Multi-omics analysis in human retina uncovers ultraconserved cis-regulatory elements at rare eye disease loci

Abstract

Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases.

Fig. 1. Integration of publicly available datasets for the characterization of the ultraconserved non-coding elements (UCNEs) library.

a Overview of the integrative multi-omics analysis for the prediction of UCNEs with potential cis-regulatory role in human retina. b Venn diagrams illustrating the CREs displaying open chromatin features based on scATAC-seq and DNase-seq (left) and their overlap of active enhancer marks (H3K27ac, H3K4me1, and H3K4me2) (right). c Barplot showing the proportions of elements from the VISTA enhancer browser (V), UCNEs (U), and putative regulatory UCNEs (characterized by retinal datasets) (U⁺) displaying reporter expression (positive). d Proportional stacked barplot showing the distribution of tissues (eye, forebrain, hindbrain, midbrain, neural tube, limb, and tail) in which the putative regulatory UCNEs display reporter expression. e, f Illustration of one of the characterized UCNEs (NR2F1_Hector_1/2) displaying open chromatin supported by DNase-seq (embryonic day 74–85, 89, and 103–125) and scATAC-seq (AC/HC/GC Precursors Cells, Early Progenitor Cells, Ganglion Precursor Cells, Late Retinal Progenitor Cells) and enhancer reporter expression in the eye. Figures obtained from VISTA (hs1170) enhancer (E) and UCSC genome (F) browsers. FW: fetal weeks; V: VISTA enhancer elements; U: UCNEs; U⁺: Putative active regulatory UCNE; ∩: intersection. Figure 1a was created with BioRender.com.

Fig. 2. Target genes under putative regulation of characterized UCNEs in retina.

a Barplot showing the number of associated genes per retinal UCNE. b Distance distribution from the TSS and its associated UCNE. c Gene ontology for the UCNE-associated target genes. An adapted Fisher’s exact test as in Chen et al. (EnrichR) was performed, see also Supplementary Data 3. GO Gene Ontology, TSS transcription start site.

Fig. 3. Contribution of UCNE genomic variation to missing heritability in rare eye diseases.

a Overview of a sub-cohort comprising n = 3206 participants of the 100,000 Genomes Project affected by posterior segment abnormalities (n = 2615), anterior segment abnormalities (n = 360), and ocular malformations (n = 245). b Variant population frequencies within putative retinal UCNEs normalized to a background composed of randomly selected sequences (see “Methods”). c Significantly lower genome-wide residual variation score (gwRVIS) values scores are observed across UCNEs and their flanking regions compared to the background composed randomly selected sequences, thereby showing the high intolerance to variation of UCNEs (*p < 0.001, Kruskal–Wallis rank sum test followed by post-hoc Dunn test with Bonferroni correction; Z-scores = −169.33 (Flanking-Random), −170.36 (UCNE-Random). Figure 3a was created with BioRender.com.

Fig. 4. Characterization of the cis-regulatory landscape of PAX6 in human retina.

a Visualization of the epigenomic context of the identified PAX6-associated UCNE (highlighted in gray) in relation to the PAX6 locus (top; chr11:31,490,261–32,075,591) and b Zoomed-in of the identified element (bottom; chr11:31,965,000–31,972,000). Figures are obtained from the UCSC genome browser.

Fig. 5. In vivo evaluation of the PAX6-associated UCNE.

a This UCNE displays reporter activity in the human embryonic forebrain (6/9 embryos) as well as in other structures including eye (3/6 embryos) and neural tube. Figure obtained from the VISTA enhancer browser (hs855). b Zebrafish enhancer assays (n = 2, see also Supplementary Data 10) at 3 different time points (1 dpf, 2 dpf, 3 dpf) showing GFP-positive tissues (optic fissure −1 dpf-, lens cells −2,3 dpf-, and forebrain −2,3 dpf-; white arrows). dpf days post-fertilization.