Transcriptional Regulation Of The Human Proliferating Cell Nuclear Antigen Gene

Proliferating cell nuclear antigen (PCNA) performs essential functions in DNA replication, DNA repair, and cell cycle control. In the present study, we comprehensively examined the putative elements in the promoter region from .538 to +60 relative to the transcription start site of the human proliferating cell nuclear antigen gene. The serum-responsive element in the PCNA 5′ promoter, the transcriptional regulation of PCNA gene in the cell cycle DNA damage checkpoint, were elucidated in detail. The synthesis of PCNA is strictly regulated during the cell cycle. To investigate the contribution of the promoter region to the upregulation of human PCNA expression at the onset of S phase, we have examined 17 putative elements with reporter assays in quiescent L-02 cells and following serum stimulation. The E2F-like sequence 5′-TTCCCCGCAA-3′ located at .84 to .75 is required for the serum-induced transactivation. In electrophoretic mobility shift assays, the nuclear extracts from asynchronous L-02 cells exhibit two binding activities toward the .75 E2F oligonucleotide, and the minor band, whose formation could be interfered with by E2F-1 antibody, represents an S phase-specific complex. This is the first 3 demonstration of the E2F site in hPCNA 5′ promoter as a serum-responsive element. 4-Nitroquinoline N-oxide (4-NQO) is a potent mutagen and carcinogen. To elucidate the responsiveness of PCNA, an essential protein in DNA replication and repair, to DNA damage induced by 4-NQO, we first examined the transcriptional regulation of hPCNA in Hep G2 cells after 4-NQO treatment with transient expression assays. It was found that hPCNA promoter was dose-dependently transactivated by 4-NQO within the concentration of 0-2 μmol/L, via the previously reported p53-binding element from –236 to –217 upstream of the transcription start site, and the transactivation depended on the presence of p53. In western blot analysis the level of Ser15 phosphorylated p53 increased drastically following 4-NQO treatment, whereas the level of total p53 increased relatively slightly. The observations that the p53 phosphorylation and the sensitivity of hPCNA promoter to 4-NQO were simultaneously blocked by staurosporine, a Ser/Thr kinase inhibitor, suggested that Ser15 phosphorylated p53 was the 4-NQO-responsive PCNA regulator. [3H]-TdR incorporation analysis and comet assays showed that after 4-NQO exposure DNA replication was inhibited, while DNA repair was induced, and the PCNA transactivation in response to 4-NQO treatment contributed to DNA repair. In conclusion, the present study provides a further insight into the mechanism involved in the cellular response to 4-NQO treatment, especially the transcriptional regulation of hPCNA, as well as the physiological significance of the regulation. hPCNA gene is considered as a housekeeping gene, since it is active in all physiological conditions, despite the differences in the transcriptional apparatus of different cell types. We have examined the contributions of the 17 putative elements with reporter assays in HeLa, Hep G2, MCF7, L-02 cells, and found that the previous reported .45 ATF site is a general transcriptional element; the six sites in the region 4 from .210 to .75, .199 RA-like site, .186 SP1 site, .139 CTF site, .122 SP1 site, .94 CTF site and .75 E2F site, make considerable contributions to the promoter activity with slight cell type specificity; .498 Ets-1-like site, .216 p53 site, .64 AP2-like site and +38 E2F-like site contribute to the promoter activity with significant cell type specificity. Additionally, we constructed the reporter plasmid with simultaneously substitutional mutations in .94 CTF site and .75 E2F site, and investigated the co-operation of the adjacent elements.