人Ska2/FAM33A核心启动子区域的初步鉴定与分析

Preliminary Identification and Characterization of Human Ska2 Core Promoter Region

Ska2(spindle and KT associated 2),也称FAM33A(family with sequence similarity 33,member A),是新近发现的一个与细胞周期调控和肿瘤发生发展均密切相关的新基因,但其表达调控机制仍不清楚。该研究是在此前对Ska2基因启动子鉴定分析的基础上,进一步对Ska2基因的核心启动子区域进行初步鉴定和分析。采用PCR技术,构建5个Ska2基因启动子的系列删除体,并对Ska2核心启动子区中的2个潜在NF-Y结合位点进行单独或联合定点突变,构建3个定点突变重组体,采用荧光素酶双报告基因分析技术检测各重组体的启动子活性。启动子活性分析结果表明,所构建删除体均具有较强启动子活性;2个潜在NF-Y结合位点单独或联合定点突变均可导致Ska2启动子活性的降低;共转染NF-Y表达质粒后,Ska2的启动子活性明显提高。该研究结果不仅将Ska2的核心启动子区域定位于一个80 bp的范围内,并且也提示NF-Y是一个调节Ska2转录的重要转录因子。

Ska2 （spindle and KT associated 2）, also known as FAM33A （family with sequence similarity 33, member A）, is a recently discovered novel gene involved in both cell cycle regulation and tumorigenesis. The molecular regulatory mechanism of its expression, however, remains unclear. We have previously identified its main promoter region, thus the present study was designed to further identify and characterize its core promoter region. PCR was used to make five different deletion mutants of Ska2 promoter reporter. Either or both of the two potential NF-Y binding sites were mutated to make three Ska2 mutant promoter reporters using PCR based site- directed mutagenesis. Dual luciferase reporter assay was used to determine promoter reporter activity. The results of promoter activity analysis showed that the five deletion mutants showed significant promoter activity, and site- directed mutagenesis of either or both NF-Y binding sites resulted in decreased promoter activity. Additionally, ectopic overexpression of NF-Y enhanced the Ska2 promoter activity. Taken together, our results suggested that thecore promoter region ofSka2 gene is mainly located in a 80 bp region and NF-Y might play an important role in the transcriptional regulation of Ska2 gene.