【作者】 朱晓宇；

【导师】 桑建利；

【作者基本信息】 北京师范大学 ， 细胞生物学， 2006， 博士

【摘要】 DNA复制调控是细胞周期调控的一个核心事件。在DNA合成过程中,细胞周期通过Cyclin、CDK、CKI及其它相关蛋白对DNA复制复合体进行复杂的活性调控,因而保证每个细胞周期中DNA只能精确地复制一次。此外,细胞周期还控制着复制体相关基因的表达。DNA合成时,DNA聚合酶δ(polδ)与PCNA、RFC构成复合体,进而完成导前导链与后滞链的延伸。真核细胞polδ由4个亚基组成,POLD1基因编码了催化亚基,其mRNA及蛋白水平在G1/S期达到高峰,但是至今尚不清楚细胞周期通过何种通路调控POLD1启动子活性。本研究运用细胞周期同步化及荧光素酶报告基因测定了人乳腺癌MCF7细胞中POLD1启动子在细胞周期不同时相的活性。结果显示启动子活性随着细胞周期进程而变化,在早S期最高。瞬时共转染实验发现,随着E2F1的积累,POLD1启动子活性逐渐升高,表现出剂量依赖效应。E2F1最大能将POLD1启动子活性提高到对照组的2.98倍。运用染色体免疫共沉淀(ChIP)技术证明E2F1能够与POLD1启动子结合。为进一步确定调控POLD1表达的细胞周期相关因子,应用质粒pXJ41-neo、pEGFP-p53、pXJ41-p21、pXJ41-as-CDK2、pXJ41-as-CyclinE、pXJ41-as-CDK4及pXJ41-as-Cyclin D1分别转染MCF7细胞,分别筛选得到稳定细胞系。结果表明增强p53或p21的表达,抑制CDK2或Cyclin E的表达都能抑制POLD1启动子活性;而抑制CDK4或Cyclin D1的表达对启动子活性没有明显影响。随着CDK2或Cyclin E表达量的降低,POLD1启动子活性逐渐降低,表现出剂量依赖效应。抑制CDK2的表达能将POLD1启动子活性降低到对照组的50%左右,而抑制Cyclin E的表达能降低到30%。此外,对POLD1启动子的抑制作用还表现出时间依赖效应,一般在转染24小时后才能表现出较强的抑制,而转染48小时后的抑制最强。用pEGFP-p53重组质粒瞬时转染MCF7细胞,RT-PCR结果显示由于p53表达增强,POLD1基因mRNA水平降低。染色体免疫共沉淀和荧光素酶实验证明p53在细胞内直接结合POLD1启动子而抑制其活性,且这种抑制具有时间依赖效应和剂量依赖效应。间接免疫荧光和荧光定位实验均显示p53在G1期进入细胞核而在S期和G2期则被转运至细胞质。但是荧光素酶实验显示p53在细胞周期各时相都能抑制POLD1启动子活性,表现出细胞周期非依赖性特点。本研究还发现,在p21表达增强的细胞中,POLD1 mRNA及蛋白水平都有所下降。p21高表达后能抑制POLD1启动子活性,且这种抑制具有剂量依赖效应,抑制最强时,启动子活性只相当于对照组的27%。同时,这种抑制也具有时间依赖效应,在转染后36小时后才能达到最大抑制。p21还可以通过影响E2F1的活性而消除E2F1对POLD1启动子的促进作用。利用p21的截取突变体和点突变体发现p21对POLD1启动子的抑制作用不依赖于Cyclin/CDK复合体或PCNA。利用一系列POLD1启动子突变体发现,p21对POLD1启动子的抑制是通过位于启动子+29～+33和+44～+48位置的CDE/CHR元件实现的。突变部分或全部的该元件后,p21几乎不能抑制启动子活性。这些结果说明p21能够以较为复杂的方式抑制POLD1启动子活性。本研究首次系统探讨了细胞周期重要调控因子对POLD1基因启动子活性的调控作用,确定出E2F1、CDK2、Cyclin E、p53和p21对POLD1启动子活性的影响及作用方式。这些结果有助于理解细胞周期进程中DNA复制的调控机理。更多还原

【Abstract】 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.更多还原