Purchase this article with an account.
Y. Yang, B.K. Chauhan, N.C. Golestaneh, K. Cveklova, A.B. Chepelinsky, A. Cvekl; Novel Regulatory Elements of the Mouse A-Crystallin Locus . Invest. Ophthalmol. Vis. Sci. 2003;44(13):429.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: αA-crystallin is expressed in lens epithelium and fibers. Dramatic induction of aA-crystallin expression is observed at the onset of lens fiber cell differentiation. The widely used promoter fragment, -366/+46, drives expression of transgenes only in lens fiber cells. This study seeks to identify distant regulatory regions (DCRs) essential for high level of expression in lens fibers and in lens epithelium. Methods: A mouse BAC clone containing αA-crystallin locus from RP23 C57/BL6/J genomic library was isolated and sequenced. Multiple sequence alignments of different αA-crystallin genomic regions were used to predict non-coding conserved sequences. Transient transfections were conducted in cultured lens epithelial(N/N1003A) and non-lens (3T3 fibroblasts) cell lines using the candidate DCRs , which were inserted upstream of the -111 to +46 promoter fragment in a luciferase expression vector. These DCRs constructions were also transfected into 3-day old rat lens epithelia explants cultured in the presence of FGF2. Presence of cis-elements in DCRs was tested using one copy of site cloned 5'-of the promoter and evaluated in transient transfections. Results: Multiple sequence alignments revealed at least 6 candidate DCRs: DCR1, 2 and 3 located 5'-, and DCR4.1, 4.2, 5.1 and 5.2, located 3'-from the start site of transcription. These DCRs contain arrays of putative transcription factor binding sites including large Maf proteins, Pax6, Prox1, CREB, AP-1, RAR/RXR, Sp1 and AP2. Transient transfections results indicated that all DCRs activated the -111/+46 αA-crystallin promoter in cultured lens epithelial cells. Studies in rat lens explants found that DCR3 contains regulatory element(s)activated during differentiation induced by FGF2. Conclusions: We have identified several novel lens-preferred distant control regions of the mouse αA-crystallin locus. DCR3 contains at least one regulatory element activated in lens explants induced to differentiate by FGF2. Ongoing studies are aimed to identify cis-regulatory elements in DCR3.
This PDF is available to Subscribers Only