Characterization of chromatin architecture established at enhancers and super-enhancers at 4 hpf. (A) Super-enhancer (SE) plot using the ROSE algorithm, which ranks enhancers based on histone H3K27ac (Zhang et al. 2018) and H3K4me1 (Bogdanovic et al. 2012). ChIP-seq data at 4 hpf in zebrafish embryos. Data are separated into four groups: Group 1 (purple), Group 2 (red), Group 3 (blue), and SE group (green). N = total number in each partition. (B) Heatmaps of insulation score at enhancers. Insulation maps at 4, 5.3, and 24 hpf ranked by insulation strength at 4 hpf, centered on enhancers from each respective group in Figure 4A. Positive insulation (red) indicates increased contacts, and negative insulation (blue) indicates a lack of contacts. (C) Comparisons of chromatin factor and attribute occupancy at enhancers. Metaplot of log2 fold enrichment of histone H3K27ac ChIP-seq (Zhang et al. 2018), RNA Pol II ChIP-seq, Rad21-cohesin ChIP-seq, ATAC-seq, and Click-iT-seq (Chan et al. 2019) signal over input are plotted, centered on enhancers from each respective group in Figure 4A: super-enhancers (Super Enh, green), Group 3 (blue), Group 2 (red), and Group 1 (purple). (D) Proportional distribution of different enhancer regions; no enhancer (No Enh, yellow), super-enhancers (SE, green) Group 3 (Grp3, blue), Group 2 (Grp2, red), and Group 1 (Grp1, purple) detected with positive insulation score 0.1–0.2, 0.2–0.3, >0.3. The proportional distribution of each positive insulation score detected over the entire genome is depicted on the right; 0–0.1 (63%), 0.1–0.2 (27%), 0.2–0.3 (8%), >0.3 (1%). The bracket highlights the positive insulation score used in the bar graph on the left. (E) Groups from A overlap with ATAC-seq peak signal across enhancers regions were analyzed using HOMER Motif Analysis to determine potential TF binding. Similarity to known binding motifs is indicated by Pearson R values in shaded red, and motif frequency is indicated by circle size. T-box transcription protein family of motifs (TBX Fam), Kruppel-like factor protein family of motifs (KLF FAM), SRY-box transcription factor protein family of motifs (SOX).
