人PRR11核心启动子区域NF-Y结合位点的定点突变分析

Site-directed Mutagenic Analysis of NF-Y-binding Sites in Human PRR11 Core Promoter

PRR11(proline-rich protein 11)是我们最近发现的一个新的肿瘤相关基因,在细胞周期和肿瘤发生等过程中起重要作用。该研究是在此前对PRR11启动子鉴定分析的基础上,对PRR11核心启动子区域中的核因子(nuclear factor Y,NF-Y)结合位点进行进一步的分析以确定其在PRR11转录调控中的作用。核苷酸序列同源性分析结果表明,PRR11核心启动子区域中的两个NF-Y结合位点在人、牛、大鼠和小鼠四个物种中均高度保守。共转染NF-Y表达质粒后,发现NF-Y的外源过表达可以明显提高PRR11的启动子活性。采用定点突变方法将PRR11启动子区域中的两个NF-Y结合位点单独或同时进行有效突变后,PRR11启动子活性明显下降,且NF-Y外源过表达对其启动子活性的激活效应也明显削弱甚至丧失。另外,对基因定点突变方法做出了改进,提出了一种更好的基于转录因子结合位点分析的碱基突变方法。这些结果表明,NF-Y结合位点是PRR11核心启动子区域中的重要的顺式作用元件,NF-Y可能通过调节PRR11的转录进而调节细胞周期和肿瘤发生等过程。

Proline-rich protein 11 （PRRll）, a novel tumor-associated gene discovered by our group, plays an important role in cell cycle and carcinogenesis, etc. Based on the previous identification and analysis of PRRll promotor, the present study focuses on determining the role of nuclear factor Y （NF-Y） binding sites in PRR11 core promoter region. Nucleotide sequence homology analysis indicated that, the human PRR11 gene promoter core sequence contains two NF-Y binding sites which are highly conservative in human, cow, rat and mouse. Co-trans- fection experiment revealed that ectopic over-expression of NF-Y could significantly increase the PRR11 promoter activity. However, efficient site-directed mutagenesis of each or both NF-Y-binding sites resulted in a significant decrease of PRRII promoter activity and a remarkable attenuation of PRRI1 promoter activation driven by the ectopic NF-Y over-expression. In addition, we also improved the conventional base-changing methods by combining the transcription factor binding prediction. Taken together, our present study strongly suggested that NF-Y binding sites are the important cis-acting elements in PRR11 core promoter region, and NF-Y might be involved in the cell cycle and carcinogenesis via regulating the transcription of human PRR11 gene.