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J.R. Taube, A.R. Yallowitz, M.K. Duncan; Regulation of ßB1 Crystallin Transcription in the Lens by EF1 and Prox1 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3482.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The –432/+30 chicken ßB1–crystallin promoter expresses at levels equivalent to endogenous ßB1–crystallin (1–2% of mRNA) in lens fiber cells of transgenic mice, and –126/+30 is 200–fold less active. These experiments seek to identify cis–elements and transcription factors that control fiber cell specific, crystallin level gene expression. Methods: Potential Prox1 sites were identified by DNaseI footprinting of the –432 to –126 region of the promoter and characterized by EMSAs with recombinant Prox1. Binding sites for other transcription factors were identified by sequence analysis. Identified cis–elements were tested functionally by site directed mutagenesis and co–transfection studies. Transcription factor localization was tested by confocal immunohistochemistry. Results: A Prox1 site was identified at –220 by DNase I footprinting. The nucleotides essential for binding in EMSA included a core AG dinucleotide similar to the OL2 element of the ßB1–crystallin promoter and a Prospero consensus sequence. In transfections of chicken lens annular pad cells, which are fiber cell precursors, a –245/+30 CAT construct was three–fold more active than –206/+30 CAT, confirming that an element activating the promoter is contained between –245 and –206. In transfections of N/N1003A cells, the activity of the –432 promoter was repressed, and truncations (–402, –282, –245, –152) caused a step–wise increase in promoter activity in the lens epithelial cells. Sequence analysis of the promoter in these regions revealed three consensus sites for ΔEF1, a repressor of Δ–crystallin expression. Co–transfection with ΔEF1 decreased activity of –432/+30 CAT in N/N 1003A cells. Removal of either the –414 or –282 site increased activity of the promoter and removed ΔEF1 responsiveness. ΔEF1 is present throughout the lenses of day 5 chick embryos and localizes primarily to the epithelium in post–hatching day 4 lenses, an expression pattern inverse to that of ßB1–crystallin, which is expressed strongly in lens fiber cells of late embryos and post–hatching chicks. Conclusions: While Prox–1, which activates the ßB1–crystallin promoter, is highly expressed in the nuclei of fiber cells containing ßB1–crystallin, the expression of ΔEF1 is high only in cells not expressing ßB1–crystallin. ΔEF1 may contribute to the lens fiber cell specific expression of ßB1–crystallin, by suppressing its expression in lens epithelial cells, while Prox–1 appears to contribute to the high level of expression of the gene in lens fiber cells, through multiple cis–elements.
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