猪ESRRB启动子克隆及其调控活性检测

Cloning and regulation of pig estrogen related receptor β gene (ESRRB) promoter

摘要:

Esrrb (Estrogen related receptor β) 属于雌激素受体家族，是一类在胚胎早期外胚层细胞中表达并对干细胞多能性维持起重要作用的基因。为了探索猪ESRRB的表达和转录调控机制，克隆了3.3 kb ESRRB启动子片段，构建了相应的报告载体。并将报告载体分别转染293T人胚肾细胞、Hela人宫颈癌细胞和小鼠C2C12成肌细胞。通过TFSEARCH和JASPER方法对ESRRB启动子潜在的转录调控位点进行分析，发现该启动子上有SMAD、STAT3、MYC、KLF4等多能转录因子的结合位点。将相应的转录因子与ESRRB启动子共转染，并检测报告基因荧光素酶的活性。结果显示猪ESRRB启动子具有明显的组织特异性调控，同时SMAD对ESRRB启动子活性有较明显的调控作用。进一步对3.3 kb片段进行了一系列的缺失，发现猪ESRRB核心区域位于5′上游的?25 bp和?269 bp之间。研究结果表明猪ESRRB启动子上潜在的转录因子结合位点及启动子核心区域是参与调控ESRRB表达的重要序列。

Abstract:

The estrogen related receptor family member Esrrb (Estrogen related receptor β) is a gene that expresses in the early stage of embryo and plays an important role in the core pluripotent network. Its function has been analyzed in human and mouse, although no report so far related to pig. Therefore, to explore its mechanism of transcriptional regulation and expression pattern, we cloned a 3.3 kb pig ESRRB promoter by PCR and constructed the green fluorescence protein (GFP) reporter vector pE3.3. We used these vectors to study the ESRRB expression pattern in 293T, H+L2ela+L2 and C2C12. Sequence was analyzed for regulatory elements that share homology to known transcription factor binding sites by TFSEARCH and JASPER program. Some pluripotency related genes such as SMAD, STAT3, MYC, KLF4 and ESRRB have been found within the 3.3 kb sequence by co-transfected pig ESRRB promoter and these potential regulators. We found that ESRRB only expressed in 293T and SMAD could activate ESRRB expression obviously. To determine the core promoter region, a series of ESRRB promoter fragments with gradually truncated 5?-end were produced by PCR and inserted into pGL3-Basic vector. After transient transfection into 293T, dual luciferase assay was used to measure these promoter activities. The result suggested that the core promoter of pig ESRRB located within ?25 bp to ?269 bp region. These results suggest that these transcription factor binding sites and the core promoter region may be essential for transcriptional regulation of pig ESRRB gene.