Pathogenic Variants in Have Distinct -regulatory Effects on Enhancers and Silencers in Photoreceptors

Dozens of variants in the gene for the homeodomain transcription factor (TF) cone-rod homeobox () are linked with human blinding diseases that vary in their severity and age of onset. How different variants in this single TF alter its function in ways that lead to a range of phenotypes is unclear. We characterized the effects of human disease-causing variants on CRX -regulatory function by deploying massively parallel reporter assays (MPRAs) in mouse retina explants carrying knock-ins of two variants, one in the DNA-binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation in these mutant retinas corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, and p.E168d2 has distinct effects on silencers. -regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are derepressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci are partially predictive of episomal MPRA activity, and distal elements whose accessibility increases later in retinal development are enriched for CREs with silencer activity. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its -regulatory function, leading to misregulation of similar sets of enhancers while having a qualitatively different impact on silencers.