Extended Data Fig. 7: MED1 IDR acetylation mutation effects on LLPS and Pol II incorporation.
From: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment
a, Colloidal blue staining for HIS-tagged EGFP-fused MED1 IDRs (6KR or 6KQ, 963-1320 aa) purified from E.coli. b, Reduced saturation concentration for 6KQ versus 6KR MED1 IDR. N = 4 (biological replicates). P value of two-sided unpaired t-test is shown. c, Colloidal blue staining for WT MED1 IDR fragment (fused to EGFP) and CBP (GST-tagged) purified from E.coli (left), and immunoblotting confirming the enforced acetylation of MED1 IDR (right). d, Droplets formed by WT MED1 IDR (fused with EGFP), with enhanced in-droplet partitioning of the acetylated versus control MED1 IDR. Representative images are shown (left) where the scale bars are 10 μm. The partition coefficient (inside versus outside droplets) is plotted, comparing control and the acetylated MED1 IDR (right). Bars report mean ± SEM. P value of two-sided unpaired t-test is shown. The parentheses show the numbers of droplets analyzed (across 3 biological replicates). e, Reduced saturation concentration for CBP-acetylated versus control WT MED1 IDR. N = 4 (biological replicates). P value of two-sided unpaired t-test is shown. f, g, Faster recovery of droplets formed by CBP-treated versus control WT MED1 IDR revealed by FRAP. Images for representative droplets (f) and recovery curves (g) are shown. Scale bar for images is 10 μm. Data points report mean ± SEM. Control and CBP-acetylated MED1 IDRs were tested for 6 and 7 droplets (across 3 biological replicates), respectively. P values (two-sided unpaired t-test) for each data point are shown. h, RNA Pol II CTD heptad sequences used in the Pol II co-condensation experiments (left) and colloidal blue staining of the mCherry-fused Pol II CTD heptads (right). i, j, Immunoblotting detection of Mediator subunits and Pol II (RPB1) remaining on beads after washing the purified Mediator complex from HeLa-S cells by buffers with increasing concentrations of salt (100/150/200/300 mM KCl) (i) or buffers with 1,6- or 2,5-Hexanediol (Hexn) at 2% or 10% concentration (j). For panels a, c, h–j, gel staining and immunoblots are representative of three independent experiments.
