Mingardo Enrico, Kalanithy Jeshurun C, Dworschak Gabriel, Ishorst Nina, Yilmaz Öznur, Lindenberg Tobias, Hollstein Ronja, Felger Tim, Angrand Pierre-Olivier, Reutter Heiko, Odermatt Benjamin
Institute of Anatomy and Cell Biology, Medical Faculty, University of Bonn, 53115, Bonn, Germany.
Institute of Human Genetics, Medical Faculty, University of Bonn, 53127, Bonn, Germany.
Sci Rep. 2024 Oct 2;14(1):22909. doi: 10.1038/s41598-024-74303-w.
Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D' = 1.0; R > 0.90) revealed marker rs2303751 (p value 8.12 × 10) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2 knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2 ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.
Isl1被描述为一种在泄殖腔周围间充质中表达的胚胎主控基因。在小鼠的生殖间充质中删除Isl1会导致异位尿道口和尿道上裂样表型。通过全基因组关联方法，我们确定ISL1是经典膀胱外翻（CBE，包括尿道上裂和膀胱外翻）的关键易感基因。在我们最近的全基因组关联研究荟萃分析中确定的最显著标记（rs6874700）在ISL1区域内的p值为1.48×10。对rs6874700以及与rs6874700处于连锁不平衡（LD）状态（D' = 1.0；R > 0.90）的所有其他全基因组显著标记进行的电子分析显示，标记rs2303751（p值8.12×10）是预测调控作用最高的标记。在此，我们描述了一个新的1.2 kb基因内启动子，它位于ISL1转录起始位点下游6.2至7.4 kb之间，位于反向DNA链上，并且在标记rs2303751的精确区域含有一个EZH2结合位点。我们发现，在HEK细胞中沉默EZH2会降低ISL1的表达。我们还发现，ezh2基因敲除（KO）的斑马鱼幼虫显示出Isl1调控的组织特异性，斑马鱼幼虫原肾区域的Isl1表达降低。此外，在ezh2基因敲除的斑马鱼Tg(wt1ß:eGFP)报告系中观察到肾管更短且畸形。我们的研究表明，Ezh2是尿路形成过程中Isl1的关键调节因子，并提示组织特异性ISL1失调是CBE形成的潜在机制。
