Developmentally Regulated Tissue–specific Expression of the Mouse A–crystallin Requires Establishment of a Broad H3K9 Acetylation Domain Including the Upstream Region DCR1, Activated via FGF2 Signaling

Abstract

Abstract: : Purpose: αA–crystallin is expressed in lens epithelium and fibers. Dramatic induction of αA–crystallin expression is observed at the onset of lens fiber cell differentiation. This study seeks to identify novel distant regulatory regions (DCRs) of αA–crystallin and to elucidate the in vivo molecular mechanism for its high level expression in lens fibers and moderate expression in lens epithelium. Methods: Multiple sequence alignments between human and mouse αA–crystallin loci were used to predict regulatory regions. Enhancer–like activities of these DCRs were tested in transient transfections in 2–day old rat lens explants in the presence and absence of FGF2 and in transgenic mice. Interactions of transcription factors/co–factors, RNA polymerase II and histone modification markers along the 16kb locus of αA–crystallin were assessed in vivo by Chromatin Immunoprecipitations (ChIPs). The expression level of αA–crystallin in the lens epithelium and fibers was determined by quantitative RT–PCR analysis. Results: A novel regulatory element, DCR1, was identified to mediate stimulation of promoter activity in the presence of FGF2. In the transgenic mouse lens, DCR1 significantly enhanced the expression level of the eGFP compared to the 1.8kb promoter alone. ChIPs revealed broad H3K9 acetylation across the 16kb locus in the lens that correlated with a high level of αA–crystallin expression. In contrast, 20–fold lower expression of αA–crystallin in lens epithelial cells was associated with a shorter 4kb H3K9 acetylated domain. The promoter activity in vivo quantitatively correlated with the recruitment of c–Maf to the promoter. Four Maf–binding sites, three of them novel, were shown in the promoter. The presence of Pax6 and c–Maf in both promoter and DCR1 was shown both in vivo and in vitro. Conclusions: Our data suggest a novel link between the αA–crystallin expression and DCR1 activated by FGF2. Spread of H3K9 acetylation across the locus and increase of c–Maf in the promoter correlated with the high level of αA–crystallin expression. Binding of both Pax6 and c–Maf to the promoter support their key roles in establishing the tissue–preferred expression of αA–crystallin in mouse lens.

Keywords: crystallins • transcription • transcription factors