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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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: Ionizing radiation (IR)-induced DNA damage causes the accumulation of DNA damage response (DDR) proteins as visible foci in cell nuclei. Despite the identified functional roles in DNA repair, the spatial relationships of different DDR proteins at foci have not been explicitly examined. This study aims to systematically compare the distribution of DDR proteins at IR-induced foci., Materials and Methods: MCF-7 cells were treated with IR, stained for γH2AX, MDC1, RNF8, RNF168, 53BP1, Abraxas (CCDC98), BRCA1, BRCC36, Merit40 (NBA1) and RAP80, and then imaged using high-resolution three-dimensional (3-D) confocal microscopy to assess the relative localization of proteins at foci., Results: All BRCA1-A complex components displayed strong co-localization, which overlapped significantly with RNF8 and RNF168, but not with γH2AX and MDC1. Intriguingly, 53BP1 co-located well with γH2AX and MDC1, but remained separate from RNF8 and RNF168. These co-localization patterns were consistent for at least 3h after IR., Conclusions: The foci formations of γH2AX-MDC1-53BP1 and RNF8-RNF168-BRCA1-A complexes are spatially independent. Such divergence was not anticipated from prior studies on the recruitment of these proteins to foci. This information indicates that individual foci may represent distinct sites of DNA repair facilitated by a specific subset of DDR proteins., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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 ~20s). 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mouse mammary tumor virus (MMTV) has long been known as a causal agent of breast cancer in mice. To date, varied MMTV-like envelope gene (env) sequences have been identified in up to 74% of human breast cancers. However, the role and origin of these MMTV-like sequences in human breast cancer remain uncertain. Our study was initiated to study the integration of MMTV-like env sequences in human breast cancer. PCR screening has identified 28 (56%) Australian breast cancer specimens and 7 (87.5%) human breast cancer cell lines to be positive for MMTV-like env sequence. In the MCF-7 genome, a fragment containing an MMTV-like env sequence of approximately 1.9 kb plus a downstream rodent-like sequence of approximately 200 bp was found to be integrated into a bacterial-like beta-lactamase sequence by insertional mutagenesis. The identified MMTV-rodent fragment is present in some MCF-7 sublines but absent in the screened specimens and other cell lines. Sporadic mutations found in this fragment indicate it has multiple copies in the MCF-7 genome. Sequence analysis has identified a novel ORF of approximately 1.6 kb which is 94-99% identical to MMTV env genes. RT-PCR was performed on the MCF-7 cDNA but no MMTV-like env transcript was detected. This is the first report to reveal the locus of MMTV-like env sequence in human cells. The MMTV-like env sequence was shown to be distinct from the human endogenous retroviral sequences and is closely related to rodents., ((c) 2008 Wiley-Liss, Inc.)

Introduction: Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited., Methods: We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V., Results: Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification., Conclusion: These data highlight the need for a range of functional studies to be performed in order to identify variants with partially compromised function. The results also raise the possibility that A1708V and R1699Q may be associated with a low or moderate risk of cancer. While data pooling strategies may provide more information for multifactorial analysis to improve the interpretation of the clinical significance of these variants, it is likely that the development of current multifactorial likelihood approaches and the consideration of alternative statistical approaches will be needed to determine whether these individually rare variants do confer a low or moderate risk of breast cancer.