Identification of a Functional In Vivo p53 Response Element in the Coding Sequence of the Xeroderma Pigmentosum Group C Gene.
Identification of a Functional In Vivo p53 Response Element in the Coding Sequence of the Xeroderma Pigmentosum Group C Gene.
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DOI：
10.1177/1947601912456288
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发表时间：
2012-02-01
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影响因子：
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通讯作者：
Ford, James M
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作者：
Hastak, Kedar;Adimoolam, Shanthi;Ford, James M
着色性干皮病C组（XPC）基因的蛋白质产物是一种DNA损伤识别因子，在全基因组核苷酸切除修复过程的早期发挥作用。XPC表达的调节在转录水平上部分由p53控制。为了鉴定参与XPC表达的p53依赖性控制的调节元件，我们在以XPC转录起始位点为中心的11 kb区域内使用多个规则间隔的引物对进行了定量PCR瓦片实验。紫外线照射后进行p53染色质免疫沉淀，并分析覆盖该区域的48个扩增子中每一个的DNA富集情况。XPC翻译起始位点上游的一个片段显著富集，而没有任何其他区域的富集。体外启动子报告基因测定和凝胶阻滞测定用于确认该区域的p53反应性，并确定具有刺激活性的最小区域。我们鉴定了一个与共有序列具有显著相似性的p53应答元件，具有3个错配。该反应元件的独特之处在于p53结合位点的部分包括XPC蛋白中前2个氨基酸的编码序列。
The protein product of the xeroderma pigmentosum group C (XPC) gene is a DNA damage recognition factor that functions early in the process of global genomic nucleotide excision repair. Regulation of XPC expression is governed in part by p53 at the transcriptional level. To identify the regulatory elements involved in the p53-dependent control of XPC expression, we performed a quantitative PCR tiling experiment using multiple regularly spaced primer pairs over an 11-kb region centered around the XPC transcriptional start site. p53 chromatin immunoprecipitation was performed following ultraviolet irradiation, and DNA was analyzed for enrichment at each of 48 amplicons covering this region. A segment just upstream of the XPC translational initiation site was significantly enriched, whereas no enrichment of any other region was noted. In vitro promoter reporter assays and gel retardation assays were used to confirm the p53 responsiveness of this region and to define the minimal region with stimulating activity. We identified a p53 response element that has significant similarity to a consensus sequence, with 3 mismatches. This response element is unique in that part of the p53 binding site included the coding sequence for the first 2 amino acids in the XPC protein.