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.