Abstract
Abstract: :
Purpose: Lens differentiation is characterized by temporally and spatially regulated expression of crystallin genes. This process is regulated at the level of transcription. Currently known transcription factors of crystallin genes include Pax6, Pax6(5a), c–Maf, MafA/L–Maf, MafB, NRL, Sox1, Sox2, RARß/RXRß, RORα, Prox1, Six3, γFBP–B and HSF2. As previous studies were conducted with only individual transcription factors or a limited number of their combinations, our goal was to employ combinations of these factors relevant to the protein composition of lens epithelial cells and differentiating lens fibers. Methods: Transient cotransfections were performed using lens and non–lens cultured cells using mouse αA–, αB–, and γF–crystallin promoters. Electrophoretic mobility shift assays (EMSAs) were used to determine binding of transcription factors from lens nuclear extracts characterized by westerns. Chromatin immunoprecipitations (ChIPs) were used to show presence of these transcription factors on crystallin promoters in chromatin. Results:A transcriptional synergism between Pax6 and Pax6(5a) , two alternatively spliced variants of the same gene, was established for all three crystallin promoters. We determined activation of αB–crystallin promoter by all four large Maf proteins (MafA, MafB, c–Maf and NRL). However, any combination of these factors resulted in weak activation of this promoter. In contrast, Pax6 highly activated αB–promoter in the presence of any combination of Maf proteins. Next, αA–crystallin promoter was strongly activated by Mafs with modest activation by Pax6/Pax6(5a). Two regions, –110/–98 and –88/–60, of the αA–crystallin promoter contain functional Maf–binding sites, MAREs. Finally, Pax6 and Six3 acted as strong co–repressors of γF–crystallin promoter activity mediated by Maf/Sox combinations. Conclusions:This study established functional synergism and antagonism between Pax6 and large Maf proteins and identified novel MAREs in the αA– and αB–crystallin promoters. In addition, our data explain lens specificity of the –88 to +46 region of the mouse αA–crystallin promoter, and demonstrate functional interaction of Pax6 and Maf proteins with two lens–specific regions, LSR1 and LSR2, of the mouse αB–crystallin gene. The results are consistent with expression patterns of these transcription factors and their crystallin target genes in lens epithelial and lens fiber cells.
Keywords: transcription factors • crystallins • transcription