Pathogenic variants in CRX have distinct cis-regulatory effects on enhancers and silencers in photoreceptors

Dozens of variants in the photoreceptor-specific transcription factor (TF) CRX are linked with different human blinding diseases that vary in their severity and age of onset. How different variants in a single TF cause a range of pathological phenotypes is not understood. We deployed massively parallel reporter assays (MPRAs) to measure changes to CRX cis-regulatory function in live mouse retinas carrying knock-ins of two phenotypically distinct human disease-causing Crx variants, one in the DNA binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). We found that the effects of CRX variants on global cis-regulatory activity patterns correspond with the severity of their phenotypes. The variants affect similar sets of enhancers but to different degrees. A subset of silencers were converted to enhancers in retinas lacking a functional CRX effector domain, but were unaffected by p.R90W. Episomal MPRA activities of CRX-bound sequences showed some correspondence with chromatin environments at their original genomic loci, including an enrichment of silencers and depletion of strong enhancers among distal elements whose accessibility increases later in retinal development. Many distal silencers were de-repressed by p.E168d2, but not by p.R90W, suggesting that loss of developmentally timed silencing caused by p.E168d2 may contribute to phenotypic differences between the two variants. Our findings indicate that phenotypically distinct disease variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to mis-regulation of similar sets of enhancers, while having a qualitatively different impact on silencers.