整合染色质三维构象和高通量实验系统解析肥胖基因组非编码易感SNP的增强子调控机制

Obesity is a common complex disease worldwide. Genome-wide association studies have successfully identified over 600 susceptibility SNPs for obesity. However, more than 90% of these SNPs are located in the non-coding region. Therefore, using effective approach to decipher the molecular mechanism from SNP to target genes to phenotype is the current urgent task. In our preliminary study, we have annotated all the susceptibility SNPs for obesity, and identified 24,623 potential functional SNPs. Among which, most of them are located in the enhancers or regions binding by active histone modification markers. Based on this, we will change the traditional “single point” research model. Instead, we will integrate multiple-omics data and high-throughput experiments to systematically analyze enhancer regulatory mechanisms of non-coding susceptibility SNPs for obesity. We have successfully constructed the chromosome 3D conformation map in adipocytes and built the STARR-seq experimental platform. Moreover, we will integrate the genomics, gene expression, and epigenomics data to systematically build the “noncoding SNP-regulatory elements-target genes” model. Then, we will conduct various functional experiments, such as dual-luciferase reporter assay, 3C/4C, CRISPR/CRISPRi, to deeply explore the mechanism of key regulatory elements and target genes. The completion of this project will provide potential effective targets for individualized diagnosis and drug development.

肥胖症是全球化多基因复杂疾病，全基因组关联研究成功鉴定出六百余个肥胖易感位点，但90%以上位于基因组非编码区，当前亟需采取有效措施，破解从非编码易感SNP到靶基因到疾病的调控机制。我们前期通过对所有肥胖位点功能注释，鉴定出24,623个潜在的功能性SNP，其中大部分位于增强子或激活型组蛋白修饰区。本项目拟在此基础上，改变传统单点研究模式，结合多层次组学数据和高通量实验技术，系统性研究非编码易感SNP的调控机制。我们已成功构建了人脂肪细胞染色质三维构象图谱，并搭建了高通量检测增强子的STARR-Seq实验平台，另外还将整合基因组、基因表达、表观调控等各类数据，系统地建立肥胖“非编码易感SNP-调控元件-靶基因”的调控模式，并利用双荧光报告系统、3C/4C、CRISPR/CRISPRi等各类功能学实验，深入解析关键调控元件和靶基因的作用机制。研究结果可为疾病精准医疗和药物开发提供潜在有效靶标。

肥胖及其相关表型危害巨大，是II型糖尿病、冠状动脉疾病等多种复杂疾病的重要诱因。全基因组关联分析已经鉴定了上千个肥胖及其相关表型的易感位点，然而大部分易感变异位点位于基因组非编码区。破解疾病非编码区易感变异的功能机制是阐明疾病致病机制的关键。大部分非编码易感位点位于基因组增强子或激活型组蛋白修饰区，因此从表观遗传学角度解析易感位点功能是重要突破口。以往研究均是基于单个位点的研究，效率低。本项目利用高通量报告分析系统STARR-seq技术（自转录激活调控区域测序），并结合多组学数据和功能学实验，系统性解析肥胖及其相关表型非编码区易感SNP的调控活性及功能。..我们利用STARR-seq实验系统性鉴定了6,014个肥胖及其相关表型非编码SNP的调控活性。在3种靶细胞中，分别鉴定了779（HepG2）、678（前脂肪细胞）和587（A673）个具有碱基特异性增强子活性的SNP片段。通过整合细胞特异性表观组、转录组、三维基因组等多组学数据和STARR-seq结果，开发了一套疾病相关SNP功能解析的方法fnGWAS，实现SNP -靶基因-疾病遗传调控网络的构建。最后，结合分子及细胞生物学实验深入探究了一个功能性SNP rs952227-A通过结合转录因子HOXC6对靶基因IRS1进行远程调控的分子机制。..本研究系统性地鉴定了肥胖及其相关表型非编码易感SNP的调控活性，并整合多组学数据构建SNP调控元件-靶基因-疾病遗传调控网络，深入解析了关键调控元件和靶基因的作用机制。研究结果可为疾病精准医疗和药物开发提供潜在有效靶标，为后GWAS时代解析非编码区疾病易感SNP的作用机制提供了新的研究思路。