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W. Cui, S.I. Tomarev, A.B. Chepelinsky, M.K. Duncan; Mafs, Pax6 and Prox1 Cooperate to Regulate ßB1 Crystallin Gene Expression in Developing Lens . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1076.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: ßB1 crystallin gene is a good marker to study lens development and fiber cell differentiation because of its spatiotemporal expression pattern. Pax6, Prox1 and Mafs are transcription factors important for crystallin gene expression and eye development. This study investigates how these transcription factors cooperate to regulate ßB1-crystallin gene expression. Methods: Distribution of MafB was investigated in mouse lens and lens cell lines by immunohistochemistry and western blotting. MafB and c-maf were cloned into the eukaryotic expression vector pcDNA3.1 Zeo (+) and transfected with wild type and mutated chicken ßB1-crystallin reporters. GST-tagged MafB, Prox1 and Pax6 were generated by using pET or pGEX recombinant protein expression system. Their DNA binding abilities were tested in electrophoresis mobility shift assays (EMSA). Results: MafB was present in both lens fiber cells and epithelial cells. Western blotting confirmed that MafB was expressed in mouse lens epithelia and some lens cell lines. In transfection assays, deletion of PL1 (-114/-102) or PL2 (-87/-76) can significantly decrease the activity of the chicken ßB1 crystallin promoter (-432/+30), when both elements were deleted, the mutated reporter lost all of its activity; individually, c-Maf, MafB and Prox1 can activate the chicken ßB1 crystallin promoter; however, Pax6 function as a repressor of the promoter by itself and was able to interfere with the transactivation abilities of MafB, c-Maf and Prox1. In EMSA, recombinant Prox1 can bind to PL2, Pax6 and MafB could bind to both PL2 and PL1. Prox1 and MafB DNA binding abilities to this promoter can be reduced and eliminated when co-incubated with increasing amount of Pax6. Conclusions: It is likely that in differentiating cortical fibers, Pax6, which binds to the chicken ßB1 promoter and inhibit its expression, is displaced by increasing concentration of Mafs and Prox1. Thus, ßB1 crystallin gene expression is initiated and maintained at high levels in order to form functional fiber cells.
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