BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection

The molecular function of BRCA1 has been the subject of intensive studies since it was cloned in 1994 [37]. Primarily in cancer, it has been characterized as a multifaceted tumor suppressor protein due to its role in cell cycle progression, DNA repair and DNA damage response processes, transcription, RNAi pathway regulation, and apoptosis [23,38]. Limited studies have linked BRCA1 to HIV-1. Initially, Zimmerman et al. [13] showed BRCA1-H2AX foci formation was required for Vpr-induced G₂ arrest. Later, this same group further elaborated on the role of BRCA1 in infection by showing Vpr-induced ATR-dependent activation of BRCA1 at serine substrate S1423 as a response to genotoxic stress, suggesting a model for Vpr-induced apoptosis via transcriptional regulation of the BRCA1 target gene, GADD45α [11]. Around the same time, Coberley et al. [12] demonstrated alterations of various genes contributing to cell cycle transition at the G₂/M checkpoint through a temporal genetic network study in mock or R5-tropic HIV-1 infected primary macrophages. Specifically, infection activated mediators of cell cycling at different times during infection including BRCA1 (intermediate), and GADD45α (late). Furthermore, BRCA1 may function as a transcriptional coactivator or corepressor, a function that varies depending on its ability to recruit both the basal transcription machinery and proteins implicated in chromatin remodeling [32,39]. Consequently, we were interested in characterizing BRCA1 function in HIV-1 transcription.

First we started our functional transcription studies in cells that have a null background for BRCA1 expression. UWB1.289 cells are derived from ovarian cancer in a germ line BRCA1 mutation carrier and lack expression of BRCA1, while UWB1.289 + BRCA1 cells are a stable UWB1.289 derivative cell line carrying a pcDNA3 plasmid coding for wild-type BRCA1 [40]. Here we co-transfected both set of cells with HIV-1 LTR-Luc, pcDNA or Tat, and Renilla reporter plasmid as a control for transfection efficiency between cell lines. Based on scored luciferase activity, results in Figure 1A indicate that there is ~13-fold difference in Tat transactivation of the HIV-1 LTR in cells expressing BRCA1 (lane 2, black bar) when compared to the BRCA1 null cells (lane 2, white bar). Next, we were interested in assessing the effect of BRCA1 overexpression in the context of integrated proviral DNA. To this end, we co-transfected pcDNA or Tat, and BRCA1 into TZM-bl cells followed by measurement of the HIV-1 LTR activity through luciferase assays. The TZM-bl cell line is a widely used reporter system for the study of Tat-dependent transcription as it harbors the luciferase gene under the control of an integrated HIV-1 5′ LTR. Results in Figure 1B show a significant transcriptional enhancement (~60% increase) when BRCA1 is overexpressed in comparison to pcDNA-transfected cells (compare lanes 5 and 4). Importantly, we did not observe any effects of BRCA1 overexpression in the absence of Tat, suggesting a Tat-dependent effect (lanes 2 and 3). Given that BRCA1 is activated by phosphorylation [23], which has also been noted to occur during HIV-1 infection [11], we assessed the importance of BRCA1 phosphorylation in the enhancement of HIV-1 transcription. To this end, we utilized a BRCA1 mutant construct with amino acid substitutions for the ATR/ATM target serine residues S1387A, S1423A, S1457A, and S1524A (BRCA1 4P). Similarly, TZM-bl cells were co-transfected with Tat or pcDNA, and BRCA1 4P for 48 hours, with subsequent Tat-dependent HIV-1 LTR-driven luciferase expression quantification. Results in Figure 1C showed no transcriptional enhancement between cells containing control DNA or BRCA1 4P (lanes 4 and 5), indicating that BRCA1 activation by phosphorylation is important for the enhancement of Tat dependent LTR transcription. Likewise, we did not see any effects of BRCA1 4P overexpression in our basal controls (lanes 2 and 3).

Based on these findings, we next wanted to examine the functional consequences of BRCA1 selective depletion in Tat-dependent transcription. To assess the effect of BRCA1 knockdown in HIV-1 transcription, we first screened various siRNAs against BRCA1 and selected the one producing more effective depletion results (data not shown). TZM-bl cells were co-transfected with Tat and siRNA against BRCA1 or GFP as a non-specific control, and luciferase assays were performed 48 hours post-transfection. Results in Figure 1D show that luciferase activity was significantly reduced by ~49% in cells transfected with siBRCA1 compared to cells transfected with siGFP (compare lanes 4 and 5). As additional controls, BRCA1 levels in these cells were assayed by qRT-PCR and western blot. BRCA1 was successfully repressed (~90%) at the transcriptional level (compare lanes 6 and 7) and protein levels were decreased by ~68% (inset western blot panel). To discard the possibility that the decrease in Tat-dependent transcription was due to cytotoxic effects upon BRCA1 knockdown, we performed viability assays on these samples. No changes in cell viability were observed between samples transfected with control siRNA or BRCA1 siRNA (compare lanes 8 and 9), indicating that the transcriptional efficiency loss is specific to BRCA1 selective depletion. Collectively, these data support the permissive role of BRCA1 for Tat-dependent HIV-1 transcription.

