Abstract
The alpha subunit of cone transducin (TalphaC) is expressed exclusively in cone photoreceptors of the eye and pineal. TalphaCisakey phototransduction protein, and inherited mutations in TalphaC cause total color blindness in humans. We use transgenic zebrafish to identify and characterize cone photoreceptor regulatory element 1 (CPRE-1) a novel 20-bp enhancer element in the TalphaC promoter (TalphaCP). CPRE-1 is located approximately 2.5 kb upstream of the translation start site and is necessary for strong cone photoreceptor-specific expression in vivo. CPRE-1 comprises of a modular arrangement of two 10-bp elements that have separate, but co-dependent transcriptional activities. In vitro, CPRE-1 specifically binds nuclear factors that are enriched in ocular tissue. Bioinformatic alignments reveal that CPRE-1 sites are evolutionarily conserved in the promoter regions of fish, rodent, and mammalian TalphaC orthologues and identify a 5'-CTGGAGTG(A/T)TGGA(G/A)GCAGGG(G/C)T-3' consensus sequence.
Highlights
Several transcription factors, including Crx, Nr2e3, Nrl, and Tr2, regulate photoreceptor-specific gene expression
An internal deletion of enhancer region 1, within the Ϫ3173 bp T␣C promoter (T␣CP) construct, results in minimal reporter activity (Fig. 1A). These results suggest that enhancer region 1 is necessary for high level cone-specific expression and that enhancer region 2 is not sufficient to compensate for loss of enhancer region 1
To develop our understanding of the molecular genetics of cones we focused on identifying cis-elements in the zebrafish T␣C promoter region
Summary
T␣C, cone transducin ␣ subunit; T␣CP, cone transducin ␣ subunit 5Ј promoter; dpf, days post fertilization; CPRE-1, cone photoreceptor regulatory element 1; TBS, Tris-buffered saline; zUVOP, UV zebrafish opsin promoter; EGFP, enhanced green fluorescent protein. We reported that a 3.2-kb promoter fragment of the zebrafish T␣C gene is sufficient to drive strong EGFP expression in zebrafish cone photoreceptors and that the most distal 1.2-kb sequence contains an enhancer-like activity [19]. To identify this enhancer of cone-specific expression, we performed an in vivo analysis of the 3.2-kb zebrafish T␣C promoter using deletion, mutant and chimeric reporter constructs, and in vitro characterization of binding to eye nuclear protein. We describe a novel, evolutionarily conserved 20-bp enhancer of the zebrafish T␣C promoter that preferentially binds eye nuclear protein, is active upstream of a heterologous promoter, and is necessary for driving strong cone-specific expression
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
