POLD1启动子区甲基化岛、转录因子PAX-5/E2F-1的发现与验证及复制性衰老过程中POLD1转录调控机制的研究

DNA polymerase δ(DNA pol δ) plays a very important role in DNA replication and DNA damage repair. Our previous study showed that the expression level of POLD1, the catalytic subunit of DNA pol δ, decreased as cell ageing, and that down-regulation of POLD1 could lead to the decline of DNA synthesis rate,the slowdown of cell cycle, the decreasing of DNA damage repair ability and the speeding up of replicative senescence.However,the mechanism of down-regulation of POLD1 level as ageing, especially,the transcriptional regulation mechanism of POLD1 is not clear.Our previous study (not published) firstly finds that there are four CpG methylation ialands in the promoter of POLD1, and that PAX-5 and E2F-1 bind with the island 3 and island 4.Human fetal lung fibroblast (2BS,WI38) is to be used in this study.The methylation level of the islands, the binding level of PAX5/E2F1 with the promoter of POLD1, and the relationship between the binding level of PAX-5/E2F-1 with the promoter and the methylation level of the islands will be analyzed in replicative senescence. The expression level of PAX-5/E2F1 , and the relationship between the binding level of PAX-5/E2F-1 with the promoter and PAX-5/E2F-1 expression level will also be analyzed in cell ageing. The co work of the methylation of the CpG islands and the expression of PAX-5/E2F-1 on the binding level of PAX-5/E2F-1 with the promoter will also be analyzed.The study above will firstly discover the transcriptional regulation mechanism of POLD1 in the replicative senescence. In the other hand,the methylation level of the promoter of POLD1, the expression level of PAX-5/E2F-1 and the binding level of PAX-5/E2F-1 with the promoter of POLD1 are to be measured in the lymphocyte of healthy people,Alzheimer disease patients and  osteoporosis patients to indicate the potential of the biomarkers of the diseases.

DNA聚合酶δ在DNA复制、损伤识别与修复中发挥重要作用，申请人前期研究发现，DNA聚合酶δ催化亚基POLD1（p125）随增龄表达下调，且下调POLD1则DNA合成率下降、损伤修复能力下降，并最终导致衰老加快。然而，POLD1增龄性表达下调的机制、尤其POLD1转录调控机制目前尚不清楚。申请人前期研究发现POLD1启动子区存在4个CpG甲基化岛，且在甲基化岛3、4位点分别结合转录因子PAX-5和E2F-1（未发表）。本研究拟分析复制性衰老过程中PAX-5/E2F-1与POLD1启动子结合水平的变化及启动子甲基化与PAX-5/E2F-1在POLD1转录调控中的作用等，以系统揭示复制性衰老过程中POLD1转录调控的分子机制。同时，分析淋巴细胞POLD1、PAX-5、E2F-1表达水平、POLD1启动子甲基化及PAX-5、E2F-1与启动子结合水平能否作为老年痴呆、骨质疏松的生物学标志物。

本研究发现，DNA聚合酶δ催化亚基（POLD1）启动子存在4个甲基化岛，并揭示了其甲基化水平增龄性变化。在甲基化增龄性变化最显著的甲基化岛3第36位点，发现并证实结合转录因子E2F1。以细胞、衰老模型鼠组织、不同年龄人群外周血淋巴细胞等材料，证实E2F1表达水平、E2F1与启动子结合水平均随龄下降；以2BS细胞、WI38细胞为材料，发现E2F1通过调节POLD1表达进而调节细胞衰老；通过甲基化、去甲基化试剂处理细胞，发现POLD1启动子甲基化有效降低了E2F1与启动子结合进而降低POLD1表达水平，从而加速细胞衰老。E2F1、POLD1启动子甲基化联合分析发现，E2F1表达下调、POLD1启动子甲基化协同加快了细胞衰老进程。. 本研究发现，PAX5在细胞和整体水平上均随龄表达降低，而下调PAX5则细胞衰老加快、POLD1表达下调。通过生信分析、CHIP、co-IP等试验，发现PAX5并未与POLD1启动子直接结合，而是与POLD1转录因子RUNX1结合，以RUNX1/PAX5复合物形式调控POLD1表达进而调控细胞衰老。. 本研究进一步（补充）研究了RUNX1对细胞衰老的影响及其分子机制。发现RUNX1结合在POLD1启动子-1005- -995、-995- -985、-905- -895三个位点；在细胞和整体水平RUNX1蛋白表达水平均随增龄而升高；在A549细胞中上调RUNX1则POLD1表达下调、细胞衰老加快，而在Bleomycin诱导的A549细胞中下调RUNX1则细胞衰老可被有效逆转。研究显示，RUNX1不仅调节POLD1表达，也有效调节了DSB及DSB损伤修复通路相关蛋白γ-H2AX、RAD51、53BP1表达，进而通过DNA合成、DNA损伤修复调节细胞衰老。通过RUNX1抑制剂RO 5-3335处理细胞、RUNX1敲减鼠的构建，充分证实了上述研究结论。. 另外，本研究观察了阿尔茨海默病（AD）患者血清及AD模型鼠脑组织中POLD1表达变化，分析了POLD1对AD神经元衰老的影响。亦观察了POLD1转录因子在骨质疏松症（OS）患者血清中的变化。结果提示，POLD1及其转录因子有成为AD、OS标志物的潜能。.综上，本研究阐明了POLD1启动子甲基化、E2F1及RUNX1/Pax5转录因子复合物对复制性衰老的影响及其分子机制，初步探讨了其在衰老相关疾病中诊