Collectively, molecular studies have shown ~1,500 interactions between HIV-1 and human host proteins [41]. In terms of Tat, the most studied interaction is its association with the pTEF-b Cyclin T1 subunit by which it exerts Tat-dependent transcriptional activation of the HIV-1 promoter [1]. Because of the fact that Tat is known to be in complex with various transcription factors and host transcriptional machinery, we were interested in determining whether these proteins associate. To this end, GST-BRCA1 constructs spanning the full length of the protein (Figure 2A) and GST-Tat were utilized in a pull-down assay from Flag-Tat transfected TZM-bl whole cell lysates. TZM-bl cells were chosen as they express robust levels of BRCA1 (see Figure 1D), and are our base cell line for our transcriptional assays. Beads bound to GST protein (lane 2, panel B) were used as a background control. Membranes were probed with antibodies against Flag (top panel) and BRCA1 (bottom panel). Western blot results in Figure 2B indicate that the only detectable association between the GST-BRCA1 fragments and Flag-Tat occurs at the aa504-802 region (lane 4, top panel), while GST-Tat inversely confirmed the BRCA1-Tat association observed with full length endogenous BRCA1 present in the TZM-bl lysate (lane 8, bottom panel). In addition, we saw GST-Tat binding to the Flag-Tat present in the cell lysate (lane 8, top panel), doubling as a positive binding control given that Tat exists as a Zn²⁺- or Cd²⁺-linked dimer [42]. To further corroborate this interaction, whole cell extracts from Tat-transfected TZM-bl cells were immunoprecipitated with BRCA1 or IgG and western blotted with anti-BRCA1, Flag, and BRG1 antibodies. Results in Figure 2C show that BRCA1-Tat interaction was observed specifically with the BRCA1 immunoprecipitation and not with the IgG. Probing against BRG1 was used as an immunoprecipitation control since it has been shown to be a BRCA1-binding partner [32]. Collectively, these results indicate that physical interaction of BRCA1-Tat is detectable at the aa504-802 region of BRCA1, suggesting that these proteins are associated and accordingly, supporting BRCA1 participation in Tat-dependent transcription.

To further discern the role of BRCA1 in Tat-dependent transcription, we generated pseudotyped HIV-1 LTR-driven reporter viral particles. Figure 3A shows the general structure of the LTR-driven reporter plasmid pNL-RRE-SA-Luc that was used to incorporate the luciferase reporter gene. To assay the impact of BRCA1 status in Tat-dependent transcription in a live infection, we utilized the UWB1.289 and UWB1.289 + BRCA1 cells for their unique ability to provide clean contrasts of BRCA1 expression. Cells were co-transfected with pcTat and Renilla reporter 24 hours prior to infection. Next, cells were infected and processed for luciferase assays 24 hours post-infection to allow for sufficient proviral integration and transcription to take place. Results in Figure 3B indicate that in vNL-Luc infected cells, LTR activation was observed at a lower level in –BRCA1 cells (lane 2, white bar) when compared to + BRCA1 cells (lane2, black bar), which exhibited a ~8-fold increase in LTR activity over its –BRCA1 counterpart. No significant background activation was detected in + Tat mock infected cells (lane 1). Collectively, these findings support our initial assays performed in these cells with a non-integrated HIV-LTR reporter plasmid (Figure 1A), where we detected a more tolerant environment for Tat-dependent transcription to occur in the presence of BRCA1. Moreover, these results provide further support that the enhancement of transcription by BRCA1 is Tat-dependent.

Given the importance of BRCA1 to the cell cycle and the fact that HIV-1 transcription has been shown to be enhanced in the G2 phase of the cell cycle [11-13,23,43,44], experiments were performed to determine if UWB1.289 cells had a difference in cell cycle distribution. It has been previously shown that UWB1.289 cells display no difference in cell cycle profile in the absence of stress, but when cells were subjected to irradiation, a G2/M arrest was observed with the BRCA1 complemented cells [40]. To determine if any alterations in cell cycle were occurring in the UWB1.289 and UWB1.289 + BRCA1 cells following HIV infection, cell cycle analysis using propidium iodine staining and flow cytometry was performed. No changes in cell cycle distribution between these two cell derivatives were observed (Figure 3C). As Sp1 is a well-known interacting partner of BRCA1, the binding of Sp1 to the LTR in the presence and absence of BRCA1 was assessed by chromatin immunoprecipitation. ChIP assays from vNL-Luc infected –BRCA1 and + BRCA1 cells were performed using antibodies against RNAP II (positive control), V5 (negative control) and Sp1. There was no significant difference in Sp1 or RNAP II binding to the LTR between the two cell populations (Figure 3D), indicating that differential binding of Sp1 to the LTR is not a contributing factor to the decreased LTR activation observed in the –BRCA1 cells. Collectively, these results suggest that BRCA1 plays a direct and critical role in Tat-dependent HIV transcription independent of cell cycle effects.

