Definition of rhodopsin regulatory elements using a retina-specific in vitro transcription system

Purpose. To identify DNA elements and protein factors which regulate the photoreceptor-specific expression of rhodopsin. Methods. The retina-specific in vitro transcription system (Chen & Zack, ARVO, 1994) was used to perform analysis of the bovine rhodopsin proxima promoter region. DNA binding factors were studied by electrophoretic mobility shift assay (EMSA). Results. The bovine retinal in vitro transcription system shows specificity for rhodopsin promoter templates as compared to non-photoreceptor-specific promoter templates. To dissect the regulatory elements within the rhodopsin promoter which are responsible for this specificity, we performed 5′-deletion analysis of the bovine rhodopsin region between -590 and -38 bp, using both retina and HeLa nuclear extracts. Deletion of the sequence from -130 to -38 bp, and particularly the -61 to -38 bp region, resulted in reduction of retinal extract-mediated transcriptional activity. In contrast, the same deletions had essentially no effect on the basal activity observed with HeLa extracts. These results suggested that the bovine rhodopsin promoter region between -130 and -38 bp contains retina-specific regulatory elements. To further identify these elements, site-specific mutations, altering 3-4 bp at a time, were introduced into the -64 to -38 bp region of the -590 and -85 rhodopsin templates. The results with the mutant templates suggested that the sequences between -64 and -58 bp as well as -51 and -40 bp contain critical elements. The former sequence is part of the binding site for the retina-specific transcription factor NRL. The proteins that recognize the latter site were studied by EMSA. The results showed that the -51 to -40 bp site is bound by both retina-specific and ubiquitously expressed protein factors. EMSA using mutant sequences further defined the putative recognition sequences for these protein factors. Cloning and characterization of these transcription factors are in progress. Conclusions. Photoreceptor-specific expression of the rhodopsin gene is controlled by interactions between multiple DNA regulatory elements and transcription factors.