Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells
© Misra et al; licensee BioMed Central Ltd. 2010
Received: 15 September 2009
Accepted: 4 March 2010
Published: 4 March 2010
BRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study.
Human BRCA2 gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G2), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm.
BRCA2 gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter in vivo and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.
The abbreviations used are
chromatin immuno precipitation
- ZAR1 :
zygote arrest 1
- ZAR2 :
upstream open reading frame.
The tumor suppressor protein BRCA2 is implicated in the regulated growth and proliferation of human breast [1–4], prostate [5, 6], ovarian [7, 8], esophageal , and pancreatic [10, 11] cells. About 25% of autosomal dominant familial breast cancers are proposed to be caused by germline mutations in BRCA2 gene [12, 13]. The mutations of BRCA2 gene predispose the cells towards neoplastic development. BRCA2 protein is over-expressed in most of the sporadic breast cancer cells [1–4]. The consequence of this over-expression of BRCA2 is not clearly understood. The notion could be that unique cellular mechanisms are triggered in the breast cancer cells to stimulate BRCA2 gene expression as a temporary measure to regulate the growth of the breast cancer cells. One potential mechanism of BRCA2 involvement in breast cancer progression may be through deregulation of the BRCA2 gene expression.
In humans, BRCA2 is a 3418-amino acid protein localized in the nucleus [14, 15]. Loss of BRCA2 function has been shown to lead to centrosome amplification, chromosomal rearrangement, aneuploidy, and reduced efficiency of homologous recombination-mediated double-strand break repair. BRCA2 is known to directly bind to RAD51, BCCIP, PALB2, and BRAF35 proteins that are involved in meiotic/mitotic recombination, DNA double-strand break (DSB) repair, and chromosome segregation [1–4, 15].
BRCA2 gene expression is stringently regulated during the cell cycle. BRCA2 expression is proportional to the rate of cell proliferation [14, 16]. While BRCA2 expression is involved in cell cycle checkpoints and DNA repair, the mechanisms of cell cycle-dependent regulation of BRCA2 gene expression remains elusive. Analysis of the minimal promoter sequence of BRCA2 recognized several conserved binding sites for transcription factors such as E-box, E2F, and Ets recognition motifs [15, 17–20]. USF1 and USF2 bind to the E-box [18, 19] and Elf1, an Ets family protein, binds to the Ets recognition motifs  to activate BRCA2 gene expression [18, 19]. Another transcription factor, NF-κB, has also been shown to bind to the promoter and induce BRCA2 gene expression . The tumor suppressor protein TP53 represses the BRCA2 promoter by blocking the binding of USF . Recently, poly-(ADP-ribose) polymerase-1 was reported to negatively regulate BRCA2 gene promoter by binding to it .
We have reported previously that the transcriptional repressor protein SLUG negatively regulates BRCA2 gene expression in SLUG-positive breast cancer cells by binding to an E2-box flanked by two Alu sequences in the -701 to -921-bp region [17, 21]. Deletion of this sequence resulted in a 5-7-fold activation of the BRCA2 promoter. But the mechanism of cell cycle dependent regulation of BRCA2 gene expression in SLUG-negative cells remains unclear.
Here, we provide experimental evidence for the bi-directional activity of human BRCA2 gene promoter. We have shown here that the reverse activity of this promoter indeed transcribes a protein (ZAR1-like, we named ZAR2) that has significant similarity (36% identity), particularly at the C-terminal amino acid sequence of the C4-type zinc finger containing homeodomain protein, zygote arrest 1 (ZAR1) [22, 23]. The similarity between ZAR1 and ZAR2 may indicate that these proteins belong to a unique family of transcriptional regulators. The chromosomal context of BRCA2 and ZAR2 genes are highly conserved among vertebrates studied. BRCA2 and ZAR2 gene expressions are reciprocally related during the cell cycle in human breast cells. Our studies suggest that negative regulation of BRCA2 gene expression by the ZAR2 at the G0/G1 phase of human breast cell growth may provide an additional mechanism of cell cycle-dependent regulation of its expression in both SLUG-positive and SLUG-negative cells.
