BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

Abstract

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.

Introduction

BRCA1 is a tumor-suppressor gene that is mutated in the germ-line of women genetically predisposed to breast and ovarian cancer [1], [2]. BRCA1 mutations are found in approximately 50% of patients with inherited breast cancer and up to 90% of families with breast and ovarian cancer susceptibility [1], [2]. BRCA1 is a 1863 amino acid protein [1] with functional domains, an N-terminal RING finger, and two tandem BRCT domains frequently targeted by genetic mutations. BRCA1 subcellular localization is regulated by nuclear-cytoplasmic shuttling, which is facilitated by nuclear localization signals (NLS; [3], [4]) and a nuclear export signal (NES; Ref. [5]). In most cases, however, cellular and ectopically expressed BRCA1 is primarily nuclear [6] due to regulation by the RING domain binding protein, BARD1, which imports BRCA1 into the nucleus and traps it there by masking its nuclear export signal [7].

BRCA1 is a multifunctional protein with proposed roles in DNA repair, transcriptional activation, cell cycle regulation, and apoptosis [8], [9]. Recently, BRCA1 was suggested to play a differential role in mediating susceptibility to apoptosis in response to different chemotherapeutic agents by promoting G2/M cell cycle arrest and cell death after treatment with antimicrotubule agents but resistance to cell death following DNA damage [10]. The role of BRCA1 as a regulator of the G2/M mitotic checkpoint [11], [12] is consistent with its loss of expression leading to increased susceptibility to genetic mutations and the triggering of p53-dependent apoptosis following DNA damage [9], [13]. In gene-targeting experiments, BRCA1-null or exon 11-deleted cells display prolonged survival only after mutational inactivation of the p53 gene [13], [14], [15]. Reducing BRCA1 levels by siRNA generated a modest increase in apoptosis [16]. The apoptosis induced by loss of BRCA1 expression may be mediated by its binding partner, BARD1, via a p53-dependent pathway [17].

The overexpression of BRCA1 in transfected cells is also known to induce apoptosis, as first described in mouse fibroblasts and breast cancer cells starved of serum [18]. The BRCA1-mediated induction of apoptosis has been linked to the DNA damage response and the H-Ras/c-Jun N-terminal kinase (JNK) pathway [8], [19], and the cellular form of BRCA1 was found to be transiently upregulated following DNA damage [20]. Several studies have focused on the ability of overexpressed BRCA1 to stimulate Gadd45/JNK signaling, which appears to be mediated specifically by the C-terminus, in that overexpression of a C-terminal BRCA1 fragment (aa 1151–1863) alone also induced apoptosis and Gadd45 expression [21]. Hsu et al. [22] showed that overexpression of a central BRCA1 sequence (aa 504–803) is also apoptotic, presumably because it disrupts normal BRCA1 association and function at centrosomes. It is therefore quite likely that overexpressed BRCA1 can disrupt or activate different signaling pathways simply because BRCA1 binds to many proteins with different functions. One neglected area of study in this regard is the possible role of the N-terminus in BRCA1 apoptotic function. The N-terminus of BRCA1 contains transport signals that control its nuclear or cytoplasmic distribution [4], [7], and a previous study by Wilson et al. [23] suggested that the cytoplasmic localization of BRCA1 correlated with cytotoxicity and aberrant nuclear morphology. We therefore wondered if proteins that regulate BRCA1 localization might also regulate its apoptotic function.

Of the many known BRCA1 binding partners, BARD1 is most often found associated with BRCA1 in vivo [24]. BARD1 co-localizes with BRCA1 in nuclear foci during S-phase [25], as well as in DNA damage-induced nuclear foci thought to be associated with DNA repair or replication [7], [26]. The expression of BRCA1 and BARD1 is coordinately regulated in different cells and tissues [27], and the two proteins co-fractionate in various DNA repair-associated nuclear complexes [28], suggesting the possibility that their association might be linked to cell survival. This notion was partly validated by our laboratory recently when we showed that the BRCA1-independent pro-apoptotic function of BARD1 [17] was suppressed by co-expression of BRCA1 [16]. In this study, we now formally demonstrate the converse situation; that is, a key role for BARD1 in the inhibition of BRCA1-mediated apoptosis. This regulation required the N-terminal dimerization domains of the two proteins and was not mediated by other N-terminal BRCA1-binding partners. In addition, our data extend the previous findings of Wilson et al. [23] and confirm a correlation between increased cytoplasmic localization of ectopic or endogenous BRCA1 and apoptotic function as defined by quantitative flow cytometric analyses and nuclear chromatin changes. This may provide an explanation for the predominant nuclear localization of BRCA1 in many cell types. We show that BARD1-mediated nuclear retention of BRCA1 reduced BRCA1 apoptotic function.