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W Cui, SI Tomarev, AB Chepelinsky, MK Duncan; MafB is Expressed in Developing Lens and Cooperates With Pax6 and Prox1 to Regulate Chicken Beta B1 Crystallin Gene Expression . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2329.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Pax6, Prox1 and Mafs are transcription factors important for crystallin gene expression and eye development. We previously showed that, in transfection assays, MafB and Prox1 could synergistically activate the chicken betaB1 crystallin promoter (-432/+30), however, both MafB and Prox1 mediated transactivation could be blocked when Pax6 was co-transfected. This study investigates how these transcription factors cooperate to regulate betaB1-crystallin gene expression. Methods: Distribution of MafB was investigated in mouse lens and lens cell lines by immunohistochemistry and western blotting. 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 differentially expressed in lens fiber cells and lens epithelial cells: in lens epithelial cells, MafB is not evenly distributed in nuclei but concentrated in a single large spot, while in fiber cells, MafB is present as multiple small spots. Western blotting confirmed that MafB was expressed in mouse lens epithelia and some lens cell lines. In EMSA, Prox1, like its Drosphila homolog Prospero, was able to bind to TDA-Pros and mPbs, two oligonucleotides used to test Prospero binding. Recombinant Prox1 can also bind to PL2 (-87/-76), an element essential for chicken betaB1 crystallin promoter activity. Pax6 and MafB could bind to PL2 and PL1 (-114/-102). Both Prox1 and MafB DNA binding abilities to this promoter can be reduced and eliminated when co-incubated with increasing amount of Pax6. Conclusion: Taken together with our previous transfection results, it is likely that in differentiating cortical fibers, Pax6, which binds to the chicken betaB1 promoter and inhibit its expression, is displaced by increasing concentration of Mafs and Prox1. Thus, betaB1 crystallin gene expression is initiated and maintained at high levels in order to form functional fiber cells.
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