Cell culture and synchronization
Human breast cancer cells were obtained from ATCC (Manassas, VA) and cultured in ATCC-recommended media [21, 24]. We synchronized the cells by serum starvation and evaluated by FACS analysis, as described earlier . For transfection and synchronization experiments, we transfected the cells with the plasmids (see below) and let them recover for 2 h in RPMI medium with 10% fetal bovine serum (FBS). This complete medium was then replaced with starvation medium (RPMI 1640, phenol red free, 0% FBS). After 36 h, the cells were stimulated to re-enter the proliferative cell cycle by replacing the starvation medium with medium containing 20% FBS. Cells were harvested at specific time points following serum stimulation and were processed. The progression of cells through the cell cycle during these experiments was monitored by flow cytometric analysis of replicate samples of propidium iodide-stained cells [18, 21]. Each transfection experiment was repeated at least twice with triplicate samples each time. We used different human cell types including human mammary epithelial cells (HMEC), human breast cancer cells like MCF7, MDA-MB-468, MDA-MB-231, BT549, as described , for further verification and confirmation experiments.
Promoter constructs, transfection and dual luciferase assay
Sequences of the oligonucleotides used in this study.
BRCA2/ZAR2 gene promoter amplification
Gene racer ZAR2 5'-RACE
PCR of C-terminal FLAG-tagged ZAR2
PCR of N-terminal FLAG-tagged ZAR2
FLAG reverse primer
GeneRacer analysis of ZAR2 gene transcription start site
The transcription start site of the ZAR2 gene was determined with the reagents from the GeneRacer Kit (Invitrogen). RNA was isolated from MCF7 cells using TRIzol reagent. The DNAse (RQ1, Promega)-treated RNA was then digested with calf intestinal phosphatase to remove 5'-phosphate from broken RNAs (if any). The 5'-caps of the intact mRNAs were removed by digestion with tobacco acid pyrophosphatase followed by ligation of a RNA oligonucleotide (44 nt) by T4 RNA ligase following suppliers protocols and using their reagents. These RNAs were used as template to make the cDNAs with GeneRacer oligo(dT) primer. The 5'-end of the ZAR2 mRNA was amplified using the GeneRacer 5'-primer and ZAR2 gene specific antisense primer (P3 and P4, Table 1) and Platinum Pfx DNA polymerase (Invitrogen). The PCR conditions were 1 cycle at 94°C for 2 min; 5 cycles at 94°C for 30 sec, 72°C for 2 min; 5 cycles at 94°C for 30 s, 70°C for 2 min; 25 cycles at 94°C for 30 sec, 60°C for 30 s, and 68°C for 2 min, and 1 cycle at 68°C for 10 min. The PCR product (814 bp: 770 bp from the ZAR2 mRNA + 44 bp from the RNA anchor) was gel purified, cloned and the nucleotide sequences were determined [21, 24].
Expression of 3X-FLAG tagged ZAR2 in MCF7 cells
Human ZAR2 (hZAR2) gene ORF (NM_001136571; 1-966 bp) was PCR amplified from the cDNAs derived from MCF7 cells with Hind III and Bam HI site-containing primers (P5 and P6, Table 1). The reverse primer did not have the endogenous stop codon. The PCR product was cloned at the Hind III/Bam HI sites of p3xFLAG-CMV-14 vector (Sigma Chemical Co. St Louis, MO) to get the C-terminal FLAG tagged construct. For the expression of N-terminal FLAG-tagged ZAR2, ZAR2 ORF was amplified from the cDNAs derived from MCF7 cells with Hind III and Xba I site-containing primers (P7 and P8, Table 1). The reverse primer retained the endogenous stop codon. The PCR product was cloned at the Hind III/Xba I sites of p3xFLAG-CMV-10 vector (Sigma). The clones were sequence verified. The plasmid DNA was transfected in the MCF7 cells using Lipofectamine 2000 (Invitrogen) following supplier's protocol. After 36 h, cells were lyzed in TRIzol (Invitrogen) for RNA isolation or in Cell lytic reagent with protease inhibitors (Sigma) for Western blotting analysis [21, 24]. Stable transfectants were also selected in G418 (1000 μg/ml) containing growth medium.
