GATA-1乙酰化对WNK4基因表达调控机制的研究

发布时间：2018-05-12 23:43

本文选题：WNK4 + 基因表达　；参考：《中国医科大学》2008年硕士论文

【摘要】： 前言 WNK4(with no lysine[K]kinase-4)基因是新近分离克隆的一个蛋白激酶基因,该基因第7外显子(exon7)和第17外显子(exon17)突变可导致常染色体显性遗传性疾病,假性低醛固酮血症(Pseudohypoaldosteronism typeⅡ,PHAⅡ)。研究表明WNK4可以作用于肾脏远曲小管和集合管上的离子转运体(Na-Cl共转运体,NCCT)和离子通道(钾离子通道,ROMK),从而起到对血压和离子平衡的调控作用。目前大量的研究集中在探索WNK4基因的功能上,但WNK4基因的上游调控因子仍不清楚。乙酰化在多种基因的表达调控中发挥重要作用,两类作用相反的酶,组蛋白乙酰转移酶(HAT)和组蛋白去乙酰化酶(HDAC)参与调节蛋白质乙酰化状态。曲古抑菌素A(TSA)能够特异性抑制HDAC活性从而引起组蛋白和一些转录相关因子的乙酰化。迄今为止尚无有关乙酰化对WNK4基因表达调控的研究。本研究以TSA作为乙酰化作用因素,探索乙酰化对人胚肾HEK293细胞中WNK4基因rnRNA和蛋白表达水平的影响,并鉴定WNK4启动子区的GATA-1反应元件及TSA对GATA-1蛋白乙酰化水平及DNA亲和力和WNK4启动子活性的影响,从而探索乙酰化调节WNK4基因表达的机制。材料与方法一、实验材料 1、人胚肾HEK293细胞系 2、胚胎(胎龄22周)肾脏组织标本 3、Real-time PCR及Western印迹杂交相关试剂 4、基因克隆及萤光素酶报告基因检测相关分子生物学试剂 5、电泳泳动迁移实验(EMSA)、染色质免疫沉淀(ChIP)及免疫沉淀(IP)相关试剂二、实验方法 1、Real-time RT-PCR检测TSA刺激前后WNK4 mRNA表达水平的变化。 2、Western印迹杂交检测TSA对WNK4蛋白表达水平的影响。 3、应用软件及萤光素酶报告基因系统分析WNK4启动子结构。 4、RT-PCR实验检测GATA-1mRNA在HEK293细胞及胚胎肾脏组织中的表达。 5、EMSA及ChIP鉴定WNK4启动子内GATA-1反应元件及TSA对GATA-1蛋白与该位点结合力的影响。 6、免疫沉淀结合Western印迹杂交检测TSA对GATA-1蛋白乙酰化水平的影响。 7、萤光素酶报告基因检测GATA-1反应元件在TSA对WNK4启动子活性影响中的作用。结果 1、Real-time RT-PCR结果显示TSA以浓度依赖方式上调WNK4 mRNA表达水平。 2、Western印迹杂交表明TSA诱导的乙酰化使HEK293细胞中WNK4蛋白水平显著增加。 3、软件分析及萤光素酶报告基因检测在WNK4启动子内发现了一些潜在的受乙酰化调控的转录因子结合位点。 4、RT-PCR的结果提示GATA-1 mRNA在HEK293细胞及胚胎肾脏组织中有明显的表达。 5、EMSA及ChIP实验在分别在体外及体内条件下证明了WNK4启动子-426～-416区为GATA-1反应元件,并且TSA使GATA-1蛋白与该位点结合增强。 6、免疫沉淀结合Western印迹杂交显示TSA可直接引起GATA-1蛋白的乙酰化。 7、应用萤光素酶报告基因系统证明了GATA-1反应元件是WNK4启动子中一个强大的正调控元件,同时它在TSA诱导的WNK4启动子的转录激活中发挥重要作用。结论 1、WNK4启动子-426～-416区为GATA-1反应元件。 2、TSA引起的GATA-1乙酰化可增强其与WNK4基因启动子GATA-1反应元件的结合,从而上调WNK4启动子转录活性,这是乙酰化调节WNK4基因表达的一种机制。
[Abstract]:Preface
WNK4 (with no lysine[K]kinase-4) gene is a protein kinase gene of newly isolated clones. The gene seventh exon (exon7) and seventeenth exon (EXON17) mutation can lead to autosomal dominant hereditary disease, pseudoaldosterone (Pseudohypoaldosteronism type II, PHA II). The study shows that WNK4 can act on renal distal curvature. The ion transporters (Na-Cl co transporters, NCCT) and ion channels (potassium channels, ROMK) on the tubules and collecting tubes play a role in regulating blood pressure and ion balance. A large number of studies have focused on the exploration of the function of the WNK4 gene, but the upstream regulator of the WNK4 gene is still unclear.
Acetylation plays an important role in the regulation of the expression of a variety of genes. Two types of enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC), are involved in the regulation of protein acetylation. Tacopalpsin A (TSA) can specifically inhibit the activity of HDAC and cause the acetylation of histone and some transcriptional related factors. So far, there is no study about the regulation of acetylation on WNK4 gene expression.
In this study, TSA was used as a factor of acetylation to explore the effect of acetylation on the expression level of WNK4 gene rnRNA and protein in human embryonic kidney HEK293 cells, and to identify the effect of GATA-1 reaction elements and TSA on the level of acetylation of GATA-1 protein and the activity of DNA affinity and WNK4 promoter in WNK4 promoter region, so as to explore the WNK4 gene of acetylation. The mechanism of expression.
