Epigenetic and Transcriptional regulation of the Human Angiotensinogen Gene by High Salt

Hypertension is caused by a combination of genetic and environmental factors. Angiotensinogen (AGT) is a component of RAAS, that regulates blood pressure. The human AGT (hAGT) gene has -6A/-6G polymorphism and -6A variant is associated with human hypertension. In this study, we have investigated the epigenetic regulation of the hAGT gene. To understand transcriptional regulation of the hAGT, we have made transgenic animals containing -6A. We show that HS affects DNA methylation and modulates transcriptional regulation of this gene in liver and kidney. HS increases hAGT gene expression in -6A TG mice. We have observed that the number of CpG sites in the hAGT promoter is decreased after HS treatment. In the liver, four CpG sites are methylated whereas after HS treatment, only two CpG sites observed. In the kidney, five CpG sites are methylated, whereas after HS treatment, only three CpG sites are observed. These results suggest that HS promotes DNA demethylation and increasing AGT gene expression. RT-PCR and immunoblot analysis show that hAGT gene expression is increased after HS treatment. Chip assay has shown that transcription factors bind strongly after HS treatment. RNA-Seq analysis identified differentially expressed genes, canonical pathways, upstream regulators. One of the plausible mechanisms for HS induced up-regulation of the hAGT gene is through IL-6/JAK/STAT3/AGT axis. Overall design: To understand the epigenetic and transcriptional regulation of hAGT gene we have made transgenic animals containing -6A. We gave high salt to mice and collected liver and kidney tissues. We isolated RNA and did RNA Seq analysis in liver and kidney tissues. We then performed gene expression profiling analysis using data obtained from RNA-seq. We further identified top canonical pathways, top diseases and functions and upstream regulators by using Ingenuity Pathway Analysis.

高血压由遗传与环境因素共同诱导产生。血管紧张素原（Angiotensinogen, AGT）是肾素-血管紧张素-醛固酮系统（RAAS）的组成组分，该系统参与血压的调控过程。人类AGT（hAGT）基因存在-6A/-6G多态性，其中-6A型变异与人类高血压密切相关。本研究针对hAGT基因的表观遗传调控机制展开了探究。为阐明hAGT的转录调控规律，我们构建了携带-6A等位基因的转基因动物。研究结果表明，高盐（High Salt, HS）处理可影响DNA甲基化水平，并调控该基因在肝脏与肾脏中的转录活性。高盐处理可上调-6A型转基因小鼠的hAGT基因表达水平。我们观察到，经高盐处理后，hAGT基因启动子区域的甲基化CpG位点数量有所减少。在肝脏组织中，原本存在4个甲基化CpG位点，而高盐处理后仅可检测到2个甲基化CpG位点；在肾脏组织中，原本存在5个甲基化CpG位点，高盐处理后仅可检测到3个甲基化CpG位点。上述结果提示，高盐处理可促进DNA去甲基化，进而提升AGT基因的表达水平。逆转录聚合酶链反应（RT-PCR）与免疫印迹分析结果显示，高盐处理后hAGT基因的表达水平显著升高。染色质免疫沉淀（ChIP）实验证实，高盐处理后转录因子的结合活性显著增强。RNA测序（RNA-Seq）分析鉴定出了差异表达基因、经典通路与上游调控因子。高盐诱导hAGT基因上调的潜在机制之一，是通过IL-6/JAK/STAT3/AGT信号轴实现的。

实验整体设计：为阐明hAGT基因的表观遗传与转录调控机制，我们构建了携带-6A等位基因的转基因小鼠。向小鼠饲喂高盐饲料后，采集其肝脏与肾脏组织。提取组织总RNA并开展肝脏、肾脏组织的RNA测序分析。基于RNA测序获得的数据，我们进行了基因表达谱分析。进一步通过Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）鉴定出了核心经典通路、核心疾病与功能模块以及上游调控因子。