detection of lung cancer. Experiments including larger patients and control group numbers, sera from patients with different types of lung cancer, as well as the comparison of this BARD1 isoform test with the standard test for lung cancer detection (CT scan), are currently ongoing, and should lead to optimized test conditions and a definition of the target patient set.
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distinct roles of BARD1 isoforms in mitosis full length BARD1 mediates aurora b degradation cancer associated BARD1β scaffolds aurora b and brca2
Cancer Research, 2009Co-Authors: Stephan Ryser, Eva Dizin, Charles Edward Jefford, Benedicte Delaval, Sarantis Gagos, Agni Christodoulidou, Karlheinz Krause, Daniel Birnbaum, Irmgard IrmingerfingerAbstract: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 BARD1β. 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 BARD1β coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1β. 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 BARD1β 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 BARD1β 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 BARD1β. [Cancer Res 2009;69(3):1125–34]
Richard Baer - One of the best experts on this subject based on the ideXlab platform.
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the brct domains of the brca1 and BARD1 tumor suppressors differentially regulate homology directed repair and stalled fork protection
Molecular Cell, 2018Co-Authors: David Billing, Matthias Szabolcs, Michiko Horiguchi, Foon Wubaer, Angelo Taglialatela, Giuseppe Leuzzi, Silvia Alvarez Nanez, Wenxia Jiang, Alberto Ciccia, Richard BaerAbstract:Summary The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here, we compare mice with mutations that ablate BRCT phospho-recognition by BARD1 (BARD1S563F and BARD1K607A) or Brca1 (Brca1S1598F). Brca1S1598F abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1 mutant tumors. Although not affecting HDR, the BARD1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, BARD1S563F/S563F and BARD1K607A/K607A mice, unlike Brca1S1598F/S1598F mice, are not tumor prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, because SFP, unlike HDR, is also impaired in heterozygous Brca1/BARD1 mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.
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the basal like mammary carcinomas induced by brca1 or BARD1 inactivation implicate the brca1 BARD1 heterodimer in tumor suppression
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Reena Shakya, Richard Baer, Ellen Mccarthy, Subhadra V Nandula, Vundavalli V Murty, Matthias Szabolcs, Elson Ospina, Katia Basso, Thomas LudwigAbstract:Women with germ-line mutations of the BRCA1 tumor suppressor gene are highly susceptible to breast and ovarian cancer. The protein product of BRCA1 is involved in a broad spectrum of biological processes and interacts with many diverse proteins. One of these, BARD1, associates with BRCA1 to form a heterodimeric complex that is enzymatically active as an ubiquitin E3 ligase. Although the BRCA1/BARD1 heterodimer has been implicated in several aspects of BRCA1 function, its role in tumor suppression has not been evaluated. To address this question, we generated mouse strains carrying conditional alleles of either BARD1 or Brca1 and used Cre recombination to inactivate these genes in mammary epithelial cells. Significantly, the conditional BARD1- and Brca1-mutant mice developed breast carcinomas that are indistinguishable from each other (and from those of double conditional BARD1/Brca1-mutant animals) with respect to their frequency, latency, histopathology, and cytogenetic features. Reminiscent of the basal-like breast carcinomas seen in human BRCA1 mutation carriers, these tumors are “triple negative” for estrogen and progesterone receptor expression and HER2/neu amplification. They also express basal cytokeratins CK5 and CK14, have an elevated frequency of p53 lesions, and display high levels of chromosomal instability. The remarkable similarities between the mammary carcinomas of BARD1-, Brca1-, and BARD1/Brca1-mutant mice indicate that the tumor suppressor activities of both genes are mediated through the BRCA1/BARD1 heterodimer.
