BACKGROUND Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expression mechanism is not known. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed to elucidate the mechanism of transcriptional regulation of SMP30 gene. RESULTS We have characterized up to -2750 bp of the promoter by DNA-protein interactions studies. Twenty eight transcription factor binding sites have been identified by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Transient transfection of 5' and 3' -deleted promoter-reporter constructs and luciferase assay illustrated the region between -128/+157 bp is sufficient to drive promoter activity. We have mapped an essential regulatory region between -513 to -352 bp which causes a drastic decline of reporter activity. This region contains CdxA, GATA2 and SRY transcription factor binding sites. Individual mutation of these three sites showed increase in reporter activity. Mutation in SRY site (-403/-368) showed maximum increase in reporter activity among these three sites. Therefore, we suggest that SRY like protein may be acting as a strong repressor of SMP30 gene along with CdxA and GATA-2. We also report that mutation of both Sp1 (172/-148 bp) and a C/EBPbeta (-190/-177 bp) transcription binding site located adjacent to each other on SMP30 gene promoter, causes a significant enhancement in reporter activity than individual mutation, thus may be causing the repression of SMP30 promoter activity. CONCLUSION These studies provide novel insights into the mechanism that regulate SMP30 gene expression.

中文翻译：

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衰老标记蛋白-30 基因启动子的分子特征：阻遏元件和功能性核因子结合位点的鉴定。

背景衰老标志蛋白30（SMP30）在大鼠肝脏衰老过程中表达下降，已被认为是一种重要的衰老标志物。除了细胞凋亡，SMP30 还通过增强膜钙泵活性来保护细胞免受各种其他伤害。这种差异基因表达机制的机制尚不清楚。已经进行了 DNA-蛋白质相互作用、突变分析和荧光素酶报告基因检测研究，以阐明 SMP30 基因的转录调控机制。结果我们通过DNA-蛋白质相互作用研究表征了高达-2750bp的启动子。28 个转录因子结合位点已通过 DNase I 足迹和电泳迁移率变化测定 (EMSA) 确定。5' 和 3' 的瞬时转染 - 删除的启动子-报告基因构建体和荧光素酶测定表明 -128/+157 bp 之间的区域足以驱动启动子活性。我们已经绘制了一个介于 -513 到 -352 bp 之间的重要调控区域，这会导致记者活动急剧下降。该区域包含 CdxA、GATA2 和 SRY 转录因子结合位点。这三个位点的个别突变显示报告基因活性增加。SRY 位点 (-403/-368) 的突变显示这三个位点中报告基因活动的最大增加。因此，我们认为 SRY 样蛋白可能与 CdxA 和 GATA-2 一起充当 SMP30 基因的强阻遏物。我们还报告了 SMP30 基因启动子上彼此相邻的 Sp1 (172/-148 bp) 和 C/EBPbeta (-190/-177 bp) 转录结合位点的突变，导致报告基因活性比个体突变显着增强，因此可能导致 SMP30 启动子活性的抑制。结论 这些研究为调节 SMP30 基因表达的机制提供了新的见解。