Cells were transfected in 6-well plate with either the C-terminal FLAG-tagged construct (p3XFLAG-CMV14 or p3XFLAG-CMV14-ZAR2) or with the N-terminal FLAG tagged constructs (p3XFLAG-CMV10 or p3XFLAG-CMV10-ZAR2) using Lipofectamine 2000 (Invitrogen). After 16 h of transfection, cells were trypsinized and plated in 8-well chamber slides for 24 h in complete growth medium, washed with PBS, fixed with ice-cold methanol for 10 min and permeabilized in 50 mM NH4Cl and 0.2% Triton X100 in PBS. After blocking with 5% goat serum in PBS, the cells were incubated with anti-FLAG M2 monoclonal antibody (Sigma) in the blocking buffer overnight at 4°C . After 16 h, the slides were washed 5 times with PBS and treated with secondary antibody conjugated with the red fluorescent dye (Alexa Fluor R555-conjugated donkey anti-mouse IgG, Invitrogen) for 1 h at room temperature. When performed dual labeling, unsynchronized MCF7 cells expressing C-terminal FLAG-tagged ZAR2 protein were processed similarly but incubated with both anti-FLAG M2 mouse monoclonal antibody (Sigma) and cyclin A monoclonal rabbit antibody (Abcam, Cambridge, MA) in the blocking buffer overnight at 4°C. After 16 h, the slides were washed 5 times with PBS and treated with secondary antibody conjugated with the red fluorescent dye (Alexa Fluor R555-conjugated donkey anti-mouse IgG, Invitrogen for FLAG) and the green fluorescent dye (Alexa Fluor R488-conjugated donkey anti-rabbit IgG, Invitrogen for cyclin A) for 1 h at room temperature. The cells were subsequently washed with PBS four times and stained with DAPI (Sigma) or Topro (Invitrogen). Finally, each slide was examined by confocal fluorescence microscopy (Nikon TE2000-U-CI confocal microscope). Each representative image was examined and digitally recorded at the same cellular level and magnification .
Real time RT-PCR
Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Isolated RNA was treated with DNase (RQ1, Promega) and then first strand cDNA was synthesized for real-Time RT-PCR (19). First strand cDNAs were synthesized using iScript cDNA synthesis kit (Biorad) with 5 μg of total RNA per reaction. For the real-time RT-PCR reaction cDNA (0.5 μl) was used per well in a total reaction volume of 25 μl. The iQSYBR green supermix (Biorad) containing the antibody-mediated hot start iTaq DNA polymerase was used for the PCR reaction. RT-PCR conditions were 1 cycle at 95°C for 3 min; 40 cycles at 95°C for 30 sec and 55°C for 1 min; 1 cycle at 95°C for 1 min; 1 cycle of 55°C for 1 min then 80 cycles for 10 sec each with 0.5°C increment after cycle two starting at 55°C, to collect the melt curve data and hold at 20°C [21, 24]. The end point RT-PCR was done using Taq PCR master mix (Qiagen). PCR conditions were 1 cycle at 94°C for 5 min; 40 cycles: 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; 1 cycle at 72°C for 10 min and then hold at 4°C.
