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Doug Chung, Ricardo F Frausto, Anthony J Aldave; Elucidating the genetic basis of PPCD3: demonstration of ZEB1 binding to COL4A3 and COL4A4 E2 box domains. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3080. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of zinc finger e-box binding homeobox 1 gene (ZEB1) gene in posterior polymorphous corneal dystrophy 3 by demonstration of binding to E2 box motifs in type IV collagen promoter regions.
Using JASPER and known published consensus sequences, putative E2 box motifs were identified within the 5 Kb promoter region upstream of the translational start sites for collagen, type IV, alpha3 (COL4A3) and collagen, type IV, alpha4 (COL4A4). In vitro DNA-protein binding assays were performed by incubating ZEB1WT-overexpressing cell lysates with DIG-labeled probes corresponding to each of the putative COL4A3 and COL4A4 E2 box domains. 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.
Nine E2 box domains were identified in the COL4A3 and seven E2 box domains were identified in the COL4A4 promoter regions. Six of the nine COL4A3 E2 box probes exhibited significant levels of binding to ZEB1 while one of seven COL4A4 E2 box probes displayed ZEB1 binding. Competitive binding against each ZEB1-bound E2 box probe using unlabeled probes and ZEB1 antibodies confirmed ZEB1-specific binding.
DNA-protein binding studies indicate that ZEB1 is capable of binding at least one of the several E2 box motifs identified in each of the promoter regions of COL4A3 and COL4A4. Though further functional investigation is needed, our results suggest that ZEB1 is likely playing a regulatory role in both COL4A3 and COL4A4 expression.
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