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The basal-like mammary carcinomas induced by Brca1 or BARD1 inactivation implicate the BRCA1/BARD1 heterodimer in tumor suppression
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Reena Shakya, Richard Baer, Subhadra V Nandula, Vundavalli V Murty, Matthias Szabolcs, Elson Ospina, Katia Basso, Ellen E. Mccarthy, Thomas LudwigAbstract:Women with germ-line mutations of the BRCA1 tumor suppressor gene are highly susceptible to breast and ovarian cancer. The protein product of BRCA1 is involved in a broad spectrum of biological processes and interacts with many diverse proteins. One of these, BARD1, associates with BRCA1 to form a heterodimeric complex that is enzymatically active as an ubiquitin E3 ligase. Although the BRCA1/BARD1 heterodimer has been implicated in several aspects of BRCA1 function, its role in tumor suppression has not been evaluated. To address this question, we generated mouse strains carrying conditional alleles of either BARD1 or Brca1 and used Cre recombination to inactivate these genes in mammary epithelial cells. Significantly, the conditional BARD1- and Brca1-mutant mice developed breast carcinomas that are indistinguishable from each other (and from those of double conditional BARD1/Brca1-mutant animals) with respect to their frequency, latency, histopathology, and cytogenetic features. Reminiscent of the basal-like breast carcinomas seen in human BRCA1 mutation carriers, these tumors are “triple negative” for estrogen and progesterone receptor expression and HER2/neu amplification. They also express basal cytokeratins CK5 and CK14, have an elevated frequency of p53 lesions, and display high levels of chromosomal instability. The remarkable similarities between the mammary carcinomas of BARD1-, Brca1-, and BARD1/Brca1-mutant mice indicate that the tumor suppressor activities of both genes are mediated through the BRCA1/BARD1 heterodimer.
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structural requirements for the BARD1 tumor suppressor in chromosomal stability and homology directed dna repair
Journal of Biological Chemistry, 2007Co-Authors: Marsha Laufer, Maria Jasin, Thomas Ludwig, Ami P Modi, Subhadra V Nandula, Shuang Wang, Vundavalli V Murty, Richard BaerAbstract:Abstract 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.
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hyperphosphorylation of the BARD1 tumor suppressor in mitotic cells
Journal of Biological Chemistry, 2005Co-Authors: Atish D Choudhury, Thomas Ludwig, Hong Xu, Ami P Modi, Wenzhu Zhang, Richard BaerAbstract:Abstract Although the BRCA1 tumor suppressor has been implicated in a number of cellular processes, it plays an especially important role in the DNA damage response as a regulator of cell cycle checkpoints and DNA repair pathways. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein, and many of its biological functions are mediated by the BRCA1-BARD1 complex. Here, we show that BARD1 is phosphorylated in a cell cycle-dependent manner and that the hyperphosphorylated forms of BARD1 predominate during M phase. By mobility shift analysis and mass spectrometry, we have identified seven sites of mitotic phosphorylation within BARD1. All sites exist within either an SP or TP sequence, and two sites resemble the consensus motif recognized by cyclin-dependent kinases. To examine the functional consequences of BARD1 phosphorylation, we used a gene targeting knock-in approach to generate isogenic cell lines that express either wild-type or mutant forms of the BARD1 polypeptide. Analysis of these lines in clonogenic survival assays revealed that cells bearing phosphorylation site mutations are hypersensitive to mitomycin C, a genotoxic agent that induces interstrand DNA cross-links. These results implicate BARD1 phosphorylation in the cellular response to DNA damage.
Tomohiko Ohta - One of the best experts on this subject based on the ideXlab platform.
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interaction of BARD1 and hp1 is required for brca1 retention at sites of dna damage
Cancer Research, 2015Co-Authors: Wenwen Wu, Hiroyuki Nishikawa, Rachel E Klevit, Takayo Fukuda, Vinayak Vittal, Masahide Asano, Yasuo Miyoshi, Tomohiko OhtaAbstract: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 non-homologous end joining, at damaged loci in S phase. UNC0638, a small molecule inhibitor of histone lysine methyltransferase (HKMT), abolished retention and cooperated with the poly(ADP-ribose) polymerase 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.