Currently, there are no therapeutic agents specifically targeting BRCA1. However recently, it has been documented that curcumin reduces expression of the BRCA1 gene by histone acetylation impairment at the BRCA1 promoter [45]. Generally, curcumin is categorized as a multifactorial compound characterized by tolerable doses linked to antioxidant therapy, anti-tumor effects, and anti-viral effects [45-50]. Moreover, curcumin has been shown to suppress pathways concomitant to HIV-1 replication and HIV-1 associated neurocognitive disorders (HAND); and when administered as adjuvant therapy to existing cART, curcumin has been shown to enhance the protease inhibitor indinavir antiretroviral activity in persistently infected cells [51-54]. In addition, a recent study demonstrated multi-pathway involvement in curcumin-induced inhibition of Tat-dependent transcription [55]. Here, the authors showed modulation of the HDAC1/NF-κB pathway used by HIV-1 to exert chromatin remodeling at the viral promoter and further promoter activation by NF-κB. Despite curcumin not being BRCA1-specific, we were interested in using the compound as a model for small molecule inhibition of BRCA1.

To confirm previously published findings in our cell-based system, TZM-bl cells were treated for 24 hours with vehicle (DMSO) or a titration of curcumin determined from concentrations used in the literature (0.5, 1, 10, and 20 μM). Western blot was then performed using anti-BRCA1 and anti-β-actin antibodies to determine the expression levels of BRCA1. Results in Figure 4A show a dose-dependent loss of BRCA1 expression with curcumin treatment when compared to DMSO (compare lanes 1 to 2–5). Next, we confirmed Tat-dependent transcription inhibition by treating Tat-transfected TZM-bl cells with 20 μM of curcumin. Results in Figure 4B show a significant decrease in transcription (~46%) with curcumin when compared to DMSO (compare lanes 3 and 4) that is specific to treatment and not due to compound cytotoxicity (compare lanes 5 and 6) and likens previously published data [55]. We next sought to determine if BRCA1 was present at the HIV-1 LTR, and if curcumin treatment could modulate its binding. ChIP assays from Tat-transfected DMSO- or curcumin-treated (20 μM) TZM-bl cells were performed using antibodies against RNAP II (positive control), IgG (negative control) and BRCA1. Interestingly, we observed the presence of BRCA1 at the viral promoter (Figure 4C). To our best knowledge, this is the first instance that BRCA1 has been detected at the HIV-1 LTR. Conversely curcumin treatment resulted in a significant ~2.9-fold loss of BRCA1 occupancy from the activated HIV-1 LTR when compared to the DMSO control (lane 3). Collectively, these results are suggestive of BRCA1 playing a role in HIV-1 Tat dependent transcription, and implies the use of chemotherapeutic agents treating BRCA1 as a druggable target.

BRCA1 contains a serine cluster domain (SCD) spanning aa1280-1524, a common motif present in ATR/ATM protein targets [56]. ATR phosphorylates BRCA1 on serine 1423 in response to UV damage or HU-induced replication arrest [57], while ATM-mediated phosphorylation events at serines 1387, 1423, 1457 and 1524, have been characterized in response to ionizing radiation-induced damage [58,59]. While the Chk2 phosphorylation site is not located within the SCD region, its phosphorylation of serine 988 occurs in response to the same stressors as with ATM [60]. Because cellular stress was shown to be highly conducive to HIV-1 replication [61], we next looked at the effect of ATR/ATM inhibition on Tat-dependent HIV-1 transcription. TZM-bl cells were transfected with Tat and treated the next day with vehicle or a titration of caffeine. Caffeine is a methylxanthine that has been used extensively to study ATR/ATM signaling as a natural inhibitor of these kinases [62]. Forty eight hours post-treatment the cells were subjected to luciferase assays. As can be observed in Figure 5A, there is a dose-dependent decrease in HIV-1 transcription with increasing concentrations of caffeine of up to ~88% inhibition when compared to vehicle (compare lane 2 to 3–5). To confirm compound inhibition specificity, we performed cell viability assays of treated cells to measure inhibitor-induced cell death. Results in Figure 5B show that caffeine was not toxic at 500 μM or 2 mM. Working concentrations in published literature use caffeine up to 10 mM, however we observed that in our system, 5 mM caffeine decreased cell viability by ~60% (lane 5). Thus, treatment with 2 mM caffeine results in a viable ~60% Tat-dependent transcriptional activity decrease without exhibiting cytotoxicity.

