Mapping the cis-regulatory architecture of the human retina reveals noncoding genetic variation in disease

Abstract

The interplay of transcription factors and cis-regulatory elements (CREs) orchestrates the dynamic and diverse genetic programs that assemble the human central nervous system (CNS) during development and maintain its function throughout life. Genetic variation within CREs plays a central role in phenotypic variation in complex traits including the risk of developing disease. We took advantage of the retina, a well-characterized region of the CNS known to be affected by pathogenic variants in CREs, to establish a roadmap for characterizing regulatory variation in the human CNS. This comprehensive analysis of tissue-specific regulatory elements, transcription factor binding, and gene expression programs in three regions of the human visual system (retina, macula, and retinal pigment epithelium/choroid) reveals features of regulatory element evolution that shape tissue-specific gene expression programs and defines regulatory elements with the potential to contribute to Mendelian and complex disorders of human vision.

Keywords: cis-regulatory element; enhancer; human; noncoding; retina.

Fig. 1.

Identification and characterization of active cis-regulatory elements (CREs) in human retina, macula, and RPE. (A) Schematic cross-section of the human eye with tissues commonly affected in inherited retinal diseases labeled. (B) Schematic of cis-regulatory control of gene expression. Transcription factors (TF1–5) bind in combination to promoters and enhancers to recruit cofactors including histone acetyl transferases (HATs) and basal transcriptional machinery such as RNA polymerase (PolII). HATs acetylate lysine residues on histones, including H3K27ac (Ac). PolII can induce transcription of enhancer RNAs (eRNAs). Both H3K27ac and eRNAs are associated with active CREs. (C) Schematic of study workflow. (D) Genome-wide DNA accessibility and H3K27ac in adult human retina, macula, and retinal pigment epithelium (RPE) and choroid, by the assay for transposase accessible chromatin (ATAC), DNase-seq, and H3K27ac ChIP-seq. Each accessible genomic region (ATAC or DNase-seq) is represented as a single horizontal line centered on the peak summit, with a window of ±1 kb. ATAC or DNase-seq signal is plotted in gray. H3K27ac ChIP-seq signal is plotted in green. For each tissue, windows of DNA accessibility and H3K27ac are ordered on highest to lowest total H3K27ac signal within the 2-kb window. For retina analyses, ATAC n = 8 samples from unrelated adults, H3K27ac n = 3, and RNA-seq n = 7. For macula analyses, ATAC n = 3, H3K27ac n = 3, and RNA-seq n = 3. For RPE analyses, DnaseI-seq n = 2, H3K27ac n = 2, and RNA-seq n = 3. (E) Expression of genes associated with DNA-accessible regions in adult human retina (seven individuals), macula (three individuals), and RPE/choroid (three individuals) as determined by sequencing total RNA from nuclei (Nuc-seq). Genes are ordered according to their corresponding accessible regions from D. (F) Representative gene loci showing custom UCSC browser tracks for ATAC-seq or DNase-seq, H3K27ac ChIP-seq, and total RNA Nuc-seq from adult human retina, macula, or RPE/choroid. (G) Enrichment of biological processes and phenotypes associated with candidate active CREs in each tissue according to analysis using the genome regions enrichment of annotations tool (GREAT) (24).

Fig. 2.

Differential CRE accessibility drives unique patterns of gene expression in human retina, macula, and RPE. (A) Overlap of DNA-accessible genomic regions in adult human retina, macula, and RPE/choroid. (B) Overlap of H3K27ac-enriched genomic regions that share DNA accessibility in adult human retina, macula, and RPE/choroid. (C and D) Representative tissue-specific gene loci showing custom UCSC browser tracks for ATAC-seq or DNase-seq, H3K27ac ChIP-seq, and total RNA Nuc-seq from adult human retina and RPE/choroid. (E) Pairwise comparison of H3K27ac signal among DNA-accessible regions from adult human retina, macula, and RPE/choroid (blue, RPE enriched; green, retina enriched; purple, macula enriched). (F) Cumulative distribution of gene expression associated with tissue-specific DNA-accessible/H3K27ac regions.

Fig. 3.

