Aberrant homeodomain-DNA cooperative dimerization underlies distinct developmental defects in two dominant CRX retinopathy models

Abstract

Paired-class homeodomain transcription factors (HD TFs) play essential roles in vertebrate development, and their mutations are linked to human diseases. One unique feature of paired-class HD is cooperative dimerization on specific palindrome DNA sequences. Yet, the functional significance of HD cooperative dimerization in animal development and its dysregulation in diseases remain elusive. Using the retinal TF Cone-rod Homeobox (CRX) as a model, we have studied how blindness-causing mutations in the paired HD, p.E80A and p.K88N, alter CRX's cooperative dimerization, lead to gene misexpression and photoreceptor developmental deficits in dominant manners. CRX^E80A maintains binding at monomeric WT CRX motifs but is deficient in cooperative binding at dimeric motifs. CRX^E80A's cooperativity defect impacts the exponential increase of photoreceptor gene expression in terminal differentiation and produces immature, non-functional photoreceptors in the Crx^E80A retinas. CRX^K88N is highly cooperative and localizes to ectopic genomic sites with strong enrichment of dimeric HD motifs. CRX^K88N's altered biochemical properties disrupt CRX's ability to direct dynamic chromatin remodeling during development to activate photoreceptor differentiation programs and silence progenitor programs. Our study here provides in vitro and in vivo molecular evidence that paired-class HD cooperative dimerization regulates neuronal development and dysregulation of cooperative binding contributes to severe dominant blinding retinopathies.

Keywords: CRX; DNA binding cooperativity; DNA binding specificity; chromatin remodeling; homeodomain; inherited retinal disease; missense mutations; photoreceptor development; transcription factor.

Figure 1.. K88N mutation significantly increased CRX HD’s cooperative binding and transactivation activity on BAT-1 sequence containing a P3 dimeric HD motif.

A. Diagrams depicting K50 HD preferred monomeric and dimeric P3 motifs. B. Alignments showing WT BAT-1 probe sequences and variants. The P3 dimeric HD motif and four monomeric HD core motifs 5’-TAAT-3’ are labelled. f and r indicates whether the core motif is on the forward or reverse strand. In BAT-1 variants, the mutated nucleotides are italicized and underlined. C.&D. EMSA gel images showing increasing amount of WT or K88N HD peptides bound to a fixed amount of BAT-1 (WT) or P5 GA control probes. The cartoon underneath each gel image shows the dimeric HD motif configuration and is labelled with the spacer length. E. Schematics and barcharts of luciferase reporter assays comparing CRX WT and K88N transactivation activity at BAT-1 and variant enhancer sequences. p-values of one-way ANOVA are annotated. ns: >5e-2; ***: <=1e-3.

Figure 2.. E80A and K88N mutations differently affect CRX HD DNA binding cooperativity at P3 sequences.

A.&B. Schematics showing the Coop-seq experimental pipelines. dsDNA oligo pools of P3 and/or P5 Coop-seq library are incubated with different HD peptides. The dimeric and monomeric binding complexes are separated from unbound DNAs by EMSA. DNAs are extracted from all three DNA bands and subjected to quantification by Illumina sequencing. Bd: dimeric band; Bm: monomeric band; U: unbound band. C. Diagram depicting the Coop-seq library design and strategy to match a P3 sequence with a P5 counterpart. Exact oligo sequences can be found in Supplementary Table S1. D. Heatmap comparing the relative cooperativity of WT and variant CRX HDs on P3 and P5 libraries (ωp3/ωp5). Note the relative cooperativity is presented in the Logarithmic scale and ordered by unsupervised hierarchical clustering. The ordered relative cooperativity matrix can be found in Supplementary Table S3.

Figure 3.. Crx^K88N/+ retinas show defective chromatin remodeling at photoreceptor CREs enriched with K50 HD motifs.