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brca1 associated protein 1 interferes with brca1 BARD1 ring heterodimer activity
Cancer Research, 2009Co-Authors: Hiroyuki Nishikawa, Wenwen Wu, Mamoru Fukuda, Ayaka Koike, Ryoko Kojima, Hiromichi Gomi, Tomohiko OhtaAbstract:The breast and ovarian tumor suppressor BRCA1 constitutes a RING heterodimer E3 ligase with BARD1. BRCA1-associated protein 1 (BAP1) is a ubiquitin COOH-terminal hydrolase that was initially identified as a protein that bound to the RING finger domain of BRCA1. However, how BAP1 contributes to the E3 activity of BRCA1/BARD1 is unclear. Here, we report that BAP1 interacts with BARD1 to inhibit the E3 ligase activity of BRCA1/BARD1. Domains comprised by residues 182-365 of BAP1 interact with the RING finger domain of BARD1, and surface plasmon resonance spectroscopy (BIAcore) analyses showed that BAP1 interferes with the BRCA1/BARD1 association. The perturbation resulted in inhibition of BRCA1 autoubiquitination and NPM1/B23 ubiquitination by BRCA1/BARD1. Although BAP1 was capable of deubiquitinating the polyubiquitin chains mediated by BRCA1/BARD1 in vitro , a catalytically inactive mutant of BAP1, C91S, still inhibited the ubiquitination in vitro and in vivo , implicating a second mechanism of action. Importantly, inhibition of BAP1 expression by short hairpin RNA resulted in hypersensitivity of the cells to ionizing irradiation and in retardation of S-phase progression. Together, these results suggest that BAP1 and BRCA1/BARD1 coordinately regulate ubiquitination during the DNA damage response and the cell cycle. [Cancer Res 2009;69(1):111–9]
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BRCA1-Associated Protein 1 Interferes with BRCA1/BARD1 RING Heterodimer Activity
Cancer Research, 2009Co-Authors: Hiroyuki Nishikawa, Wenwen Wu, Mamoru Fukuda, Ayaka Koike, Ryoko Kojima, Hiromichi Gomi, Tomohiko OhtaAbstract:The breast and ovarian tumor suppressor BRCA1 constitutes a RING heterodimer E3 ligase with BARD1. BRCA1-associated protein 1 (BAP1) is a ubiquitin COOH-terminal hydrolase that was initially identified as a protein that bound to the RING finger domain of BRCA1. However, how BAP1 contributes to the E3 activity of BRCA1/BARD1 is unclear. Here, we report that BAP1 interacts with BARD1 to inhibit the E3 ligase activity of BRCA1/BARD1. Domains comprised by residues 182-365 of BAP1 interact with the RING finger domain of BARD1, and surface plasmon resonance spectroscopy (BIAcore) analyses showed that BAP1 interferes with the BRCA1/BARD1 association. The perturbation resulted in inhibition of BRCA1 autoubiquitination and NPM1/B23 ubiquitination by BRCA1/BARD1. Although BAP1 was capable of deubiquitinating the polyubiquitin chains mediated by BRCA1/BARD1 in vitro , a catalytically inactive mutant of BAP1, C91S, still inhibited the ubiquitination in vitro and in vivo , implicating a second mechanism of action. Importantly, inhibition of BAP1 expression by short hairpin RNA resulted in hypersensitivity of the cells to ionizing irradiation and in retardation of S-phase progression. Together, these results suggest that BAP1 and BRCA1/BARD1 coordinately regulate ubiquitination during the DNA damage response and the cell cycle. [Cancer Res 2009;69(1):111–9]
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down regulation of brca1 BARD1 ubiquitin ligase by cdk2
Cancer Research, 2005Co-Authors: Ryosuke Hayami, Ko Sato, Wenwen Wu, Toru Nishikawa, Mamoru Fukuda, Junya Hiroi, Ritsuko Ohtanikaneko, Tomohiko OhtaAbstract:BRCA1, a breast and ovarian tumor suppressor, is a phosphoprotein whose cellular expression level is regulated in a cell cycle–dependent manner. BRCA1 interacts with BARD1 to generate significant ubiquitin ligase activity which catalyzes nontraditional Lys-6-linked polyubiquitin chains. However, it is not clear how the activity is regulated and how this affects BRCA19s multiple cellular functions. Here we show that the ubiquitin ligase activity of BRCA1-BARD1 is down-regulated by CDK2. During the cell cycle, BARD1 expression can largely be categorized into three patterns: moderately expressed in a predominantly unphosphorylated form in early G1 phase, expressed at low levels in both phosphorylated and unphosphorylated forms during late G1 and S phases, and highly expressed in its phosphorylated form during mitosis coinciding with BRCA1 expression. CDK2-cyclin A1/E1 and CDK1-cyclin B1 phosphorylate BARD1 on its NH2 terminus in vivo and in vitro. Intriguingly, the BRCA1-BARD1–mediated in vivo ubiquitination of nucleophosmin/B23 (NPM) and autoubiquitination of BRCA1 are dramatically disrupted by coexpression of CDK2-cyclin A1/E1, but not by CDK1-cyclin B1. The inhibition of ubiquitin ligase activity is not due to the direct effect of the kinases on BARD1 because an unphosphorylatable mutant of BARD1, S148A/S251A/S288A/T299A, is still inhibited by CDK2-cyclin E1. Alternatively, BRCA1 and BARD1 are likely exported to the cytoplasm and their expressions are remarkably reduced by CDK2-cyclin E1 coexpression. Recognizing the importance of cyclin E1 overexpression in breast cancer development, these results suggest a CDK2-BRCA1-NPM pathway that coordinately functions in cell growth and tumor progression pathways.
