小鼠IRF-3基因核心启动子的鉴定及其调控元件的初步鉴定

发布时间：2018-05-02 15:06

本文选题：mIRF-3 + 启动子　；参考：《南京医科大学》2012年硕士论文

【摘要】：目的：克隆小鼠干扰素调节因子3（murine Interferon regulatory factor3，mIRF-3）基因启动子并寻找其核心功能区域，鉴定与mIRF-3基因启动子结合的转录因子，为阐明其表达调控机制奠定基础。方法：以小鼠NIH3T3细胞DNA为模板，采用PCR方法获取mIRF-3基因5’侧翼序列，测序正确后插入pGL3-Basic载体。运用双荧光素酶报告基因检测系统鉴定其启动子活性。采用缺失分析法分别从mIRF-3基因5’侧翼的5’端逐段缺失，克隆得到不同长度的截短片段，插入pGL3-Basic载体，使用双荧光素酶检测系统定位mIRF-3基因启动子核心功能区域，鉴定其启动子活性。利用点突变技术、染色质免疫沉淀（ChIP）和RNA干扰等实验，分析mIRF-3核心启动子区域的转录因子结合位点。结果：核酸序列测定结果显示，克隆到的mIRF-3基因5’侧翼序列与Genbank中记录完全一致，荧光素酶活性分析结果表明其在NIH3T3细胞中具有较强的启动子活性。结果表明，自5’端缺失的截短片段其活性与pGL3-Basic相比，表现为先升高后降低，-301～+1bp截短片段表现出接近最高的启动子活性，而-196～+1bp截短片段的活性基本消失，提示mIRF-3的核心启动子区域位于-301～-196bp之间。并且-301/-196区域间可能存在核心调控元件。生物信息学预测该区域包含了Sp1、IK1、E2F1、C-MYB、Egr2和YY1等结合位点。点突变结果表明Egr2和YY1具有维持mRF-3的基本转录活性的作用。RNA干扰实验表明Egr2和YY1增强了mIRF-3的启动子活性。染色质免疫沉淀(chromatin immunoprecitation，ChIP)实验结果表明YY1转录因子与mIRF-3的启动子区有结合。结论：mIRF-3基因的核心启动子区域位于转录起始位点上游-301与-196bp之间，Egr2和YY1在核心启动子调控方面具有关键的调控作用，，且二者起正调控作用。
[Abstract]:Aim: to clone the promoter of mouse interferon regulatory factor 3(murine Interferon regulatory factor3 mIRF-3 and to identify the transcription factors binding to the promoter of mIRF-3 gene, so as to lay a foundation for elucidating the mechanism of its expression and regulation. Methods: the 5 'flanking sequence of mIRF-3 gene was obtained by PCR using DNA of mouse NIH3T3 cells as template, and then inserted into pGL3-Basic vector after correct sequencing. The promoter activity was identified by double luciferase reporter gene detection system. Different length truncated fragments were cloned from 5'flanking end of mIRF-3 gene by deletion analysis and inserted into pGL3-Basic vector. The core functional region of promoter of mIRF-3 gene was located by double luciferase detection system. Its promoter activity was identified. Point mutation technique, chromatin immunoprecipitation and RNA interference were used to analyze transcription factor binding sites in the core promoter of mIRF-3. Results: the results of nucleic acid sequencing showed that the 5'flanking sequence of the cloned mIRF-3 gene was identical with that recorded in Genbank, and luciferase activity analysis showed that it had strong promoter activity in NIH3T3 cells. The results showed that the activity of the truncated fragment from 5 '-terminal deletion was higher than that of pGL3-Basic, and the activity of the truncated fragment from -301 to 1bp showed the highest promoter activity, while the activity of the truncated fragment of -196~ 1bp was almost disappeared. It suggested that the core promoter region of mIRF-3 was between -301 and 196 BP. And there may be core regulatory elements between-301 /-196 regions. Bioinformatics predicted that the region contained binding sites such as Sp1CIK1E2F1, C-MYBNEgr2 and YY1. The results of point mutation showed that Egr2 and YY1 could maintain the basic transcriptional activity of mRF-3. RNAi experiments showed that Egr2 and YY1 enhanced the promoter activity of mIRF-3. Chromatin immunoprecipitation assay revealed that YY1 transcription factors bind to the promoter region of mIRF-3. Conclusion the core promoter region of the 1: mIRF-3 gene is located between -301 and -196bp upstream of the transcription initiation site. Egr2 and YY1 play a key role in the regulation of the core promoter, and both play a positive role in the regulation of the core promoter.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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