Materials and methods
First, experimental materials
1, human embryonic kidney HEK293 cell line
2, fetal (22 weeks of gestational age) kidney tissue specimen
3, Real-time PCR and Western blot related reagents
4, molecular cloning and detection of luciferase reporter gene related molecular biological agents.
5, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and immunoprecipitation (IP) related reagents.
Two, experimental method
1, Real-time RT-PCR was used to detect the change of WNK4 mRNA expression level before and after TSA stimulation.
2, Western blot hybridization was used to detect the effect of TSA on the expression level of WNK4 protein.
3, application software and luciferase reporter gene system were used to analyze the structure of WNK4 promoter.
4, RT-PCR assay was used to detect the expression of GATA-1mRNA in HEK293 cells and embryonic kidney tissues.
5, EMSA and ChIP identified the effects of GATA-1 response elements in WNK4 promoter and TSA on the binding capacity of GATA-1 protein to this locus.
6, immunoprecipitation combined with Western blot hybridization was used to detect the effect of TSA on the acetylation level of GATA-1 protein.
7, luciferase reporter gene was used to detect the role of GATA-1 response element in the effect of TSA on WNK4 promoter activity.
Result
1, Real-time RT-PCR results showed that TSA increased the level of WNK4 mRNA expression in a concentration dependent manner.
2, Western blot hybridization showed that TSA induced acetylation significantly increased the level of WNK4 protein in HEK293 cells.
3, software analysis and luciferase reporter gene detection revealed potential binding sites for transcription factors binding to acetylation in the WNK4 promoter.
4, the results of RT-PCR suggest that GATA-1 mRNA has obvious expression in HEK293 cells and embryonic kidney tissues.
5, EMSA and ChIP experiments showed that the WNK4 promoter -426 ~ -416 region was a GATA-1 reaction element in vitro and in vivo, and TSA enhanced the binding of GATA-1 protein to the site.
6, immunoprecipitation combined with Western blot hybridization showed that TSA could directly cause acetylation of GATA-1 protein.
7, the use of the luciferase reporter gene system demonstrated that the GATA-1 reaction element is a powerful positive regulatory element in the WNK4 promoter, and it plays an important role in the transcription activation of the WNK4 promoter induced by TSA.
conclusion
1, the WNK4 promoter -426 to -416 region is the GATA-1 reaction element.
2, TSA induced GATA-1 acetylation can enhance its binding with the WNK4 gene promoter GATA-1 reaction element, thereby up-regulation the WNK4 promoter transcriptional activity, which is a mechanism for acetylation to regulate the expression of WNK4 genes.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R394

【共引文献】