A. Heatmaps depicting the normalized ATAC-seq or CRX ChIP-seq signal intensities at Crx^K88N-reduced accessible ATAC-seq peaks. B. PWM logo and enrichment significance E-value of the STREME de novo discovered HD motif. C. Line plot showing the average developmental accessibility kinetics of Crx^K88N-reduced ATAC-seq peaks. The developmental ATAC-seq data is from Aldiri et al. 2017. D. Barchart showing Biological Process (BP) Gene Ontology (GO) term enrichment of differentially expressed genes adjacent to Crx^K88N-reduced ATAC-seq peaks. E. Heatmap comparing the P10 expression changes of Crx^K88N-reduced ATAC-seq peaks associated genes in different Crx mutant retinas. The gene set is identical to that in D. F. Schematics depicting chromatin remodelling defects at photoreceptor regulatory regions in the Crx^K88N/+, Crx^K88N/N, and Crx^R90W/W retinas.

Figure 4.. CRX K88N ectopic activity at Q50 HD motifs impedes the silencing of progenitor regulatory programs in developing photoreceptors.

A. Heatmaps depicting the normalized ATAC-seq or CRX ChIP-seq signal intensities at Crx^K88N-increased accessible ATAC-seq peaks. B. PWM logo and enrichment significance E-value of STREME de novo discovered HD motifs. C. Line plot showing the average developmental accessibility kinetics of Crx^K88N-increased ATAC-seq peaks. The developmental ATAC-seq data is from Aldiri et al. 2017. D. Heatmap depicting the log odds ratio enrichment of embryonic day 14.5 (e14.5) or adult retinal VSX2 binding sites under Crx^K88N-increased ATAC-seq peaks. p-values of Fisher’s exact tests are indicated. The VSX2 ChIP-seq data is from Bian et al. 2022. E. PWM logo and significance E-value of STREME de novo discovered basic helix-loop-helix (bHLH) motif under Crx^K88N-increased ATAC-seq peaks. PWM logos of selected retinal progenitor/neurogenic bHLH TFs are given for comparison. JASPAR IDs of the selected TFs can be found in Methods. F. Barchart showing Biological Process (BP) Gene Ontology (GO) term enrichment of genes adjacent to Crx^K88N-reduced ATAC-seq peaks.

Figure 5.. Crx^E80A retinas show defective chromatin remodelling at CREs enriched for dimeric K₅₀ HD motifs.

A. Heatmaps depicting the normalized ATAC-seq or CRX ChIP-seq signal intensities at Crx^E80A differentially accessible ATAC-seq peaks. B.&C. PWM logos and enrichment significance E-values of STREME de novo discovered HD motifs under Crx^E80A differentially accessible ATAC-seq peaks. D.&E. Line plots showing the average developmental accessibility kinetics of Crx^E80A differentially accessible ATAC-seq peaks. The developmental ATAC-seq data is from Aldiri et al. 2017.

Figure 6.. CRX E80A differential activity at monomeric and dimeric K50 HD motifs contributes to gene mis-expression at different stages of photoreceptor development.

A. Diagrams of photoreceptor genes regulated solely by monomeric K50 HD motifs (top) or combinationally by monomeric and dimeric K50 HD motifs (bottom). For simplicity, representative motif logos are shown. The relative position of the motifs is arbitrary. B. Heatmap comparing the CRX-DAG expression changes in the Crx^E80A/A retinas at ages of post-natal day 10 (P10) and day 21 (P21). The gene sets in the heatmaps are as defined in A. C.&D. Schematics demonstrating the K50 HD division-of-labor model in regulating photoreceptor epigenome and transcriptome at different stages of development.

Figure 7.. Monomeric and dimeric K50 HD motifs associated with E80A dosage dependent activity changes in retinal ex plant MPRAs.

A. Schematics showing ex plant retinal MPRA experimental pipeline. B.&C. Box and strip plots comparing monomeric (B) or dimeric (C) K₅₀ HD motif activities in ex plant cultured WT, Crx^E80A/+ and Crx^E80A/A retinas. In panel B, CREs overlapped with ATAC-seq peaks that were not significantly reduced in the Crx^E80A retinas are plotted. In panel C, CREs overlapped with ATAC-seq peaks that were significantly reduced in the Crx^E80A retinas are plotted. p-values of Mann-Whitney-Wilcoxon tests are annotated.

Figure 8.. Model schematics.