Supplementary Figure 5: Cluster enhancer determination and allocation.

(a) Spearman correlations for individual ATAC-seq samples (n = 2 each) based on the combined peaks of unstimulated (WT u) and 1-hour C. albicans-stimulated (WT Ca) HoxER-PU.1^WT neutrophils. (b) Expression of AP-1 transcription factors in the indicated neutrophils. All samples were n = 3 except for HoxER-PU.1^ΔNeu which was n = 2. Expression is shown as in reads per kilobase per million (RPKM) obtained from mRNA-seq. Bars represent mean ± s.e.m. expect for HoxER-PU.1^ΔNeu which is only shown as mean. (c) Scheme of the used enhancer determination strategy and their allocation to genes. Enhancers were determined as promoter distal regions marked with H3K4me1. Enhancers with differential H3K27ac occupancy in one condition were allocated to the next differential expressed gene within one TAD. Box indicates the active enhancer, which was allocated to the differentially expressed gene. (d) PU.1 occupancy on gene regulatory elements (promoters and enhancers) of the cluster genes. (e) Number of genes dedicated to clusters I-IV, number of promoters of cluster genes with PU.1 occupancy, number of enhancers which could be allocated to the cluster genes, the number of these enhancers with PU.1 occupancy and the number of expressed genes that could be allocated to the PU.1 bound enhancers. (f) Reporter activities of putative enhancers measured by luciferase assay in transiently transfected THP-1 cells. Data represent the mean of two (E3–5, negative regions), three (E2) or four (E1) independent experiments. (g,h) Integrative genomics viewer images showing tracks of normalized tag counts of H3K4me1 (K4me1), H3K27ac (K27ac) and PU.1 ChIP-seq as well as ATAC-seq in HoxER-PU.1^WT and HoxER-PU.1^ΔNeu neutrophils. Shown are the PU.1 activated gene loci of the cluster I gene Cd34 and the cluster II gene Il1b (g), and PU.1 inhibited loci of the cluster III gene Spp1 and the cluster IV gene Cd63 (h). Grey boxes indicate relevant enhancer positions. Sown are representative tracks of two independent samples each.