Fig. 6

blf and drl cluster genes restrict the expression of vasculogenesis- and monocytopoiesis-promoting genes. (A) Read densities and signal distribution heatmap of Blf and Drl CUT&Tag sequencing at gene-coding regions, with 3 kb upstream and 3 kb downstream of transcription start site (TSS) shown. (B) Genome-wide distribution of Blf and Drl CUT&Tag sequencing peaks. Promoter regions are defined as sequences within 3 kb of the TSS of annotated genes. Downstream regions are defined as sequences within 3 kb of the gene end. (C) Quantitative Venn diagram showing overlapping peaks and peak-associate genes between Blf and Drl in hematopoietic progenitors at 20 hpf. (D) De novo motif prediction on Drl-binding sites was performed on CUT&Tag data using HOMER. The top three motifs are shown. (E) Activating and repressive function prediction of Drl in hematopoietic progenitors. BETA software was used with default parameters (peaks within ±100 kb of TSS) to integrate Drl CUT&Tag binding sites with expression data from the RNA-seq profile in hematopoietic progenitors from control and blf−/−; drl 4KO animals (n=3). The yellow, blue and green lines represent genes activated, repressed or unaffected by Drl, respectively. The percentage of genes is cumulated by the rank of genes based on their regulatory potential scores. P-values (in parentheses) were derived from Kolmogorov–Smirnov tests. (F) Circular plot of 33 representative Drl direct target genes, simultaneously presenting a detailed view of the relationships between expression changes (left semicircle perimeter) and processes (right semicircle perimeter). Color code represents the log2(fold change) value. (G) Top: Drl CUT&Tag binding profiles at the etsrp locus. Bottom: RNA in situ hybridization of etsrp expression in control (n=37), blf−/−; drl 4KO (n=36) and drl overexpressing (n=30) embryos at the 22-somite stage; lateral view. Red arrowheads indicate ectopic etsrp expression in the intermediate cell mass. (H) Top: Drl CUT&Tag binding profiles at the irf8 locus. Bottom: RNA in situ hybridization of irf8 expression in control (n=25), blf−/−; drl 4KO (n=37) and drl overexpression (n=36) embryos at the 22-somite stage; frontal view. (I) RNA in situ hybridization of mpx expression in control (n=34) and blf−/−; drl 4KO embryos injected with control (n=26) or irf8 morpholino (n=30). Samples were collected at the 22-somite stage and are displayed as frontal view. Scale bars: 100 μm.