Fig. 4: Functional coupling of enhancer and promoter bursting.

a Transcriptional burst fractions of the Il12b HSS1 enhancer (left, 3–4 independent biological replicates, n = 14,759 cells) and Il12b (right, four independent biological replicates, n = 17,038 cells). b log₂-transformed burst fractions of the indicated enhancers and promoters in resting (UT) and LPS-activated macrophages (LPS). Two-way ANOVA formula (BF_Gene ~ BF_Enhancer*Gene). P value (P) for whether there is a relationship between the bursting of enhancers and genes (frequency_enhancer) = 1.94e-09. At least three independent biological replicates per gene and condition, n = 38093 cells. Error band: 95% confidence interval. c Bi-allelic enhancer (left) and promoter bursts (right) occur in individual cells more often than expected based on the burst fractions of enhancers and promoters (3–4 independent biological replicates, n = 14759 cells for Il12b HSS1 and n = 17038 cells for Il12b). Cochran–Mantel–Haenszel test (replicate as strata) with Bonferroni correction for multiple testing, two-sided considering a random population of the same size where the sampled frequency equals the expected frequency in case of bursting events happening independently. d Simultaneous application of enhancer (magenta) and target gene probes (green) illustrated for the Il12b locus. The position of probes is shown in Fig. 3a. More than eight biological and four technical replicates. e Experimentally observed (observed) co-occurrence of eRNA and mRNA transcriptional bursts over co-occurrence expected by chance (expected) at the level of individual cells. Cochran–Mantel–Haenszel test (replicate as strata) with Bonferroni correction for multiple testing, two-sided. At least three independent biological replicates per gene and condition, n = 38093 cells. f The distance distribution of Il12b intron - Il12b intron pairs, Il12b HSS1 enhancer—Il12b HSS1 enhancer pairs and Il12b intron—Il12b HSS1 enhancer pairs is plotted. A horizontal red line indicates a distance of 1 μm. The percentages of Il12b intron— Il12b intron pairs (1.2%), Il12b HSS1 enhancer—Il12b HSS1 enhancer pairs (4.1%) and Il12b intron—Il12b HSS1 enhancer pairs below 1 μm (46.8%) is indicated. Tukey HSD test (ANOVA design formula distance ~ pair + replicate), two-sided. g Box plots of experimentally observed (observed) co-occurrence of eRNA and mRNA transcriptional bursts over co-occurrence expected by chance (expected) at the level of individual alleles (i.e. at distances below 1 μm). Cochran–Mantel–Haenszel test (replicate as strata) with Bonferroni correction for multiple testing, two-sided. At least three independent biological replicates per gene and condition, n = 38,093 cells.