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. 2017 Feb;27(2):259-268.
doi: 10.1101/gr.203679.115. Epub 2016 Dec 13.

Comparative Analyses of Super-Enhancers Reveal Conserved Elements in Vertebrate Genomes

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Free PMC article

Comparative Analyses of Super-Enhancers Reveal Conserved Elements in Vertebrate Genomes

Yuvia A Pérez-Rico et al. Genome Res. .
Free PMC article

Abstract

Super-enhancers (SEs) are key transcriptional drivers of cellular, developmental, and disease states in mammals, yet the conservational and regulatory features of these enhancer elements in nonmammalian vertebrates are unknown. To define SEs in zebrafish and enable sequence and functional comparisons to mouse and human SEs, we used genome-wide histone H3 lysine 27 acetylation (H3K27ac) occupancy as a primary SE delineator. Our study determined the set of SEs in pluripotent state cells and adult zebrafish tissues and revealed both similarities and differences between zebrafish and mammalian SEs. Although the total number of SEs was proportional to the genome size, the genomic distribution of zebrafish SEs differed from that of the mammalian SEs. Despite the evolutionary distance separating zebrafish and mammals and the low overall SE sequence conservation, ∼42% of zebrafish SEs were located in close proximity to orthologs that also were associated with SEs in mouse and human. Compared to their nonassociated counterparts, higher sequence conservation was revealed for those SEs that have maintained orthologous gene associations. Functional dissection of two of these SEs identified conserved sequence elements and tissue-specific expression patterns, while chromatin accessibility analyses predicted transcription factors governing the function of pluripotent state zebrafish SEs. Our zebrafish annotations and comparative studies show the extent of SE usage and their conservation across vertebrates, permitting future gene regulatory studies in several tissues.

Figures

Figure 1.
Figure 1.
Identification of typical enhancers and SEs in vertebrate genomes. (A) Workflow for the identification of vertebrate typical enhancers and SEs. Schematic representations depict the cells and tissues analyzed. (B) Saturation curves of H3K27ac density across brain data sets (whole brain for zebrafish, olfactory bulb for mouse, and middle frontal lobe for human). The number of ranked typical enhancers and SEs by H3K27ac density (x-axis) and their densities (y-axis) are plotted. Horizontal dotted lines represent density cutoffs used for the classification of SEs and vertical dotted lines demark SEs from typical enhancers. The total number of predicted SEs is noted on the right side of each graph.
Figure 2.
Figure 2.
Genomic distribution of typical enhancers and SEs. (A) Density plots representing the proportion of genes (y-axis) covered by typical enhancers and SEs in the vicinity of TSSs (x-axis) in zebrafish brain, mouse cerebellum, and human angular gyrus. (B) Proportion of gene bodies overlapping with typical enhancers, SEs, and control regions (y-axis) in different zebrafish, mouse, and human cells and tissues (x-axis). The mean and the standard deviation (black bars) calculated from bootstrap analyses of control regions are shown. All comparisons between typical enhancers and SEs and their controls have significant differences (P-values from z-scores ≤3 × 10−4), with the exception of zebrafish pluripotent state and heart typical enhancers. (NS) Not significant. (C) Distribution of typical enhancer and SE sequences across genomic features. The y-axis shows the percentage of total brain typical enhancer or SE base pairs overlapping the different genomic features represented in the legend. Adult brain data sets for mouse and human correspond to olfactory bulb and cingulate gyrus, respectively.
Figure 3.
Figure 3.
Cell and tissue specificity of vertebrate typical enhancers and SEs. (A) Distribution of H3K27ac at selected genes (genomic position represented on the x-axis) in both pluripotent state and adult brain of zebrafish, mouse, and human (raw tag counts represented on the y-axis). Typical enhancers and SEs are denoted by gray bars and red bars, respectively. (B) Chow-Ruskey diagrams representing the overlap between pluripotent state (orange), brain (green), heart (purple), intestine (red), and testis (blue) typical enhancers and SEs in zebrafish. Color-coded tables show the percentages of cell- or tissue-specific and nonspecific regions for each data set.
Figure 4.
Figure 4.
SE conservation in vertebrates. (A) Metagenes of sequence conservation of typical enhancers and SEs from zebrafish whole brain, mouse olfactory bulb, and human middle frontal lobe. The x-axis depicts the start and end of typical enhancers and SEs flanked by 3 kb of adjacent sequence. The y-axis represents sequence conservation calculated by PhastCons. (B) Venn diagrams show the number of orthologous genes associated with brain typical enhancers (left) and SEs (right) in zebrafish (green), mouse (blue), and human (purple). Color-coded tables show the percentages of intersection and difference for each species. The observed differences in overlap between typical enhancers and SEs in the three species are significant (P-values ≤5.497 × 10−8) based on G-tests of independence. (C) ChIP-seq binding profiles for H3K27ac at the indicated loci in zebrafish, mouse, and human brain (raw tag counts represented on the y-axis). Typical enhancers and SEs are denoted by gray bars and red bars, respectively. Gene positions are noted along the x-axis. (D) Box plots depicting average sequence conservation of brain SEs with maintained orthologous association in zebrafish, mouse, and human and with no maintained orthologous association. The y-axis shows sequence conservation calculated by PhastCons. The box bounds the interquartile range divided by the median, and the notch approximates a 95% confidence interval for the median. All observed differences in conservation between SE categories are significant (P-value ≤9.1 × 10−3) based on Wilcoxon rank-sum tests.
Figure 5.
Figure 5.
Analysis of zebrafish SE composition by ATAC-seq. (A) Venn diagrams representing the overlap between ATAC-seq peaks (purple) and Nanog peaks (orange) genome-wide (left) and within pluripotent state SEs (right). (B) Cluster, consensus motif sequence, and logos of SOX-related de novo–found motifs in ATAC-seq peaks within SEs (left). JASPAR matrix models (right) of SOX2, SOX9, and ESRRA. (Ncorr) Normalized correlation between identified motifs and JASPAR models. (C) Top molecular function and wiki pathway GO terms enriched for the ATAC-seq peaks containing sites of the de novo identified oligos_7nt_m2 (left) and oligos_6nt_m3 (right) motifs shown in B. Binomial FDR q-values for the GO terms are displayed in a color-scale (q-values ≤6.7 × 10−4).
Figure 6.
Figure 6.
Functional analysis of vertebrate SEs. (A) Genomic context and conservation of the zebrafish (left) and mouse (right) irf2bpl and Irf2bpl loci. Horizontal bars represent SEs (red). Raw H3K27ac ChIP-seq, ATAC-seq, and Nanog ChIP-seq profiles are shown in tag counts (y-axis). The TFBS track represents the TFBS enrichment along the mouse locus. The Vertebrate Cons tracks represent conservation scores calculated by PhastCons. Gray and green highlighted regions correspond to the regions tested in reporter assays. Regions driving specific GFP expression are indicated in green. (B) GFP expression driven by the zebrafish SE-irf2bpl D region (left) and the mouse K region (right) in transgenic zebrafish embryos at 48 hpf. White arrows indicate the olfactory placode (op). (C) Genomic context and conservation of the zebrafish and mouse zic2a and Zic2 loci as described in A. Horizontal bars represent typical enhancers (gray) and SEs (red). (D) GFP expression driven by the zebrafish P, Q, and S regions (left) and the mouse T region (right). (h) Hindbrain, (nt) notochord, (r) retina, (rp) roof plate, (sc) spinal cord, (t) telencephalon.

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