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Doug Chung, Ricardo F Frausto, Stephan Chiu, Benjamin Lin, Anthony J Aldave; Characterization of ZEB1-dependent Regulation of COL4A3 Expression. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4896. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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 collagen, type IV, alpha3 (COL4A3) gene transcription via binding to putative E2 box motifs in the COL4A3 promoter region.
Putative E2 box motifs were identified by in silico analysis within the promoter region of COL4A3. Electrophoretic mobility shift assays were performed by incubating ZEB1WT-overexpressing cell lysates with DIG-labeled probes corresponding to each of the identified COL4A3 E2 box motifs. Competitive binding with unlabeled probes and anti-ZEB1 antibodies were performed to validate ZEB1-specific binding for each of the DIG-labeled E2 box probes. Dual-luciferase reporter assays were performed to test the effects of: ZEB1WT overexpression on the luciferase activity of COL4A3 promoter constructs that contain or are void of the identified E2 box motifs; and PPCD3-associated ZEB1 mutations on the activity of COL4A3 promoter constructs.
Nine E2 box motifs were identified by in silico analysis within the promoter region of COL4A3. An in vitro DNA binding assay demonstrated ZEB1 binding to six of the nine probes corresponding to the COL4A3 E2 boxes. Competitive binding against each ZEB1-bound E2 box probe using unlabeled probes and ZEB1 antibodies confirmed ZEB1-specific binding. The COL4A3 promoter reporter construct containing all E2 box motifs showed significant reduction in reporter gene expression with ZEB1 overexpression, while the expression of the reporter construct containing only the core promoter region and none of the identified E2 box motifs was unchanged.
DNA-protein binding studies indicate that ZEB1 is capable of binding several E2 box motifs in the promoter region of COL4A3. Thus, ZEB1 likely plays a regulatory role in COL4A3 expression, as evidenced by our demonstration that the expression of COL4A3 is negatively regulated by ZEB1 binding to E2 box motifs in the COL4A3 promoter region.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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