Enhancer-promoter interaction frequencies and spatial separation depend on the ERα ligand.

A) ChIP-qPCR for ERα at the indicated regions in MCF-7 cells treated with vehicle (veh), E2 or tamoxifen (4OH) for 30 min. T-test, mean of 2 biological replicates shown as dots. B) As in (A), but for at RpolII occupancy. C) RT-qPCR of NRIP1 and GREB1 pre-mRNAs in MCF-7 cells treated with veh, E2 or 4OH for 1h. T-test, Bonferroni correction for multiple testing, mean +/- SD of 3 biological replicates. D) C-TALE heatmaps at GREB1 (left; 5kb resolution) and NRIP1 (right; 10kb resolution) loci from MCF-7 cells treated with veh, E2 or 4OH for 30 min. ERα +Veh, ERα +E2, ERα +4OH ChIP-seq data [23] and TT-seq +E2 30 min tracks are shown. Enhancer and promoter regions highlighted with yellow and grey bars, respectively. In (left) red-green-blue heatmaps, each pixel represents the normalized contact frequency between a pair of loci. In the red-white-blue heatmaps to the right each pixel represents the ratio between 4OH-treated samples and veh- or E2-treated samples. Arrowheads indicate pixels corresponding to enhancer-promoter interaction frequencies. Arrows indicate contacts across the GREB1 gene body. E) Virtual 4C plots derived from normalized contact frequencies shown in (D) and using the enhancer (e, left) and promoter (p, right) regions as viewpoints. Arrowheads indicate the promoters (left) and the enhancers (right). Genome coordinates: hg19. Data for a biological replicate are in S7A and S7B Fig. F) Representative images of nuclei (DAPI, blue) from cells treated with veh, E2 or 4OH and hybridised with fosmid probes targeted to the enhancer (e, green) and promoter (p, red) regions of GREB1 or NRIP1. Scale bar, 5μm. G) Violin plots showing the distribution of DNA FISH interprobe distances in cells treated with veh, E2 or 4OH using e-p fosmid probes at GREB1 or NRIP1 loci. Boxes indicate median and interquartile distances. Two-sided Mann-Whitney test, Holm-Bonferroni correction for multiple testing. n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data from a biological replicate are shown in S7C Fig. Statistical data for Fig 3 are in S3 Table.