Transcriptional regulation of the human DNA polymerase δ catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1
Transcriptional regulation of the human DNA polymerase δ catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1
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DOI：
10.1074/jbc.m101167200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Lee, MYW
中科院分区：
文献类型：
--
作者：
Li, BQ;Lee, MYW
DNA聚合酶δ催化亚基基因（POLD 1）作为p53的转录靶点进行了研究。北方印迹显示，POLD 1 mRNA的稳态水平显着降低与甲基磺酸甲酯处理的细胞中野生型p53表达增加相关。当异位野生型p53的表达被诱导到生理相关的水平，在“tet-off”培养的细胞中，其中p53的表达受到四环素的严格调控，它被发现，POLD 1稳态mRNA被抑制约65%。使用POLD 1启动子荧光素酶报告基因构建体的瞬时共转染实验表明：（i）POLD 1启动子活性被转染的野生型p53质粒抑制至最大约86%;（ii）p53通过与POLD 1启动子的P4位点的序列特异性相互作用介导大部分转录抑制;（iii）肿瘤来源的p53 DNA结合结构域中的p53突变完全消除了p53反式阻遏活性。此外，转染试验表明，p53能够抑制Sp1刺激的POLD 1启动子活性，这种抑制主要是由于Sp1蛋白和P4 Spl-binding site（与P4 p53结合位点重叠）之间序列特异性相互作用的丧失。最后，凝胶迁移分析表明，p53与Sp1蛋白竞争结合POLD 1启动子的P4序列。
The DNA polymerase delta catalytic subunit gene (POLD1) was studied as a transcriptional target of p53. Northern blotting showed that a significantly decreased steady-state level of POLD1 mRNA was associated with increased wild-type p53 expression in cells treated with methyl methanesulfonate. When ectopic wild-type p53 expression was induced to a physiologically relevant level in "tet-off" cultured cells in which p53 expression was tightly regulated by tetracycline, it was found that POLD1 steady-state mRNA was repressed by about 65%. Transient cotransfection experiments using a POLD1 promoter luciferase reporter construct showed that: (i) POLD1 promoter activity was inhibited by transfected wild-type p53 plasmid to a maximum of about 86%; (ii) p53 mediated a large part of the transcriptional repression through a sequence-specific interaction with a site identified as the P4 site of the POLD1 promoter; (iii) tumor-derived p53 mutations in the p53 DNA-binding domain completely abolished the p53 transrepression activity. Moreover, transfection assays demonstrated that p53 was able to repress Sp1-stimulated POLD1 promoter activity and that this repression was largely due to the loss of the sequence-specific interaction between Sp1 protein and the P4 Spl-binding site, which overlaps the P4 p53-binding site. Finally, gel shift assays suggested that p53 competes with Sp1 protein for binding to the P4 sequence of the POLD1 promoter.