Journal: Nature structural & molecular biology
Article Title: Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly
Figure Lengend Snippet: Acutely active E 2 -responsive MegaTrans enhancers concentrate a protein complex that can undergo phase transition. a , Schematic representation of the ERα/MegaTrans complex recruited to E 2 -activated enhancers, which transcribe eRNAs and recruit the condensin I/II complexes. b , Western blot analyses showing that ERα, several MegaTrans components and condensin component SMC4 are precipitated by biotinylated isoxazole (b-isoxazole). FUS and GAPDH proteins are used as a positive and negative control for the assay, respectively. WCL, whole-cell lysate. c,d , FRAP data on phase-separated droplet formed in vitro by purified recombinant GATA3-maltose binding protein (MBP) ( c ) and ERα-MBP ( d ). Top, charts show individual data points represented by dots, lines represent fitting to an exponential model to estimate the half-time of recovery. Bottom, representative images of in vitro droplets before and after photobleaching. e , Fluorescence microscopy images of a representative nucleus from MCF7 cells transfected with ERα-mCherry, before (−E 2 ) or after (+E 2 , 5 or 15min) E 2 treatment. Scale bar, 2 μm. f , Mean intensity and photobleaching normalized fluorescence of ERα-mTurquoise foci in E 2 treated MCF7 cells relative to pre-bleaching signal. Error bars represent s.e.m. of n ≥10 cells per time point. g , Levels of eRNA from indicated enhancers, measured by reverse transcription PCR, from MCF7 cells depleted of endogenous GATA3 and expressing either wild type or IDR-deleted GATA3 (GATA3-IDR mut), after 1 h E 2 stimulation. shCTL indicates non-targeting control shRNA. The IDR (aa 2–250) is shown in the schematics on top. Results are shown as individual data points (circles), mean±s.d. (lines). P values were calculated with an unpaired Student’s t -test. Data are representative of three independent experiments.
Article Snippet: Protein supernatant was collected by centrifugation at 4 °C, 16,500g for 15 min. Then, 5% lysates were saved for whole cell extract control and remaining were aliquoted equally and incubated with various concentrations (10, 30 and 100 μM) biotinylated isoxazole (Sigma, catalog no. 900572) at 4 °C for 1 h with rotation.
Techniques: Sublimation, Western Blot, Negative Control, In Vitro, Purification, Recombinant, Binding Assay, Fluorescence, Microscopy, Transfection, Polymerase Chain Reaction, Expressing, shRNA