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nucleophosmin b23 is a candidate substrate for the brca1 BARD1 ubiquitin ligase
Journal of Biological Chemistry, 2004Co-Authors: Ko Sato, Ryosuke Hayami, Wenwen Wu, Toru Nishikawa, Hiroyuki Nishikawa, Yoshiko Okuda, Haruki Ogata, Mamoru Fukuda, Tomohiko OhtaAbstract:Abstract The breast and ovarian tumor suppressor BRCA1 forms a heterodimeric RING-type ubiquitin ligase with BARD1 to catalyze untraditional Lys-6-linked polyubiquitin chains. It is not clear how the BRCA1-BARD1 ligase regulates various cellular processes such as DNA repair, cell-cycle progression, transcriptional regulation, and centrosome duplication. Here we report that BRCA1-BARD1 catalyzes the polyubiquitination of nucleolar phosphoprotein nucleophosmin/B23 (NPM). Two different mass spectrometry screens for protein ubiquitinated by BRCA1-BARD1 both identified NPM. NPM interacts with N-terminal fragments of BRCA1 and BARD1 in a manner dependent upon BRCA1-BARD1 heterodimer formation. NPM colocalizes with BRCA1 and BARD1 in mitotic cells suggesting the possibility of NPM regulation by BRCA1-BARD1 during mitosis. BRCA1-BARD1 catalyzes the ubiquitination of NPM in vitro and in vivo, and BRCA1-BARD1 co-expression in cells causes NPM stabilization rather than degradation. This is consistent with the notion that this ligase catalyzes untraditional polyubiquitin chains. Given the many overlapped functions between NPM and BRCA1, we propose that NPM is a strong candidate as a substrate of the BRCA1-BARD1 ubiquitin ligase.
Beric R Henderson - One of the best experts on this subject based on the ideXlab platform.
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BARD1 splice variants display mislocalization in breast cancer cells and can alter the apoptotic response to cisplatin
Cancer Letters, 2016Co-Authors: Kamila A Marzec, Irmgard Irmingerfinger, Estefania Martinoecharri, Beric R HendersonAbstract:Abstract We previously showed that BARD1 is a shuttling protein with pro-apoptotic activity in MCF-7 breast cancer cells. BARD1 is expressed as splice variant isoforms in breast cancer. Here we characterized YFP-tagged BARD1 splice variants (beta, omega, phi, ΔRIN, epsilon) for subcellular localization and apoptotic efficacy. We found that loss of nuclear localization (NLS) or export (NES) sequences influenced cellular distribution. The beta and omega variants (+NLS/−NES) shifted exclusively to the nucleus. In contrast, BARD1-epsilon (−NLS/+NES) was mostly cytoplasmic. Variants that lacked both NLS and NES were evenly distributed. Interestingly, the more nuclear isoforms (omega and beta) were least apoptotic in MCF-7 cells as measured by FACS. The cytoplasmic localization of BARD1 isoforms correlated with increased apoptosis. This relationship held in cells exposed to low dose (5 µM) of cisplatin. At 20 µM cisplatin, the main observation was a protective effect by the omega isoform. Similar analyses of HCC1937 cells revealed less pronounced changes but a significant protective influence by BARD1-epsilon. Thus BARD1 variants differ in localization and apoptotic ability, and their expression profile may aid prediction of drug efficacy in breast cancer.
