Antagonistic action of Six3 and Prox1 at the γ-crystallin promoter

gamma -Crystallin genes are specifically expressed in the eye lens, Their promoters constitute excellent models to analyse tissue-specific gene expression, We investigated murine Cryge/f promoters of different length in lens epithelial cell lines. The most active fragment extends from position -219 to +37, Computer analysis predicts homeodomain and paired-domain binding sites for all rodent Crygd/e/f core promoters. As examples, we analysed the effects of Prox1 and Six3, which are considered important transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a weak stimulation of Cryge/f promoter activity was found for PROX1, In contrast, PROX1 stimulated the Crygf promoter 10-fold in CD5A cells without endogenous PROX1, In both cell lines Six3 repressed the Crygf promoter to 10% of its basal activity. Our cell transfection experiments indicated that Cryg expression increases as Six3 expression decreases. Prox1 and Six3 act antagonistically on regulation of the Crygd/e/f promoters, Functional assays using randomly mutated gammaF-crystallin promoter fragments define a Six3-responsive element between -101 and -123 and a Prox1-responsive element between -151 and -174, Since Prox1 and Six3 are present at the beginning of lens development, expression of Crygd/e/f is predicted to remain low at this time. It increases as Six3 expression decreases during ongoing lens development.