Quantitative Chromatin Immunoprecipitation (ChIP)-PCR Analyses (qChIP-PCR)
MCF7 cells stably transfected to over express C-terminal FLAG-tagged ZAR2 were used for this study. ChIP assays were done with the EZ MagnaChIP kit reagents and protocols (Upstate-Millipore). Briefly, cells were treated with formaldehyde (1% final concentration) for 10 min at 37°C. Cross-linking was terminated with addition of glycine (0.125 M final concentration). Cells were washed twice with ice-cold PBS containing protease inhibitor cocktail (Sigma). The chromatin pellets were sonicated in SDS lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris (pH 8.1)] to an average DNA size of 500 bp with a Fisher model 50 Sonic Dismembranator using an optimized sonication condition. The sonicated extract was centrifuged for 10 min at maximum speed and diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl). The diluted ChIP lysates were pre-cleared with Magna beads for 30 min at 4°C. Immunoprecipitations were performed at 4°C overnight with either FLAG (Sigma) or normal mouse IgG (Santa Cruz Biotechnology). After 1 h incubation with 20 μl Protein A-Magna beads suspension, the conjugates were collected by magnetic separator. Immunocomplexes were washed twice sequentially in low salt immune complex wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], high salt immune complex wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl], LiCl immune complex wash buffer [0.25 M LiCl, 1% IGEPAL-CA-630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris (pH 8.1)], and TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 8.0)]. Elution of the immunocaptured chromatin complexes were performed with ChIP elution buffer provided in the kit. The DNA-protein cross-linking was reversed by incubating at 62°C for 2 h with Proteinase K. DNA fragments were obtained using Qiagen DNA purification column. DNA samples and standards were analyzed using real-time PCR system (BioRad) and iQSYBR Green PCR Master Mix (BioRad). Primers used to amplify BRCA2/ZAR2 promoter were as described  (Table 1). The following cycling parameters were used: 95°C for 3 min, 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 1 min. Dissociation curve analyses were performed to confirm specificity of the amplification products. All samples were run in triplicate and all data were normalized with control IgG and 1% input DNA amplification [21, 24].
Knockdown of ZAR2 gene expression
ZAR2 mRNA was knocked down in MCF7 cells using 50 pmol/ml of ZAR2 specific stealth siRNA#1 (P15 and P16, Table 1) and stealth siRNA#2 (P17 and P18, Table 1). Corresponding control stealth siRNAs used were (#1: P19/P20 and #2: P21/P22, Table 1). Stealth siRNAs were custom designed and synthesized by Invitrogen. Transfection of the cells with siRNAs was done by lipofection following Invitrogen-provided protocol. After 68 h cells were lysed in TRIzol for RNA isolation. First strand cDNAs were synthesized using iSCRIPT cDNA synthesis kit (BioRad). Evaluation of the levels of ZAR2 and BRCA2 mRNAs was done by real-time RT-PCR using iQSYBR green Supermix (BioRad). To evaluate the effect of ZAR2 mRNA knockdown on the activities of the forward (BRCA2) and the reverse (ZAR2) promoters, MCF7 cells were transfected with 50 pmol/ml of the ZAR2 or control siRNA for 68 h. The cells were then transfected with the BRCA2 forward or reverse promoter containing reporter constructs (Fig. 1). Dual luciferase assay was performed after 24 h as described previously  following the supplier's protocol (Promega).
Human BRCA2 gene promoter has bi-directional activity
Relative activities of the forward and the reverse promoters of BRCA2 gene in different unsynchronized human breast cancer cells at 95% confluency.
Relative luciferase activity* in
0.9 ± 0.2
1.6 ± 0.2
3.0 ± 0.5
22.0 ± 2.1
5.4 ± 0.3
8.7 ± 0.8
15.4 ± 0.1
The forward and the reverse promoter activities are differentially regulated during the cell cycle
Ratio of the forward (BRCA2) and the reverse (ZAR2) activity in the G0/G1 and S/G2 growth phases of different breast cancer cells using transient transfection with the dual reporter/promoter construct (see Fig. 1B).
Ratio of Rluc/Fluc activities* in
0.125 ± 0.03
0.23 ± 0.06
0.33 ± 0.01
0.28 ± 0.07
3.25 ± 0.14
3.12 ± 0.11
2.5 ± 0.12
3.31 ± 0.13
The bi-directional promoter of BRCA2 gene produces overlapping transcripts
The genetic arrangements of BRCA2 and ZAR2 genes are highly conserved among the vertebrates studied
A new exon and a new intron are identified for human ZAR2 gene
The 5'-UTR of human ZAR2 mRNA is riddled with upstream AUG codons
The nucleotide sequence of the ZAR2 cDNA is shown in Fig. 4B. The 676 nt 5'-untranslated region (UTR) of the ZAR2 mRNA has several upstream AUG codons (uAUGs) and out of frame upstream open reading frames (uORFs). Thus, translation of ZAR2 mRNA may potentially be regulated by these uAUGs and uORFs [30, 31].