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the BARD1 brct domain contributes to p53 binding cytoplasmic and mitochondrial localization and apoptotic function
Cellular Signalling, 2015Co-Authors: Varsha Tembe, Kirsty M Brodie, Estefania Martinoecharri, Kamila A Marzec, Kate Mills, Anna Defazio, Helen Rizos, Emma Kettle, Ross A Boadle, Beric R HendersonAbstract:Abstract BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425–525) and modest (525–567) to strong (551–777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function.
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Characterization of BARD1 targeting and dynamics at the centrosome: The role of CRM1, BRCA1 and the Q564H mutation
Cellular Signalling, 2011Co-Authors: Kirsty M Brodie, Beric R HendersonAbstract:Abstract BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time > 500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~ 20 s). The ~ 25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128–550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.
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differential modulation of brca1 and BARD1 nuclear localisation and foci assembly by dna damage
Cellular Signalling, 2010Co-Authors: Kirsty M Brodie, Beric R HendersonAbstract:The BRCA1/BARD1 heterodimer regulates genomic maintenance and contributes to the DNA damage checkpoint response. We previously reported that BRCA1 and BARD1 can shuttle between nucleus and cytoplasm. In this study, we evaluated the localisation patterns of BRCA1 and BARD1 in response to different types of DNA damaging agents and chemotherapeutic drugs. In MCF-7 cells, endogenous BRCA1 increased transiently in the nucleus at 2 h after ionising radiation (IR), whereas BARD1 was unaffected. IR treatment did not induce nuclear export of either protein, in contrast to previous reports. DNA damage by UV radiation, etoposide or camptothecin caused a preferential down-regulation of nuclear BARD1 at 6 h post-treatment. The UV-dependent loss of nuclear BARD1 was blocked by the proteasome inhibitor MG132, but not by leptomycin B, indicating a change in BARD1 nuclear degradation rather than nuclear export. MG132 also blocked the dispersal of BARD1/BRCA1 nuclear foci at 6 h after UV, implicating the proteasome in repair foci disassembly. In the cytoplasm, BRCA1 and BARD1 were detected at centrosomes but their distribution was not altered by DNA damage. BARD1 displayed a stronger mitochondria accumulation than BRCA1, and became phosphorylated at mitochondria in response to DNA damage. The mitotic spindle poisons vincristine and paclitaxel had no effect on BRCA1 or BARD1 subcellular distribution. We conclude that BARD1 phosphorylation, expression and localisation patterns are regulated in the nucleus and at mitochondria in response to different forms of DNA damage, contributing to the role of BRCA1/BARD1 in DNA repair and apoptotic responses.
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BARD1 regulates BRCA1-mediated transactivation of the p21WAF1/CIP1 and Gadd45 promoters.
Cancer Letters, 2008Co-Authors: Megan Fabbro, Beric R HendersonAbstract:BRCA1 regulates gene transcription as part of its tumor suppressor function. Prior studies on BRCA1 transactivation did not account for the impact of its binding partner, BARD1. Here we tested the effect of BARD1 on BRCA1 transactivation of the p21 and Gadd45 promoters. We show that BARD1 promoted nuclear accumulation of BRCA1, but repressed BRCA1-mediated transactivation by up to 75% in transfected cells normalized for nuclear BRCA1 levels. The BRCA1 (C61G) RING mutant transactivation function was not regulated by BARD1. We propose that BARD1 reduces BRCA1 transcriptional activity, and that this at least partly involves BRCA1/BARD1 E3 ubiquitin ligase activity, which is disrupted by the C61G mutation.
Jeffrey D Parvin - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of BARD1 missense variants in homology directed repair and damage sensitivity
PLOS Genetics, 2019Co-Authors: Aleksandra I Adamovich, Tapahsama Banerjee, Margaret Wingo, Kathryn Duncan, Jie Ning, Fernanda Martins Rodrigues, Kuanlin Huang, Feng Chen, Li Ding, Jeffrey D ParvinAbstract:The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants.
