Padhy Biswajit, Hayat Bushra, Nanda Gargi Gouranga, Mohanty Pranjya Paramita, Alone Debasmita Pankaj
School of Biological Sciences, National Institute of Science Education and Research (NISER) Bhubaneswar, P.O. Jatni, Khurda, 752050, India, Homi Bhabha National Institute, Training school complex, Anushakti Nagar, Mumbai 400094, India.
JPM Rotary Club of Cuttack Eye Hospital and Research Institute, Cuttack 753014, India.
Hum Mol Genet. 2017 Nov 15;26(22):4519-4529. doi: 10.1093/hmg/ddx329.
Genetic variants at PTK2B-CLU locus pose as high-risk factors for many age-related disorders. However, the role of these variants in disease progression is less characterized. In this study, we aimed to investigate the functional significance of a clusterin intronic SNP, rs2279590, that has been associated with pseudoexfoliation, Alzheimer's disease (AD) and diabetes. We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 bp intronic region flanking the rs2279590 variant through CRISPR-Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele 'A' constitutes a transcription factor binding site for heat shock factor-1 (HSF1) but not with allele 'G'. By binding to allele 'A', HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus has a widespread enhancer effect on two nearby genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2); both of which have been previously associated with AD as risk factors. To summarize, our study unveils a mechanistic role of the common variant rs2279590 that can affect a variety of aging disorders by regulating the expression of a specific set of genes.
PTK2B-CLU基因座上的遗传变异是许多与年龄相关疾病的高危因素。然而，这些变异在疾病进展中的作用尚未得到充分表征。在本研究中，我们旨在探究一个与假性剥脱、阿尔茨海默病（AD）和糖尿病相关的聚类蛋白内含子单核苷酸多态性（SNP）rs2279590的功能意义。我们之前已经表明，rs2279590的等位基因在晶状体囊组织中差异调节聚类蛋白（CLU）基因的表达。这种多态性存在于一个以H3K27Ac和DNase I超敏位点为标志的活性调控区域，并且是CLU表达的一个表达数量性状位点（eQTL）。在此，我们报告rs2279590周围区域存在一个增强子元件。通过在HEK293细胞中进行CRISPR-Cas9基因组编辑，缺失rs2279590变异侧翼的115 bp内含子区域，结果显示聚类蛋白基因表达下降。电泳迁移率变动分析和染色质免疫沉淀试验表明，携带等位基因“A”的rs2279590构成热休克因子-1（HSF1）的转录因子结合位点，而携带等位基因“G”的则不然。通过与等位基因“A”结合，HSF1消除了该位点的增强子效应，这一点已通过报告基因试验得到验证。有趣的是，rs2279590位点对附近的两个基因——蛋白酪氨酸激酶2β（PTK2B）和环氧化物水解酶2（EPHX2）具有广泛的增强子效应；这两个基因之前都已被确定为AD的风险因素。总之，我们的研究揭示了常见变异rs2279590的机制作用，它可以通过调节一组特定基因的表达来影响多种衰老相关疾病。
