Transcriptional regulation of mouse alphaA-crystallin gene

Abstract

The expression of mammalian alphaA-crystallin ranks among the most abundant tissue-specific protein. Its expression in ocular lens is controlled at the level of transcription. Increased expression of alphaA-crystallin commences with lens fiber cell differentiation.;Here, we identified four Maf- and two Pax6-binding sites in the -111/+46 promoter fragment by EMSAs and DNase I footprinting. Site-directed mutagenesis of these sites resulted in reduced promoter activities in lens cells. Co-transfections using Pax6 and c-Maf revealed moderate and strong activations of this promoter, respectively. Pax6 has a neutral effect on c-Maf-mediated alphaA-crystallin promoter activation. Chromatin immunoprecipitations (ChIPs) established in vivo interactions of Pax6 and c-Maf with the alphaA-crystallin promoter in lens cells.;We identified three putative distal control regions (DCR1-3). All DCRs augmented the alphaA-crystallin promoter in cultured lens cells; however, only DCR1 responded to FGF2 in lens epithelial primary culture. In transgenic mice, both DCR1 and DCR3 stimulated expression of EGFP from the 1.8 kb promoter (containing DCR2) in lens fiber cells albeit with 24--36h later in DCR3 transgenic mouse. DCR1 also supported expression of EGFP in lens epithelium. Quantitative ChIPs in new born mouse lens, lens epithelial cells and fibroblasts revealed that high level of alphaA-crystallin expression was correlated with the increased recruitment of c-Maf and CREB to the promoter and the simultaneous presence of Pax6 both in the promoter and DCR1. Expanded distribution of H3K9 acetylation as well as Snf2h was detected across 16kb locus in mouse lenses compared to limited promoter region in lens epithelium. Increased occupancy of Brg1 is associated with high level of alphaA-crystallin expression in the lens.;In summary, our data suggested that tissue-specificity of alphaA-crystallin is established by its interactions with Pax6 and c-Maf Temporal and spatial regulation of alphaA-crystallin is controlled through DCR1 and DCR3. DCR1 is required to initiate expression in lens vesicle and enhanced expression in primary lens fibers in response to FGF2. In contrast, DCR3 is later active in primary lens fibers and functionally associated with the increased binding of CREB. High and moderate levels of alphaA-crystallin expression are also controlled by chromatin remodeling including HATs, SWI/SNF and ISWI.