Fig. 3: Putative enhancer regions have characteristic enhancer features and activity.

a Heat map representation of RPM normalized Pol II occupancy measured by HiS-NET-seq for individual nucleotides at putative enhancer regions. Signal outliers above the 99.99-quantile were excluded. b Distribution of transcriptional activity at putative enhancers for two biological HiS-NET-seq replicate measurements. c Spearman’s correlation analysis of Pol II occupancy per putative enhancer for HiS-NET-seq and ENCODE’s H3K27ac data (¹⁰³; ρ = 0.6). d Quantification of TPM normalized Pol II occupancy measured by HiS-NET-seq at putative enhancer (n = 12,637, described in Methods) and promoter regions (n = 9454, two-sided Wilcoxon rank sum test; ****p < 2.2e−16). The boxplot shows the median values as the center. The box is defined by the first to the third interquartile range. The whiskers extend this interquartile range by a factor of 1.5, not exceeding the minimum or maximum values. Outlier measurements that exceed the whiskers are not shown. e Schematic view of the enhancer assay. The putative enhancer sequence was tested in sense (S) and antisense (AS) orientation for its ability to increase the expression of the Firefly luciferase (Luc) compared to the control. As a negative control, an early SV40 promoter (P) driven luciferase reporter was used. f Luciferase reporter assay measuring enhancer activity in K562 cells. Putative enhancer sequences were tested in sense (S)- and antisense (AS) directions. Luciferase (Luc) activity was normalized to that of a co-transfected Renilla luciferase and further normalized to the negative control. The HS2 minimal enhancer sequence¹¹⁰ served as a positive control. Mean fold change +/− standard deviation was calculated and statistical significance was determined using a two-sided t-test (***p < 0.001, **p < 0.002, *p < 0.033, ns: 0.12, ns: not significant). All constructs were tested in biological triplicate, except control construct HS2 S, #2, #3, #5, #6, #8, and #9S were tested in biological quadruplicate. Source data are provided as a Source Data file. a–f Data were obtained for human K562 cells. a, c, d Mean HiS-NET-seq signals from two biological replicate measurements are shown.