骨质疏松症易感区域1p36.12非编码SNP通过增强子远程调控靶基因的机制研究

Since more than 90% SNPs identified by genome-wide association studies (GWAS) are located in non-coding regions, it becomes difficult and important to discover the underlying mechanism of non-coding SNPs - target genes - diseases. 1p36.12 is a susceptibility locus firstly identified by large-scale GWAS on osteoporosis in white population. We have also validated the significant associations in Chinese population. However, most susceptibility SNPs in this locus are in intergenic region, the function of these SNPs remains unknown. Our preliminary study have shown that, SNPs in 1p36.12 could act as enhancer to regulate a long non-coding RNA (LINC00339) through long-range loop formation. Based on these work, we propose our plan: first, we will conduct a comprehensive bioinformatics analyses on susceptibility SNPs in 1p36.12 through integrating multiple epigenomic regulatory data, gene expression data, and three-dimension genomic data. Second, we will validate the results by conducting various functional experiments in cellular and mouse model, such as luciferase assay, chromosome conformation capture (3C), CRISPR/Cas9, and so on. Finally, we will investigate the relationship between LINC00339 and its target gene CDC42, which is an important gene involved in bone metabolism. Our aim is to identify the long-range regulation pattern of “non-coding susceptibility SNPs – disease”. The completion of this project will provide potential effective target for precision medicine and drug development.

全基因组关联研究（GWAS）发现的疾病易感SNP位点90%以上位于非蛋白编码区，破解从非编码SNP到靶标基因到疾病的内在关系是当前研究的关键和难点。1p36.12是国际大规模GWAS报道的第一个白人骨质疏松易感区域，申请人在中国人中也验证到显著关联，然而，该区域的易感SNP多位于基因间隔区，功能机制至今未知。我们前期研究提示，该易感SNP区域可作为增强子远程调控长链非编码RNA（LINC00339）的表达。本项目拟在此基础上，通过整合各类表观调控、基因表达和三维基因组数据，对1p36.12上易感SNP进行系统生物信息学分析，并结合双荧光报告系统、3C、CRISPR/Cas9等各类功能学实验在细胞和小鼠上验证，同时研究LINC00339对其Cis调控的骨代谢重要基因CDC42的作用机制，旨在深入解析“非编码易感SNP—疾病”的远程调控模式。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。

GWAS研究发现1p36.12是骨质疏松的重要易感区，但该区域的易感位点均位于基因组非编码区，真正的功能性易感位点及分子调控机制未知。本项目通过整合基因组、转录组、表观组合三维基因组数据，进行系统生信息学分析，并开展多种功能实验验证，致力于破译“非编码易感SNP-骨质疏松”的远程调控机制。我们成功鉴定出1个功能性易感SNP（rs6426749），作为等位基因特异型增强子（rs6426749-G），结合转录因子TFAP2A，激活增强子元件活性，与长链非编码RNA基因LINC00339的启动子形成染色质远程互作，来增强LINC00339的表达，CTCF蛋白在远距离成环过程中发挥重要作用。进一步研究发现，LINC00339主要表达在细胞质中，会抑制成骨细胞分化。我们发现，LINC00339作为支架，可招募蛋白PARP1，对骨质疏松的重要明星基因CDC42具有负调控效应。综上，本研究完整解析了一个“非编码易感SNP-LINC00339-CDC42-骨质疏松症”的远程调控新模型，证明了非编码区易感SNP对于骨质疏松症发病机制的重要调控作用，可为骨质疏松的精准医疗和药靶开发提供潜在有效靶标。同时，本项目的研究模式也可为其它复杂疾病非编码区易感SNP功能研究提供研究参考和有效手段。在本项目的支持下，现已发表 SCI 收录论文 11篇，其中第一标注的 SCI 收录论文4篇，包括遗传学领域权威期刊American Journal of Human Genetics和内分泌领域顶尖期刊Nature Reviews Endocrinology，中文核心期刊2篇，授权发明专利1项。