ZAR2 protein has strong similarity in amino acid sequence with ZAR1 and is highly conserved among vertebrates
ZAR2 protein is predominantly located in the cytosol of unsynchronized human breast cells
Expressions of BRCA2 and ZAR2 during cell cycle are inversely related
ZAR2 is predominantly located in the nucleus of G0/G1 phase human breast cells
We synchronized C-terminal FLAG-tagged ZAR2-expressing MCF7 cells and evaluated the subcellular location of this protein at G0/G1 and S/G2 phase by immunofluorescence confocal microscopy using FLAG antibody. Our data suggest that ZAR2 protein is predominantly concentrated in the nucleus of these cells at the G0/G1 phase whereas it is mainly present in the cytosol at the S/G2 phase cells (Fig. 8C). Evaluation of ZAR2 protein distribution in the subcellular fractions by Western blotting analysis also revealed similar localization pattern (data not shown). These data suggest that not only the expression of ZAR2 gene is strictly regulated cell cycle-dependently but its subcellular localization is also controlled in a growth stage-dependent manner. ZAR2 and BRCA2 gene expressions are thus inversely related.
ZAR2 binds to the BRCA2/ZAR2 gene promoter in vivo
Knockdown of ZAR2 in the G0/G1 phase stimulated the expression of BRCA2
BRCA2 levels go up in many aggressively growing breast cancer cells [17–21]. It appears that the level of BRCA2 protein in the cell must commensurate with the need of the cells to avoid detrimental consequences in the cellular physiology. No BRCA2 in the dividing breast cells will predispose them to non-homologous end joining mode of DNA double strand break repair thus to potential oncogenesis . Understanding the mechanisms of this stringent mechanism of BRCA2 gene expression regulation is critical to evaluate etiology of human breast cancer.
Human genome is riddled with bi-directional promoters [23, 35, 36]. In this study we characterized the bidirectional promoter that expresses BRCA2 and ZAR2 genes. Human BRCA1 gene and about 11% of the total other human gene promoters have bi-directional activities . While assessing the activities of human BRCA2 gene promoter (Fig. 1A) in both orientations, reverse orientation serving as a negative control, we made three significant observations: (i) The human BRCA2 gene promoter is active in both the forward and the reverse orientations; (ii) The BRCA2 gene promoter is more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1), and (iii) when the cells are in the dividing state (S/G2), the forward activity of the promoter is higher than the reverse activity (see below). The reverse activity was insignificant when we did not include the exon 1 and part of the intron 1 sequence of the BRCA2 gene (26). We have repeated this experiment with different human cell types including human mammary epithelial cells (HMEC), human breast cancer cells like MDA-MB-468, MDA-MB-231, BT549, immortalized human breast cells like MCF10A, MCF10AT, human liver cells HepG2 and human monocytes U937. In all these cells the promoter behaved similarly. Thus, we believe that this cell cycle dependent differential bi-directional promoter activity of the BRCA2 gene is an intrinsic property of BRCA2 and ZAR2 genes. Recently, a ZAR2 paralog, Xzar2, has been cloned from the African clawed frog Xenopus laevis . Xzar2 was shown to be involved in epidermal fate determination mainly through signaling pathways distinct from that of BMP-Smad during early embryogenesis .
As mentioned above, BRCA2 gene expression is tightly regulated in human breast cells [14, 15, 17–21]. The BRCA2 mRNA and protein are only significantly expressed in the S/G2 phase cells and they are undetectable in the G0/G1 phase cells [18–21]. Over expression of BRCA2 protein was shown to be lethal for the survival of human pancreatic cancer cell line Capan-1 .
