Relationship Between Cell Cycle Regulatory Genes And Transcription Of POLD1 Gene

DNA replication regulation is one of the core events of cell cycle regulation. During DNA synthesis, cell cycle system controls the activity of DNA replication complex(RC), through Cyclins, CDKs, CKIs and other relative proteins, as ensures that DNA is exactly doubled only once in one cell cycle. Furthermore, the expression of genes relative to RC assembling is regulated by cell cycle system as well. To synthesize DNA, DNA polymeraseδ(polδ), PCNA and RFC consist of a complex, completing the elongation of leading and lagging strands. In eukaryotic cells, polδcontains four subunits, and the catalytic subunit is encoded by POLD1 gene, whose mRNA and protein levels reach peak at G1/S phase. However, how the cell cycle system regulates POLD1 promoter activity is unknown so far.In this study, cell cycle synchronization and luciferase reporter system were used to investigate POLD1 promoter activity, in human breast cancer cell MCF7, in different phases of cell cycle. Results showed that the promoter activity altered following the cell cycle progression, and was the highest at early S phase. Transient cotransfection assay revealed that the gradually accumulation of E2F1 caused an ascendant promoter activity, suggesting a doze-dependent stimulation effect. Furthest, E2F1 could raise the activity of POLD1 promoter up to 2.98 folds, compared with control group. Besides, a directly binding of E2F1 to POLD1 promoter was displayed by chromatin immunoprecipitation (ChIP) assay.In search of cell cycle relative factors, which were able to regulate the expression of POLD1 gene, variety of plasimids, including pXJ41-neo, pEGFP-p53, pXJ41-p21, pXJ41-as-CDK2, pXJ41-as-CyclinE, pXJ41-as-CDK4 and pXJ41-as-Cyclin D1, was transfected into MCF7 cells, respectively, and stable cell lines were obtained. Results indicated that POLD1 promoter activity was inhibited by over-expressed p53 or p21, as well as by low-expressed CDK2 or Cyclin E, while not affected by low-expressed CDK4 or Cyclin D1. The promoter activity of POLD1 was declined with the degradation of CDK2 or Cyclin E, suggesting a doze-dependent effect. Compared with control group, POLD1 promoter activity was deceased to 50% by the inhibition of CDK2 expression, and 30% by the inhibition of Cyclin E. Furthermore, the inhibition represented a time course-dependent effect also, and the distinct inhibition occurred at 24 hours after transfection and peaked at 48 hours.PEGFP-p53 plasmid was transient transfected into MCF7 cells, and RT-PCR revealed a decrease of POLD1 mRNA level, as a result of the enhancement of p53 expression. Using ChIP and luciferase assay, it was demonstrated that p53 bound to POLD1 promoter in vivo, exerting doze- and time course-dependent inhibition. Immunofluoresence assay and fluorescent observation showed that p53 was recruited into nucleus at G1 phase and excluded to cytoplasm at S and G2 phases. However, POLD1 promoter activity was suppressed at each phase of cell cycle, suggesting a cell cycle independent character.It was also shown in this study that in the cells overexpressing p21, mRNA and protein levels of POLD1 were decreased. P21 could inhibit POLD1 promoter activity by doze-dependent manner, decreasing the promoter activity to 27%, and time course-dependent manner, as well, reaching a maximum suppression at 36 hours after transfection. Additionally, the promotion effect resulting from E2F1 was reduced by p21. Truncation and site mutations of p21 were employed and it was revealed that the inhibition effect of p21 on POLD1 promoter did not reckon on Cyclin/CDK complex or PCNA. Through a series of mutants derived from POLD1 promoter, the judgment was demonstrated that p21 inhibited POLD1 promoter through the CDE/CHR element, localized at +29～+33 and +44～+48. If the element was mutated partly or entirely, p21 could hardly inhibit POLD1 promoter. These results displayed that p21 suppressed POLD1 promoter through complicated way.This study systemically investigated regulation of POLD1 promoter activity through important cell cycle factors, for the first time. It was identified that E2F1, Cyclin E, CDK2, p53 and p21 participated in the regulation of POLD1 promoter activity, and the regulatory mode was explored. These results would contribute to the insight into the DNA replication mechanism regulated by cell cycle.