James L. Shepherdson1,2,5, Ryan Z. Friedman1,2,5, Yiqiao Zheng3, Chi Sun3, Inez Y. Oh3, David M. Granas1,2, Barak A. Cohen1,2, Shiming Chen3,4 and Michael A. White1,2 1Department of Genetics, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA; 2Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA; 3Department of Ophthalmology and Visual Sciences, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA; 4Department of Developmental Biology, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA

↵5 These authors contributed equally to this work.

Corresponding authors: chenshimingwustl.edu, mawhitewustl.edu Abstract

Dozens of variants in the gene for the homeodomain transcription factor (TF) cone-rod homeobox (CRX) 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 cis-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 Crx retinas corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, and p.E168d2 has distinct effects on silencers. Cis-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 cis-regulatory function, leading to misregulation of similar sets of enhancers while having a qualitatively different impact on silencers.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.278133.123.

Freely available online through the Genome Research Open Access option.

Received May 27, 2023. Accepted February 1, 2024.