Padhy Biswajit, Kapuganti Ramani Shyam, Hayat Bushra, Mohanty Pranjya Paramita, Alone Debasmita Pankaj
School of Biological Sciences, National Institute of Science Education and Research (NISER) Bhubaneswar, P.O. Bhimpur-Padanpur, Jatni, Khurda, Odisha 752050, India; Homi Bhabha National Institute (HBNI), Training School Complex, Anushaktinagar, Mumbai 400094, India.
Sri Sri Borda Hospital, Dhauli, Bhubaneswar, Odisha 751002, India.
Gene. 2023 Feb 20;854:147096. doi: 10.1016/j.gene.2022.147096. Epub 2022 Dec 5.
Polymorphisms in the PTK2B-CLU locus have been associated with various neurodegenerative disorders including pseudoexfoliation glaucoma, Alzheimer's and Parkinson's. Many of these genomic variants are within enhancer elements and modulate genes associated with the disease pathogenesis. However, mechanisms by which they control the gene expression is unknown. Previously, we have shown that clusterin enhancer element surrounding rs2279590 intronic variant, a risk factor in the pathogenesis of pseudoexfoliation glaucoma modulates gene expression of clusterin (CLU), protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase 2 (EPHX2). Here, we explored the mechanism by which rs2279590 enhancer regulates their gene expression through chromosome conformation capture assays. 3C assays revealed a strong enhancer-promoter chromatin interaction between rs2279590 enhancer and promoters of genes CLU, PTK2B and EPHX2 in the HEK293 wild type cells. Moreover, genomic knockout of rs2279590 element significantly decreases the chromatin-chromatin cross-linking frequency suggesting gene regulation at transcriptional level through formation of chromatin loop. In addition, molecular assays showed a significantly decreased expression of EPHX2 but not PTK2B at both mRNA and protein level in the lens capsule of pseudoexfoliation affected patients in comparison to control subjects implying a role of EPHX2 in the pathogenesis of pseudoexfoliation.
PTK2B - CLU基因座中的多态性与多种神经退行性疾病相关，包括假性剥脱性青光眼、阿尔茨海默病和帕金森病。这些基因组变异中的许多位于增强子元件内，并调节与疾病发病机制相关的基因。然而，它们控制基因表达的机制尚不清楚。此前，我们已经表明，围绕rs2279590内含子变异的簇集蛋白增强子元件，这是假性剥脱性青光眼发病机制中的一个危险因素，可调节簇集蛋白（CLU）、蛋白酪氨酸激酶2β（PTK2B）和环氧水解酶2（EPHX2）的基因表达。在此，我们通过染色体构象捕获分析探索了rs2279590增强子调节其基因表达的机制。3C分析揭示了在HEK293野生型细胞中，rs2279590增强子与CLU、PTK2B和EPHX2基因的启动子之间存在强烈的增强子 - 启动子染色质相互作用。此外，rs2279590元件的基因组敲除显著降低了染色质 - 染色质交联频率，表明通过染色质环的形成在转录水平上进行基因调控。此外，分子分析显示，与对照受试者相比，假性剥脱性青光眼患者晶状体囊膜中EPHX2在mRNA和蛋白质水平的表达均显著降低，但PTK2B没有，这意味着EPHX2在假性剥脱性青光眼的发病机制中起作用。