Given that caffeine is a generic inhibitor, we wanted to explore treatment with a more specific compound. To confirm the importance of ATM in this process, the small molecule ATM inhibitor, KU55933 (which we will refer to henceforth as ATMin) was utilized. This inhibitor has been shown to specifically inhibit ATM in the low IC₅₀ of 12.9 nM without inhibiting ATR at doses of up to 100 μM [63]. As Chk2 also phosphorylates BRCA1 in response to DNA damage [64], a Chk2 inhibitor (Chk2in) was also tested. TZM-bl cells were transfected with Tat and treated the next day with vehicle or a titration of ATMin or Chk2in. Forty eight hours post-treatment the cells were subjected to both luciferase (Figure 5C) and viability assays (Figure 5D). The results showed a dose-dependent Tat-dependent LTR transcriptional inhibition of up to ~55% in the presence of ATMin but not Chk2in (compare lanes 6 and 9, panel C), demonstrating ATM kinase specificity in this inhibitory process. Both inhibitors showed no cytotoxicity. To further confirm the specific participation of ATM in Tat-dependent transcription, we performed ATM selective depletion in Tat-transfected TZM-bl cells. Results in Figure 5E indicate that upon ATM knockdown, Tat transactivation of the viral promoter is decreased by ~45%. As additional controls, ATM levels in these cells were assayed by qRT-PCR and western blot. ATM expression was successfully decreased (~40%) at the transcriptional level (compare lanes 3 and 4) and protein levels were decreased by ~54% (inset panel) as confirmed by western blot using antibodies against ATM and β-actin as a loading control.

In order to functionally link the inhibitor study findings to BRCA1, ChIP assays from Tat-transfected DMSO- and ATMin-treated TZM-bl cells were performed using antibodies against Histone H3 phosphorylated at serine 10 [pS10-H3 (positive control)], V5 (negative control), and BRCA1. Results in Figure 5F reveal total BRCA1 (lane 3, compare white and black bars) occupancy loss (~2-fold) from the activated HIV-1 LTR with ATMin treatment when compared to the DMSO control. It is important to note that we also observed a decrease in pS10-H3 binding to the LTR following ATMin treatment as well as a decrease in RNAP II binding following curcumin treatment (Figure 4C). These results indicate that these treatments are affecting more than just BRCA1 binding to the LTR. As we hypothesize that BRCA1 may be acting as a scaffold protein, there are likely multiple LTR binding factors that will be influenced if BRCA1 leaves the LTR. However, we cannot rule out a global change at the promoter after treatments that is affecting multiple binding partners. Taken together, these results suggest that BRCA1 phosphorylation plays a role in Tat-dependent transcription as observed indirectly by the inhibition of upstream BRCA1 activators. Also, they are suggestive of ATM-dependent BRCA1 phosphorylation requirement for its recruitment to the HIV-1 LTR in the presence of Tat. Moreover, these results show that LTR transcription is potently and specifically inhibited by ATMin. These observations are correlated with various studies implicating these kinases in HIV-1 infection [14,65-68]. For example, caffeine and caffeine-related methylxanthines, including FDA-approved theophylline, have been used to inhibit HIV-1 integration in primary cells [65]. Similarly, HIV-1 IN has been shown to stimulate an ATM-dependent DNA damage response and that in the absence of this enzyme, cells are sensitized to retroviral-induced death [67]. Thus, treatment with ATMin suppressed viral replication, not only of wild-type, but drug-resistant HIV-1. Of interest for the present study, caffeine has been found to prevent pTEF-b dissociation from its 7SK snRNP inactive complex, illustrating an additional mechanism for its inhibition of Tat-dependent transcription [69].

Finally we aimed to confirm our findings in a T-cell model of infection. To this end, CEM T-cells were infected with HIV and BRCA1 occupancy of the LTR assessed by chromatin immunoprecipitation. ChIP assays from HIV (NL4-3) infected CEM T-cells were performed using antibodies against pS10-H3 (positive control), V5 (negative control), and BRCA1. Indeed, we observed BRCA1 present at the LTR in HIV infected T-cells (Figure 6A). Next we examined if inhibition of BRCA1 phosphorylation through the use of the ATMin would alter BRCA1 binding to the LTR. CEM cells were pre-treated with ATMin for 2 hours prior to infection. ATMin was also added following infection. Interestingly, no significant difference in BRCA1 binding to the LTR was observed after ATMin treatment (Figure 6B). However, a decrease in p-BRCA1 (S1423) was observed after ATMin treatment, indicating that there was a shift in the form of BRCA1 bound at the LTR following ATMin treatment. Collectively these results indicate that BRCA1 is present at the HIV-1 LTR in a highly relevant model of HIV infection.