Several mechanisms are known to be operative in breast cancer cells to regulate BRCA2 gene expression [15, 18–21]. We reported previously that cell cycle stage-dependent regulation of BRCA2 gene expression in SLUG-positive breast cells occurs through a distal E2-box/Alu repeat containing silencer element located upstream of the BRCA2 gene transcription start site [17, 21]. The zinc-finger transcriptional repressor, SLUG, binds to the uniquely located E2-box sequence in the silencer element in the non-dividing cells and blocks the expression of BRCA2 gene by chromatin remodeling . We recently found that peroxiredoxin 5 competes with SLUG for the binding to the BRCA2 gene silencer in the dividing cells and thus de-silences the expression of BRCA2 gene in the dividing human breast cells (Misra, S. and Chaudhuri, G., unpublished). Transcription factors other than SLUG that have been reported to regulate human BRCA2 gene expression include USF1 and 2 [18–20], P53 , NFkB , ElF1 , and PARP1 . A recent report indicated the presence of a SNP (G to A) at the -26 position of human BRCA2 gene . This SNP is in the exon 1 of ZAR2 gene (Fig. 2B). Whether TP53 also regulates ZAR2 gene expression and whether this SNP affects its promoter activity is yet to be determined.
The bi-directional promoter of BRCA2/ZAR2 gene produces two partially overlapping transcripts. Whether these RNAs hybridize with each other and form double-stranded (ds) RNA and whether this ds-RNA has any role in regulating the activities of the promoter is yet to be determined. One of the potential roles of the ds-RNA could be siRNA-mediated transcriptional gene silencing through DNA methylation [40, 41].
The biological function of ZAR2 protein is not known. It has two putative C4-type zinc fingers and potentially could be a transcription factor. We found BRCA2 and ZAR2 gene expressions have inverse relationships during the cell cycle. It is possible that ZAR2 protein somehow inhibits BRCA2 gene expression. Although ZAR2 protein has two putative NLS sequences, in the dividing stage of the human breast cells ZAR2 is trapped predominantly in the cytoplasm. Thus, ZAR2 in the dividing breast cells may not have any significant effect on the BRCA2 gene expression. At the non-dividing (G0/G1) phase ZAR2 protein predominantly accumulates in the cell nucleus, binds to the BRCA2/ZAR2 gene promoter and consequently, both ZAR2 and BRCA2 gene expressions are inhibited. While it is tempting to speculate that ZAR2 represents a mechanism of cell cycle dependent regulation of BRCA2 gene expression, direct involvement of ZAR2 in BRCA2 gene transcription is yet to be determined.
As ZAR2 over expression decreased the levels of BRCA2 in the cells, this gene, if disregulated, and over expressed in the cells, it may promote the growth of the tumor. On the other hand, ZAR2 may be needed to suppress BRCA2 expression in the quiescent cells. Expression of BRCA2 in these cells could be detrimental for the cell growth and survival . We made an interesting observation while knocking down ZAR2 mRNA levels in different breast cancer cells. Out of four cell lines tested (MCF7, MDA-MB-231, MDA-MB-468 and BT549), only BT549 died at the G0/G1 phase in the ZAR2 knocked down cells. We found that ZAR2 knockdown in the quiescent cells leads to the elevation of the levels of BRCA2 which should be detrimental to the cells . But the ability to suppress the growth of the cells by BRCA2, the cell may need to have high MAGE-D1 level . Our explanation for the essentiality of ZAR2 in the BT549 cells is that only these cells among the four cells tested have high levels of MAGE-D1 . ZAR2 protein thus may have multiple balancing roles in the biology of BRCA2 and perhaps other molecules in the cells.
BRCA2 gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger-containing transcription factor ZAR2. BRCA2 and ZAR2 levels in the cells are inversely related with respect to the cell cycle. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 accumulates in the nucleus of the cells at the quiescent stage of the cells and binds to the BRC2 gene promoter in vivo. ZAR2 is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.
We appreciate Dr Tanu Rana for reading the manuscript and providing valuable critiques. Confoal microscopy was performed through the use of the MMC Morphology Core which is supported in-part by NIH grant U54NS041071. Supported in part by the DOD-CDMRP IDEA Grant# W81XWH-06-1-0466 and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC.
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