EZH2 specifically regulatesISL1during embryonic urinary tract formation

Abstract

AbstractIsl1has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion ofIsl1from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identifiedISL1as 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 apvalue of 1.48 × 10-24within theISL1region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D’ = 1.0; R2> 0.90) revealed marker rs2303751 (pvalue 8.12 x 10-20) 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 theISL1transcription starting site, which is located in the reverse DNA strand and harbors a binding side for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reducesISL1expression. We show thatezh2-/-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 inezh2-/-ko zebrafishTg(wt1ß:eGFP)reporter lines. Our study shows, that Ezh2 is a key regulator ofIsl1during urinary tract formation and suggests tissue specificISL1dysregulation as an underlying mechanism for CBE formation.