Abstract
Purpose: :
Although the mechanisms whereby cis-regulatory elements (CREs) establish spatiotemporal patterns of gene expression are becoming better understood, relatively little is known about the quantitative determinants of gene expression level, particularly within living mammalian tissues. This deficiency in our understanding is attributable to the non-quantitative nature of most available transcription factor binding data and the lack of quantitative assay systems for enhancer activity in vivo. In order to circumvent these difficulties for cis-regulatory analysis in mouse photoreceptors, we have developed a novel system for quantifying promoter activity in living retinas.
Methods: :
We employed the QuMFRA assay (Nucleic Acids Res. 29:2471-8) to quantify the relative affinity of the photoreceptor transcription factor, Crx (cone-rod homeobox), for its cognate sites. We then used site-directed mutagenesis to create variants of several endogenous photoreceptor-specific CREs carrying a variety of mutations in their Crx binding sites. Lastly, we measured the activity of these variant promoters by quantifying the fluorescence driven by promoter-reporter fusion constructs that had been electroporated into living retinas.
Results: :
We quantitatively determined the relative binding affinity of all single-nucleotide variants of the consensus binding site of Crx. We then showed that it is possible to use these data to accurately predict the relative binding affinity of Crx for all possible 8 bp binding sites. Next, we quantitatively fine-tuned the expression of a minimal rod-specific promoter in vivo over a 100-fold range, by rationally adjusting the binding affinity of only three Crx sites. Lastly, we demonstrate that this approach to fine-tuning promoter activity is applicable to a range of photoreceptor-specific promoters including one which drives expression specifically in cones and another which drives expression in both rods and cones.
Conclusions: :
This approach will facilitate the quantitative engineering of photoreceptor-specific CREs to be used as drivers in gene therapy vectors for the treatment of blindness and may be generally applicable to engineering other cell type-specific promoters.
Keywords: photoreceptors • transcription factors • opsins