Study On Promoter Cloning, Identification And Transcriptional Regulation Of NFBD1 And Its Function Involvement In Cell Proliferation And Apoptosis

NFBD1 (a nuclear factor with BRCT domain 1), also known as MDC1 (mediator of DNA damage checkpoint protein 1), is a large nuclear protein that mainly participates in DNA damage response. We have previously found that, NFBD1 is transcriptionally regulated after DNA damage, which further interacts with p53 to regulate cell survival and death. However, the molecular mechanism underlying it is still not clear. In addition, our previous results also suggest the involvement of NFBD1 in cell proliferation and apoptosis. In the present study, we have systematically investigated the transcriptional regulation of NFBD1 and its role in cell proliferation and apoptosis. The results are summarized as followings.(1) Characterization and identification of NFBD1 promoter.At first, the transcriptional start sites for NFBD1 gene have been identified by 5' RACE and bioinformatic analysis. Results indicated that human NFBD1 gene has at least four transcriptional variants with distinct transcriptional start sites. Several overlapping genomic fragments from the 5'- flanking region of NFBD1 gene have been then coloned into pGL3-basic vector to construct NFBD1 promoter reporters. Luciferase reporter assay indicated that NFBD1 promoter region is mainly located in a 1.5-kb region nearby the major transcriptional start site. Transcriptional factor binding analysis indicated that, NFBD1 promoter lacks of TATA box, but contains classical CCAAT box and GC box as well as other putative transcriptional factor binding sites, suggesting that transcriptional factors such as Sp1 and NF-Y might be invovled in the transcriptional regualtion of NFBD1 gene.(2) Identification and functional analysis of NFBD1 core promoter.To further identify the NFBD1 core promoter region, serial deletions of NFBD1 promoter reporter have been constructed by DNA blunting method. Luciferase reporter assay indicated that NFBD1 core promoter is mainly located in a 325-bp region nearby the major transcriptional start site, which contains consensus binding sites for both Sp1 and NFY. Chromatin Immunoprecipitation Assay (ChIP) and gel shift assay demonstrated that Sp1 and NFY, but not STAT1, are associated with the NFBD1 promoter region. Site-directed mutations of Sp1-binding sites, but not NFY-binding sites, completely abrogated the NFBD1 promoter activity, indicating a critical role of Sp1 in the transcriptional regulation of NFBD1.(3) Transcriptional downregulation of NFBD1 after DNA damage.Adriamycin treatment significantly decreased the NFBD1 promoter activity as well as the mRNA expression levels of NFBD1. On the other hand, Sp1 is phosphorylated and its binding to NFBD1 promoter is significantly reduced after adriamycin treatment although its expression kept unchanged. Serial deletion analysis of NFBD1 promoter reporter also indicated that Sp1 mediated the transcriptional downregulation of NFBD1 after DNA damage. In support with this notion, mithramycin A (MA, Sp1 inhibitor) treatment resulted in a significant down-regulation of NFBD1. Furthermore, siRNA-mediated knockdown of the endogenous Sp1 in HeLa cells reduced the expression levels of NFBD1, which renders cells sensitive to adriamycin (ADR). Taken together, those findings strongly suggest that Sp1 mediates the transcriptional downregulation of NFBD1 after DNA damage.(4) The role of STAT1 in the transcriptional regulation of NFBD1.In response to DNA damage, STAT1 as well as its transcriptional targets are activated or repressed. However, the protein level, phosphorylation and subcellular localization of STAT1 showed little change in response to DNA damage. On the other hand, both interferon gamma-activated endogenous STAT1 and siRNA mediated STAT1 knockdown had undetectable effects on the promoter activity and mRNA level of NFBD1 although fludarabine(Flu, STAT1 inhibitor) showed a nonspecific effect of reducing the promoter activity and mRNA level of NFBD1. Those results indicated that STAT1 is not involved in the direct transcriptional redulation of NFBD1.(5) The role of NFBD1 in cell proliferation and apoptosis.siRNA-mediated NFBD1 knockdown caused a significant growth retardation in HeLa cells. Analysis of the NFBD1 expression profile during cell cycle indicated that NFBD1 as well as Plk1 is a novel G2/M phase gene which shows a higher expression level during G2/M phase. In support with this notion, siRNA-mediated NFBD1 knockdown caused the accumulation of DNA damage, activation of G2/M checkpoint and G2/M arrest. In addition, NFBD1 depletion also induced apoptosis as shown by the sub-G1 peak, cleavage of caspase 3 and PARP (Poly-ADP-ribose Polymerase). Furthemore, results also suggested that p53-independent noxa transactivation is involved in this apoptotic process. Finally, NFBD1 siRNA also significantly enhanced the cellular sensitivity to DNA damaging agents such as adriamycin and cisplatin. Taken together, those results indicated that 1) NFBD1 plays an important role in cell proliferation and apoptosis and 2) NFBD1 may be used as an attractive target to develop novel therapeutic chemosensitizers and gene therapeutic drugs against cancer.