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. The widely used promoter fragments, i.e. -366/+46 and -111/+46, to express transgenes are active only in lens fiber cells. This study seeks a) to identify distant control regions (DCRs) essential for high level of αA-crystallin expression in lens fibers and in lens epithelium and b) to perform fine mapping of regulatory sites present in the -111 to +46 promoter fragment. Methods: A mouse BAC clone containing the αA-crystallin locus from RP23 C57/BL6/J genomic library was isolated and sequenced. Multiple sequence alignments (MSA) of different genomic loci (human, mouse, chicken, hamster, and mole rat) were used to predict non-coding conserved sequences. Transient transfections were conducted in cultured lens and non-lens cells using the candidate DCRs combined with the -111 to +46 promoter fragment. Protein-DNA binding studies were used to study binding of Pax6 and c-Maf. Results: MSA revealed at least six candidate DCRs. These DCRs contain arrays of putative transcription factor binding sites including c-Maf, Pax6, Prox1, CREB, RAR/RXR, Sp1 and AP2. Transient transfections have identified at least three DCRs activating the αA-crystallin promoter in lens cells but not in fibroblasts. The promoter fragment -111 to +46 contains three Pax6 and two c-Maf binding sites. Conclusion: We have identified several novel lens-specific distant control regions of the mouse αA-crystallin promoter. The -111 to +46 promoter fragment contains an array of regulatory sites interacting with Pax6, c-Maf and CREB.
Keywords: 604 transcription • 417 gene/expression • 476 molecular biology