Abstract
PURPOSE: To investigate the role of the zinc finger e-box binding homeobox 1 (ZEB1) transcription factor in posterior polymorphous corneal dystrophy 3 by demonstrating its ability to regulate type IV collagen gene transcription via binding to putative E2 box motifs. METHODS: Putative E2 box motifs were identified by in silico analysis within the promoter region of collagen, type IV, alpha3 (COL4A3) and collagen, type IV, alpha4 (COL4A4). To test the ability of ZEB1 to bind to each identified E2 box, electrophoretic mobility shift assays were performed by incubating ZEB1-enriched nuclear extracts with DIG-labeled probes containing one of each of the identified E2 box motifs. Dual-luciferase reporter assays were performed to test the effects of ZEB1 on the luciferase activity of COL4A3 and cadherin 1 (CDH1) promoter constructs, and to determine the effect of a ZEB1 truncating mutation on CDH1 promoter activity. RESULTS: ZEB1 exhibited binding to six of the nine COL4A3 E2 box probes, whereas no binding was observed for either of the two COL4A4 E2 box probes. ZEB1 overexpression resulted in reduced activity of the COL4A3 promoter construct containing all identified E2 box motifs, whereas a truncating ZEB1 mutation led to the loss of ZEB1-dependent repression of the CDH1 promoter. CONCLUSIONS: COL4A3 gene expression is negatively regulated by ZEB1 binding to E2 box motifs in the COL4A3 promoter region. Therefore, the altered expression of type IV collagens, particularly COL4A3, in the corneal endothelium in individuals with PPCD3 is likely due to reduced transcriptional repression in the setting of a single functional ZEB1 allele.