A 350 bp region of the proximal promoter of Rds drives cell-type specific gene expression
Introduction
Rds is one of the most common ocular genes to carry pathogenic mutations. Over 80 different disease causing mutations in Rds have been identified and are responsible for a wide range of degenerative phenotypes including autosomal dominant retinitis pigmentosa (RP) and various forms of macular dystrophy (Berson, 1993, Keen and Inglehearn, 1996). We and others have successfully delivered wild-type murine Rds to the retinas of mice with an Rds-associated haploinsufficiency RP phenotype (the rds+/−) and reported structural and functional rescue of the diseased retina (Ali et al., 2000, Cai et al., 2009b). For our studies we used both ubiquitous (chicken beta-actin, CBA) and photoreceptor specific (human interphotoreceptor retinoid binding protein, IRBP) promoters and while they represented satisfactory preliminary choices, further knowledge of the native Rds promoter will be a great advantage for our future studies. However thus far, no reports have specifically addressed regulation of this gene. Characterization of the Rds promoter region will give a better understanding of the native regulation of the Rds gene and may enable us to enhance our gene therapy studies by incorporating critical regulatory elements in our vector design.
Gene therapy has been a popular and promising therapeutic approach for the treatment of inherited retinal degenerations in various animal models and patients [rodents (Ali et al., 2000, Cai et al., 2009b, Weber et al., 2003), dogs (Acland et al., 2005, Acland et al., 2001, Le Meur et al., 2006), primates (Jacobson et al., 2006, Lotery et al., 2003, Weber et al., 2003) and humans (Bainbridge et al., 2008, Cideciyan et al., 2008, Hauswirth et al., 2008)], and optimization of ocular gene therapy by expanding promoter choices is advantageous. Due to the prevalence of inherited retinal degenerations associated with photoreceptor and retinal pigment epithelial (RPE) defects, these cell types have often been targets of gene delivery studies. While tissue-specific promoters like vitelliform macular dystrophy 2 (VMD2) and rhodopsin (MOP) and ubiquitously expressed promoters like chicken beta-actin (CBA) have been successfully used to direct expression in the retina (Allocca et al., 2007, Cai et al., 2009a, Cai et al., 2009b), strong promoters that can direct proper levels of gene expression in rods and cones have been lacking. The strongest currently used ocular promoter (the MOP promoter) is typically thought to be rod specific, or to drive very low levels of gene expression in cones (Glushakova et al., 2006). For the treatment of rod-based diseases, this promoter is a good choice, however, many diseases target both cones and rods. The most commonly used promoters to target both rods and cones have been the promoter for the photoreceptor transcription factor Crx and the IRBP (interphotoreceptor retinoid binding protein) promoter (Nour et al., 2004, Oh et al., 2007) although other promoters such as the rhodopsin kinase promoter, have also been studied for this purpose (Khani et al., 2007). To expand the available options for strong rod/cone promoters, we chose to characterize the promoter region for a gene that is expressed robustly in both photoreceptor types; Rds (retinal degeneration slow, also referred to as Peripherin/rds, P/rds, or Prph2). Our goals were first, to characterize a novel promoter that could be potentially used to direct high levels of expression of any gene (but particularly Rds) in rods and cones in gene therapy studies; and second, to study regulation of the Rds gene to better understand the expression and regulation of this key outer segment protein. We isolated a 3.5 kb fragment of the 5′ flanking region of the mouse Rds gene from wild-type C57BL/6 genomic DNA, identified regulatory factor binding sites in the promoter, and characterized the in vitro activity and cell-type specificity of various promoter fragments.
Section snippets
Cloning of the 5′ flanking region and identification of regulatory sequences
3.5 kb of the 5′ flanking region of the murine Rds gene was isolated from C57BL/6 genomic DNA using the PromoterFinder™ DNA Walking kit (Clontech Laboratories, Inc., Palo Alto, CA, for details see Supplementary Methods and Supplementary Fig. 1). Products were cloned into the pBluescriptKS+ vector and sequenced. Sequences were blasted against the ensembl database (www.ensembl.org). Analysis for the presence of known transcription factor binding sites (cis-elements) in the 3.5 kb murine RDS 5′
Sequence analysis of the 5′ flanking region
The 5′ flanking region of murine RDS was sequenced and analyzed as described in the methods (Supplementary Fig. 2). The GC content in the 400 bp immediately 5′ to the ATG initiation codon is 55%. The observed/expected CpG ratio was 0.49 indicating that the immediate upstream promoter region is not CpG rich. Sequence analysis of the flanking region showed the absence of typical TATA and CCAAT elements. Over 800 potential cis-regulatory elements were detected in the analyzed region. Those
Discussion
In this study, we evaluated the 3.5 kb flanking region of the Rds gene due to its structural importance in the photoreceptor outer segment, functional necessity for proper vision, and experimental value in the design of optimized gene therapy vectors. Unlike many other dual-photoreceptor-specific genes, it lacks typical TATA and CAAT promoter elements but contains several other regulatory elements. This region is not CpG rich. Notably, several critical photoreceptor transcription factor binding
Acknowledgements
This work was supported by the National Eye Institute (EY10609-MIN, EY018656-MIN, EY14052-MRA, EY018512-SMC), and the Foundation Fighting Blindness (MIN, MRA).
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These two authors contributed equally to this work.