Enhancer-driven 3D chromatin domain folding modulates transcription in human mammary tumor cells

(A) Experimental flow used to interrogate 3D genome organization in MCF7 cells treated or not with E2. (B) Interaction frequency 5C heatmaps of the progesterone receptor gene domain in estrogen starved (−E2) and stimulated (+E2) for 45 min and 3 h MCF7 cells. TAD boundaries do not change in MCF7 cells (red arrows) and ESRBs are indicated (orange arrows). Increased (green arrowhead) and lost (red arrowhead) contact frequencies are indicated. (C) Quantification of estrogen receptor binding site (ERBS–ERBS) interactions is calculated from 5C heatmaps. Representing the gain (first row) and lost (second row) of interactions between specific ERBSs. (D) Quantification of ERBS–transcription start site interactions showing variation in interaction with different ERBSs.

(A) Genomic position of fosmid probes used for 3D DNA FISH analysis. (B) Representative images from 3D imaging of dual-fosmid labeled and DAPI co-stained nuclei of MCF7 cells stimulated (+E2) or not (−E2) with 100 nM estradiol. Fos4 labeled with Alexa488 (green), Fos5 in red (labeled with Atto 647). Maximal projection of three planes is presented (0.2 μm per single plane). See MM for details. Scale bar 5 μm. (C) The violin plots representing inter-probe distances for different pairs of fosmids (n = 80–130 nuclei). Fisher’s test: *P-values: >0.05 (ns), <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). (D) The pie charts illustrating the proportion of inter-probe distances >200 nm, interval from 200–100 nm (≤200 nm) and interval from 0–100 nm (≤100 nm) for different pairs of fosmids in MCF7 cell stimulated or not by E2. Fisher’s test was used for statistics. (E) Representative images from 3D imaging of triple-fosmid–labeled and DAPI co-stained nuclei of MCF7 cells stimulated (+E2) or not (−E2) with 100 nM estradiol. Maximal projection of three planes for Fos1 (in red), Fos3 (in green), and Fos5 (in blue) are presenting. Scale bar 5 μm. (F) Single-cell analysis of relative position of three loci simultaneously representing as survival zone distribution of Fos1, Fos3, and Fos5. See MM for details. (G) Angle around the Fos3 measured from mutual position of the three loci in single cell. Fisher’s test was used for statistics.

(A) 3D models 5C contact frequencies in MCF7 cells exposed (+E2) or not (−E2) to estradiol, 20% of the most frequent models using TADbit (Serra et al, 2017) with distances (d) between the PGR gene body and its upstream ERE region are displayed. (A, B) 5C map (normalized counts) of the PGR domain on Chr 11. Color bar below the map corresponds to domains in (A). (A) Distances derived from models shown in (A): gray arches link segments <200 nm in the maps, red and blue arches link segments for which distances increased (red) or decreased (blue) in at least 50% of the cell population after 45 min or 3 h of estradiol exposure.

(A) Kinetics of estrogen receptor α progressive binding on estrogen receptor binding sites within the regulatory domain of the progesterone receptor gene. Data analyzed from Guertin et al (2014). (B) Time-course of chromatin-associated activators (GATA3, P300, c-Fos, c-jun, MYC, FoxA1), necessary for estrogen receptor α-dependent gene activation showing their presence at early or late E2 response. (C) Time-course after estradiol addition to hormone-starved MCF7 cells indicating RNA Pol2 pausing index (in red), mRNA production measured by RNA-seq (in blue) and by GRO-seq (in gray).