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abstract 1367 brca1 and BARD1 protein interactions that are required for dna repair function
Cancer Research, 2018Co-Authors: Aleksandra I Adamovich, Tapahsama Banerjee, Margaret Wingo, Miranda Gardner, Michael A Freitas, Jeffrey D ParvinAbstract:Breast and ovarian cancers are prevalent among women, and hereditary breast and ovarian cancers (HBOCs) have been associated with germline mutations in genes such as BRCA1 and BARD1. BRCA1 and BARD1 form an obligate heterodimer, and the BRCA1/BARD1 complex is required for tumor suppression functions. Our lab has tested hundreds of BARD1 and BRCA1 variants and identified many that are deficient in homologous recombination, which we have shown to accurately predict cancer predisposition in the clinic. Several of these functionally defective BRCA1 mutants map to a pocket of amino acids on the surface of the protein that does not have any known binding partners. Repair-deficient mutations are also present on the surface of the BARD1 protein in domains that are not known to be associated with DNA repair. We hypothesize that the DNA repair deficiencies mediated by these BRCA1 and BARD1 mutants are due to differences in protein binding when compared to wild-type protein. We have found that BRCA1 mutants in this protein pocket do not phosphorylate and do not localize to the nucleus following DNA damage, both of which are characteristic of the DNA damage response. However, BARD1 repair-deficient mutants still bind phosphorylated BRCA1, indicating that their deficiencies are not due to loss of BRCA1 function. To investigate protein interaction differences we are creating fusion proteins of wild-type and mutant BRCA1 with BioID2, which will biotinylate proteins that bind to BRCA1. We will then be able to identify, via avidin purification and mass spectrometry, protein interactions that are present in the wild-type but absent in the mutant BRCA1. Novel proteins that we identify will be tested for DNA repair and tumor suppressor function. This information will allow us to better understand both BARD1 and BRCA1 function and the mechanism of homologous recombination. Citation Format: Aleksandra Adamovich, Margaret Wingo, Tapahsama Banerjee, Miranda Gardner, Michael Freitas, Jeffrey Parvin. BRCA1 and BARD1 protein interactions that are required for DNA repair function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1367.
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functional analysis of BARD1 missense variants in homology directed repair of dna double strand breaks
Human Mutation, 2015Co-Authors: Tapahsama Banerjee, Lea M Starita, Jessica L Gillespie, Amanda Ceravolo, Matthew R Parvinsmith, Stanley Fields, Amanda E Toland, Jeffrey D ParvinAbstract:Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.
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brca1 dna binding activity is stimulated by BARD1
Cancer Research, 2006Co-Authors: Amanda M Simons, Scott R Williams, J Mark N Glover, Andrew A Horwitz, Lea M Starita, Karen Griffin, Jeffrey D ParvinAbstract:The breast- and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including transcription, ubiquitination, and DNA repair. Its tumor suppression activity is tightly linked to that of BARD1, a protein that heterodimerizes with BRCA1. It has been previously shown that BRCA1 binds to DNA, an interesting functional observation in light of the genetic data linking BRCA1 to DNA repair pathways. In this work, we reexamine the DNA-binding properties of BRCA1, comparing them with the DNA-binding properties of the BRCA1/BARD1 heterodimer. Because nuclear BRCA1 exists as a heterodimer with BARD1, it is likely that in vitro studies of the heterodimer will provide a more accurate model of physiologic conditions. Our results indicate that whereas BARD1 cannot directly bind DNA, it does enhance DNA binding by BRCA1. This is a surprising observation as both DNA-binding domains are distal to the BARD1-interacting RING domain of BRCA1. Further analysis of the dimerization reveals that the BRCA1/BARD1 interaction is not limited to the amino-terminal RING domains of each protein. The carboxyl terminus of BRCA1 contributes significantly to the stability of the heterodimer. We also show that the presence of BARD1 has a secondary effect, as autoubiquitination of BRCA1/BARD1 heterodimers additionally enhances the affinity of BRCA1 for DNA. Together, these data suggest that BRCA1 and BARD1 heterodimerization is stabilized via domains not previously thought to interact and that BARD1 acts in both ubiquitination-dependent and ubiquitination-independent ways to influence the role of BRCA1 in DNA repair. (Cancer Res 2006; 66(4): 2012-8)
