内含子区的肥胖易感SNP通过影响增强子活性调控BCL2的作用机制研究

Obesity is a typical complex disease. Deciphering the molecular mechanism of susceptibility loci located in noncoding region remains a key problem in current studies for complex diseases. A previous genome-wide association study published in Nature suggested that rs12454712, which is located in the intron of BCL2, was associated with obesity. We also validated the association between rs12454712 and obesity related traits in Han Chinese population. However, its regulation mechanism is still unclear currently. Our previous epigenomic annotation results showed that rs12454712 was located in an enhancer region. Dual-luciferase reporter assay experiment confirmed the activity of the enhancer. Based on the preliminary results, we plan to carry out the following experiments: ① Integrate Expression Quantitative Trait Loci (eQTL) and Hi-C data analyses to predict the target gene, use dual-luciferase reporter assay and chromosome conformation capture to confirm the interaction between the enhancer and the target gene. ② Use the CRISPR/Cas9 system to establish SGBS cell lines with different genotypes and confirm the effect of the SNP on the target gene; ③ Predict the transcription factor and verify that different alleles of the SNP may affect the binding of transcription factor, leading to different target gene expression levels. In summary, we will integrate bioinformatics analyses and biological experiments to investigate the molecular mechanism of rs12454712 polymorphism on obesity phenotype. Our results may provide targets for individualized diagnosis and drug development. This study would also provide a reference avenue for the investigations on the molecular mechanism of noncoding variants.

肥胖症是典型的复杂疾病。破译非编码区易感位点的调控机制是复杂疾病研究的难点和关键。位于BCL2基因内含子区的rs12454712是Nature一项大规模全基因组关联研究发现的肥胖易感位点，我们在汉族群体中也验证到其与肥胖表型关联，然而其功能机制至今未知。我们前期通过表观调控元件注释发现该位点位于增强子区，双荧光报告酶系统实验也证实该区域具有增强子活性。本项目拟在此基础上：①结合表达数量性状基因座（eQTL）和染色质互作数据分析鉴定靶基因，并利用双荧光报告酶系统和3C实验验证；②利用CRISPR/Cas9系统构建不同基因型的人前体脂肪细胞系并检测其对靶基因的影响；③预测并验证该位点可能影响的与增强子结合的转录因子。本研究将结合生物信息学分析和生物学实验解析rs12454712多态性影响个体表型的调控机制，为疾病精准医疗与药物开发提供潜在的靶标，并为非编码区易感位点调控机制研究提供参考思路。

肥胖症是典型的复杂疾病，寻找其遗传易感性位点并研究作用机制可能为肥胖及相关疾病的治疗提供新的思路。然而目前发现的肥胖易感位点多位于非编码区，为后续转化带来了挑战。非编码区易感位点的调控机制也是复杂疾病研究的难点和关键。多项全基因组关联研究报道了BCL2基因内含子区的SNP rs12454712与体重指数校正后的腰臀比的显著相关性。我们在汉族人群中也验证到了该位点与肥胖表型的相关性，但是其作用机制尚不清楚。本项目在原有工作的基础上对该位点的调控机制进行了探索。.首先，分析显示rs12454712与其他位点不存在连锁不平衡（r2 > 0.8），且源自脂肪组织的表观数据注释显示该点位于增强子区，说明该位点是一个独立的功能性位点。其次，多个肥胖相关组织包括脂肪、肝脏、脑和肌肉的染色质互作数据分析表明该增强子区与BCL2的启动子区存在互作。脂肪组织表达数量性状基因座分析显示该位点不同的基因型与BCL2的表达水平显著相关（β = 0.13，P = 9.03 × 10-4），说明该位点的靶基因是BCL2。再次，双荧光报告实验显示rs12454712所在区域是一个增强子，且SNP不同的碱基型会影响增强子调控BCL2启动子表达的活性。特别地，rs12454712-C等位基因可与转录因子ZNF329结合，敲低ZNF329会显著降低BCL2的表达水平，而过表达ZNF329则会显著增加BCL2的表达水平。ZNF329的shRNA与带有C碱基的BCL2启动子增强子质粒共转时才显示出对BCL2表达水平的降低效果。这说明，rs12454712的碱基改变可能影响增强子与ZNF329的结合，进而影响BCL2的表达水平。综上，我们的研究结果表明rs12454712可能作为一个碱基特异性的增强子来影响其所在基因BCL2的表达。.本研究整合了生物信息学分析和功能学实验来探索肥胖非编码区易感位点的调控机制，该研究模式可为类似的研究提供可参考的思路。

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