Fig. 4: Most TGFβ-dependent genes are in the TGFβ-regulated enhancers neighborhood.

a Change of mRNA levels (RNA-seq signal of 2 h TGFβ versus vehicle) for genes upstream or downstream of an early-activated (left) or early-repressed (right) enhancer, as in the scheme (red boxes), or of a randomly selected enhancer (gray boxes). For randomization, see Methods. Eg±n (whereby n = 1, 2, 3 or 4) depicts genes that occupy the first, second, third, or fourth positions, respectively, in the chromosomal order, upstream (−) or downstream (+) of the enhancer. Plots for late-enhancer categories are shown in Supplementary Fig. 8a. b Change of mRNA levels (RNA-seq signal of 2 h TGFβ versus vehicle) of genes located at the indicated distance (kb) upstream or downstream of an early-activated (top left) or early-repressed (top right) enhancer. Inclusion of the genes in the interval was determined by the position of their TSSs. Lower panels represent the corresponding data when enhancers were randomized. Plots for other enhancer categories are shown in Supplementary Fig. 8b. c Correlation plot between changes of mRNA levels (RNA-seq signal of 2 h TGFβ versus vehicle) of the first, second, third, or fourth closest genes respect to an enhancer, versus the change of H3K27ac of the enhancer (ChIP-seq signal of 2 h TGFβ versus vehicle). Data were binned into 20 intervals. Spearman correlation coefficient and slope of the regression line are shown. d Change of mRNA levels (RNA-seq signal of 2 h TGFβ versus vehicle) for genes that are located around an early-activated enhancer. Only enhancers for which the second-closest gene (marked in red) upstream (Eg-2) or downstream (Eg+2) was robustly upregulated (log₂ FC > 1; adjusted p < 0.05) were considered. Gray color boxes correspond to change of mRNA levels of genes around a random enhancer. Plots for other enhancer categories are shown in Supplementary Fig. 9. a, b, d Statistical significance between real and random distributions were determined with the two-tailed Mann–Whitney test, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Sample size of each distribution and exact p-values are provided in Supplementary Data 5. The horizontal black line of the boxplot represents the median value, the box spans the 25th to 75th percentiles, and whiskers indicate 5th and 95th percentiles. Source data are provided as a Source Data file.