Aberrant DNA cooperative binding underlies defective photoreceptor differentiation in mouse models of dominant CRX diseases

Abstract

Purpose : Transcription factor CRX regulates photoreceptor development and maintenance. We previously reported that two dominant human disease associated mutations in the CRX homeodomain (HD), p.E80A and p.K88N, alter CRX DNA binding specificity and lead to distinct photoreceptor deficits in mutation knock-in mouse models. In the current study, we further elucidate how mutations affect CRX’s DNA binding cooperativity and impact chromatin remodeling in the mutant mouse retinas.

Methods : Coop-seq characterized CRX HD’s cooperative binding on dimeric HD DNA motifs in vitro. Bulk ATACseq in P14 Crx mutant and WT mouse retinas found shared and genotype-specific alterations. Motif analysis found enrichment of distinct dimeric HD DNA motifs in mutant-specific differentially accessible regions. Analysis of epigenome and transcriptome data revealed correlation of chromatin accessibility changes and gene mis-expression.

Results : CRX binding is essential for chromatin remodeling at photoreceptor regulatory regions. p.E80A reduced CRX HD’s cooperative binding in vitro. A specific set of developmentally regulated genomic regions enriched for a K₅₀ HD dimeric motif showed reduced CRX binding and accessibility in the Crx^E80A/A retinas. Defective remodeling at these regions explained photoreceptor gene down-regulation in the adult Crx^E80A/+ and Crx^E80A/A retinas. In contrast, CRX K88N HD showed enhanced cooperative binding in vitro. The Crx^K88N/+ and Crx^K88N/N retinas lost ATAC signals at regions normally bound by WT CRX and gained ATAC signals at ectopic regions enriched for a Q₅₀ HD dimeric motif. The Crx^K88N-associated ectopic regions gradually lose accessibility in the post-natal WT retinas. Mutant CRX K88N activity likely impeded the silencing of progenitor regulatory programs and affected development progression in a dominant manner.

Conclusions : CRX’s cooperative DNA binding at specific dimeric HD motifs is essential in photoreceptor development. Our study affords new insights into the distinct pathogenic mechanisms of two dominant CRX HD mutations: p.E80A and p.K88N differently affect CRX’s cooperative binding on dimeric HD motifs, impair remodeling of photoreceptor regulatory regions, and disrupt photoreceptor differentiation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.