Supplementary Figure 3: In vivo evidence supporting transcription-factor condensate on MegaTrans enhancers and role of IDR.
From: Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly
a. RNA FISH showing localization of transcribing TFF1 locus to mCherry-ERα foci. The non-E2 target loci are not recruited to the foci b. Quantitation of RNA FISH measuring median distance between ERα-mCherry signal and an E2 target (TFF1 intron, n = 32) and non-E2 target (DYRK1, n = 42). Boxes represent interquartile ranges (IQRs). Whisker represents points in lower and upper quartiles within 1.5 IQR from lower and upper edges of IQR. P-values were calculated using Wilcoxon Rank Sum test with continuity correction. c. Representative FRAP experiment images of ERα-Turquoise transfected MCF7 cell, treated with 1hr of E2 at indicated time periods. Regions outlined in circles indicate bleached foci. Data representative of >10 cells in each time point d. Efficient shRNA-dependent depletion of GATA3 mRNA, measured using Q-PCR using GATA3 mRNA primers, in a clonal MCF7 cell line. Data shown are individual values as mean ± s.d. P-value calculated with unpaired Student’s t-test. Data are representative of 3 independent experiments. shCTL represent non-targeting control shRNA. e. Western blot showing equivalent expression of HA-tagged GATA3-WT and GATA3-IDR mut in MCF7 cells. f. GATA3-WT but not GATA3-IDR mutant can rescue expression of FOXC1 mRNA. Results are shown as individual data points (circles), mean ± s.d. Data is representative of 3 independent experiments.
