Non-Coding Variants of Angiotensinogen Gene and Hypertension

 描述（由申请人提供）：高血压是心肌梗死、心力衰竭、血管疾病、中风和肾衰竭的严重风险因素。肾素-血管紧张素系统（RAS）在血压调节中起重要作用。最近的全基因组关联研究（genome wise association studies，GWAS）表明，位于人血管紧张素原（angiotensinogen，hAGT）基因内含子-I +1164的A/G SNP（rs 2004776）与高血压相关。含+1164A等位基因的hAGT基因的核苷酸序列与HNF-3结合位点的同源性较+1164G高。HNF 3家族属于“先锋”转录因子，其与启动子和增强子的结合使得其他组织特异性转录因子能够进入染色质。hAGT基因的启动子和内含子I中的SNPs可以分为2种主要的单倍型：单倍型-I（含有+1164 A）和II（含有+1164 G）。与单体型II相比，单体型I中的核苷酸变体更强地结合转录因子，并且在瞬时转染时，与单体型II相比，含有单体型I的报告构建体具有增加的启动子活性。通过在HPRT基因座处敲入方法产生含有hAGT基因的单倍型I或II的转基因小鼠。初步研究表明：（a）与单倍型II相比，含有单倍型I的TG小鼠的hAGT启动子中的CpG二核苷酸是低甲基化的并且可接近转录因子，（B）与单倍型II相比，含有单倍型I的雄性TG小鼠在肝、肾和脂肪中具有增加的基础和GR诱导的hAGT基因表达，（c）来自含有单倍型-I的转基因动物的肝和肾的染色质与单倍型-II相比更强地结合HNF 3 β、C/EBPβ、STAT-3和GR，（d）含有hREN基因和hAGT基因的单倍型-I的双TG小鼠与单倍型-II相比具有增加的BP，其通过含有高碳水化合物：高脂肪：高盐饮食。在本申请中，将通过DNA甲基化测定、染色体构象捕获（3C）测定、ChIP测定和瞬时转染测定来分析内含子-I中+1164处的SNP和其它顺式作用DNA元件对hAGT基因的转录调控的作用。将使用含有hREN基因和hAGT基因的单倍型I或单倍型II的雄性/雌性双转基因小鼠分析西方饮食对hAGT基因表达、血压调节和终末器官损伤的影响。

 DESCRIPTION (provided by applicant): Hypertension is a serious risk factor for myocardial infarction, heart failure, vascular disease, stroke, and renal failure. The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure (BP). Recent genome wise association studies (GWAS) have shown that an A/G SNP (rs2004776) located at +1164 in intron-I of the human angiotensinogen (hAGT) gene is associated with hypertension. The nucleotide sequence of hAGT gene containing +1164A allele has stronger homology with HNF-3 binding site as compared to +1164G. HNF3 family belongs to "pioneer" transcription factors whose binding to promoters and enhancers enables chromatin access for other tissue-specific transcription factors. SNPs in the promoter and intron I of the hAGT gene can be divided in 2 major haplotypes: haplotype-I (containing +1164A) and II (containing +1164G). Nucleotide variants in haplotype-I bind more strongly to transcription factors as compared to haplotype-II and reporter construct containing haplotype-I has increased promoter activity as compared to haplotype-II on transient transfection. Transgenic mice containing either haplotype-I or II of the hAGT gene were generated by knock-in approach at the HPRT locus. Preliminary studies have shown that: (a) CpG dinucleotides in the hAGT promoter of TG mice containing haplotype- I are hypo-methylated and accessible to transcription factors as compared to haplotype-II, (b) male TG mice containing haplotype-I have increased basal and GR induced expression of the hAGT gene in liver, kidney and fat as compared to haplotype-II, (c) chromatin from liver and kidney of transgenic animals containing haplotype-I binds more strongly to HNF3β, C/EBPβ, STAT-3 and GR as compared to haplotype-II, (d) double TG mice containing hREN gene and haplotype-I of the hAGT gene have increased BP as compared to haplotype-II which is further increased by Western diet containing high carbohydrate: high fat: high salt diet. In the present application, th role of SNP at +1164 and other cis-acting DNA elements in intron-I on transcriptional regulation of the hAGT gene will be analyzed by DNA methylation assay, chromosome conformation capture (3C) assay, ChIP assay and transient transfection assay. The effect of Western diet on the expression of the hAGT gene, blood pressure regulation, and end organ damage will be analyzed using male/female double transgenic mice containing hREN gene and either haplotype-I or haplotype-II of the hAGT gene.