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A. J. Aldave, V. S. Yellore, J. Z. Lee, S. A. Rayner, S. Bhat; Analysis of the Role of ZEB1 in the Pathogenesis of Posterior Polymorphous Corneal Dystrophy Through Transcriptional Control of COL4A3 and COL2A1 Expression. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4642.
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Electromobility shift assays were performed using mouse thymus nuclear extracta known rich source of ZEB1. Different labeled oligonucleotide probes were synthesized based on the 5’ upstream sequences (in COL4A3 and COL2A1 genes) that include the conserved E2 box (CACCTG) known to be recognized by ZEB1.
EMSA studies demonstrated that the mouse thymus nuclear extracts contained protein(s) that formed a major complex (complex-1, lower mobility) and a minor complex (complex-2, higher mobility) with mouse 32P-COL4A3 probe. A complex, comparable in mobility to complex-2 was the predominant complex obtained with mouse 32P-COL2A1 probe. Mutant probes that varied from the wild type sequence within the E2 box, as well as in the flanking sequences, were poor competitors. In addition, an unlabeled human COL4A3 oligonucleotide (derived from homologous region of the human COL4A3 gene promoter and predicted to bind ZEB1) competed out the complex-2 (higher mobility complex) obtained with mouse 32P-COL2A1, suggesting that the complex-2 contains ZEB1. Interestingly, this probe did not compete out complex-1 obtained with 32P-COL4A3.
This data suggest that the human COL2A1 and the mouse COL4A3 sequences bind to similar factors in the mouse thymus nuclear extract and thus can be exploited to dissect the transcriptional activities that impact collagen (COL4A3) expression in the context of its regulation by ZEB1 and its eventual role in the pathogenesis of PPCD. We are in the process of using antibodies to further characterize the two complexes, one of which (the higher mobility shift complex) we believe contains ZEB1.
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