Coregulation of human retinal enhancers by combinatorial binding of TFs. (A) Position weight matrices (PWMs) of TF binding motifs enriched within accessible/H3K27ac+ genomic regions from adult human retina, macula, and RPE/choroid. (B) Genome-wide distribution of DNA accessibility, H3K4me2, H3K27ac, and TF binding in adult human retina by the assay for transposase accessible chromatin (ATAC) (n = 7) and H3K4me2 (n = 3), H3K27ac (n = 3), or TF ChIP-seq (see Materials and Methods for n). Each genomic region is represented as a single horizontal line centered on the peak summit, with a window of ±1 kb. Genomic regions are ordered on highest to lowest total H3K27ac signal. (C) ABCA4 gene locus showing custom UCSC browser tracks for ATAC-seq, H3K27ac, and TF ChIP-seq and total RNA Nuc-seq from adult human retina. Individual candidate CREs (e1-e7, promoter) are highlighted in gray. (D) A candidate enhancer (e1) upstream of the ABCA4 gene showing custom UCSC browser tracks for TF binding motif mutations (black), TF binding motifs (purple, CRX; red, OTX2; blue, MEF2; green, ROR), ATAC-seq, H3K27ac, H3K4me2, and TF ChIP-seq and vertebrate conservation from adult human retina. Area highlighted in gray corresponds to sequence assayed in E. (E) Luciferase reporter assay comparing activity of the consensus human enhancer sequence highlighted in D to induced mutations of individual TF binding motifs (*P < 0.05).

Fig. 4.

Deconvolution of cell type-specific regulatory elements. (A) t-Stochastic neighbor embedding (tSNE) plot of global gene expression of 4,763 single nuclei from adult human retinal cell nuclei; nuclei are colored according to 14 unsupervised clusters. (B) Proportion of cell classes in the human retina identified according to known marker gene expression in single retinal nuclei (n = 3 biological replicates) (SI Appendix, Fig. S4A). (C) Marker gene expression of individual photoreceptor cell types (RP1, rod; PDE6H, pan-cone; OPN1LW, long-wavelength [red] cones; OPN1SW, short-wavelength [blue] cones). (D) Epigenetic features at photoreceptor cell type-specific marker gene loci shown by custom UCSC browser tracks for ATAC-seq, H3K4me2, H3K27ac, and TF ChIP-seq and total RNA Nuc-seq from adult human retina.

Fig. 5.

Conservation and divergence of CRE function in the human and mouse retina. (A) Rhodopsin gene locus showing custom UCSC browser tracks for ATAC-seq, H3K4me2, H3K27ac, and TF ChIP-seq and total RNA Nuc-seq from adult human or mouse retinas. Individual candidate CREs are highlighted in green. (B) CABP5/Cabp5 gene locus showing custom UCSC browser tracks for ATAC-seq, H3K4me2, H3K27ac, and TF ChIP-seq and total RNA Nuc-seq from adult human or mouse retinas. Individual candidate CREs are highlighted in green. (C and D) t-Stochastic neighbor embedding (tSNE) plot of 31,528 single cells from the mouse retina from ref. (C) or 4,763 single-cell nuclei from adult human retinas (D) according to global gene expression; nuclei are colored by unsupervised clusters (BPCs, clusters composed of bipolar cells; rods, clusters composed of rod photoreceptor cells). (E and F) Expression of Cabp5/CABP5 among single retinal cells/nuclei in the mouse and human retina, respectively.

Fig. 6.

Developmentally dynamic DNA accessibility underlies distinct biological functions, pathologies, and regulatory mechanisms. (A) Dynamic DNA accessibility during human retinal development. Each accessible genomic region (ATAC or DNase-seq) is represented as a single horizontal line centered on the peak summit, with a window of ±1 kb. ATAC (adult) or DNase-seq (developing) signal is plotted in black. For each developmental stage, windows of DNA accessibility are ordered on highest to lowest total accessibility signal. (B and C) Developmentally dynamic DNA accessibility at the ATOH7 (B) or Rhodopsin (C) gene loci displayed as custom UCSC browser tracks for DNase-seq (developing) or ATAC-seq (adult) from a time course of human retinal development and CTCF, H3K4me2, and H3K27ac ChIP-seq and total RNA Nuc-seq from adult human retina (red box, 6.5 kb deleted ATOH7 enhancer region resulting in inherited human nonsyndromic congenital retinal nonattachment; NCRNA; ref. 16). (D) Enrichment of biological processes and phenotypes associated with developmentally dynamic candidate CREs in the human retina according to analysis with the genome regions enrichment of annotations tool (GREAT). (E) TF binding motif enrichment at regions that are accessible in developing or adult human retina.

Fig. 7.

Disease gene-associated CREs and noncoding variation. (A) Heatmap of human retinal disease gene expression by tissue type. (B) SNP and indel frequency at disease gene-associated CREs (gnomAD, MAF 0.01) (8,685 indels, 15,306 SNPs). (C) The RDH12 gene locus showing custom UCSC browser tracks for ATAC-seq, H3K4me2, H3K27ac, and TF ChIP-seq and total RNA Nuc-seq from the adult human retina. Presumptive promoter region highlighted in gray. (D) Consensus DNA sequence centered around promoter ATAC-seq peak summit. Single-nucleotide variants (SNVs) (red with black highlight) found in unrelated individuals with Leber congenital amaurosis/retinitis pigmentosa. (E) Luciferase reporter assay comparing the relative activities of RDH12 consensus and variant promoter constructs (***P < 0.0005).