Abstract
Purpose :
Precisely regulated gene expression is essential for retinal development and maintenance. Over and under-expression of Rhodopsin (Rho) in mouse retina affect rod function and survival. The Rho 5’-regulatory region contains two enhancers, RER and CBR, each capable of driving high expression levels of reporter genes in mouse rods. However, little is known about the in vivo role of each enhancer. In this study, we aimed to determine the importance of these two enhancers in regulating the expression of the mouse Rho gene during rod development and maintenance.
Methods :
CRISPR-Cas9 was used to create mouse lines lacking the Rho promoter, each individual enhancer, or both enhancers together. The resulting mice were characterized for retinal gene expression by qRT-PCR or RNA-seq, the morphology of the photoreceptors by IHC and H&E, and the chromatin state by ATAC-seq.
Results :
The CBR-/- retinas showed significantly reduced Rho expression compared to WT during postnatal development, but recovered by adulthood, suggesting CBR has a specific role during rod development. In contrast, the RER-/- retinas displayed very little defects at any time. However, the retinas of CBR/RER double knockout mice displayed developmental defects similar to CBR-/-, but failed to recover, suggesting a distinct role for the RER in regulating Rho in the mature rod photoreceptor.
Conclusions :
The CBR and RER have both overlapping and distinct roles in controlling Rho expression. Our results highlight the importance of detailed analysis of the regulatory logic of enhancer-promoter pairs. Since non-coding region mutations can alter gene expression important for retinal development or maintenance, our study provides a model for functional analysis to predict the effects of human mutations on disease risk and progression to inform treatment.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.