Figure 6. Increasing the levels of local RNA synthesis reduces condensate formation and transcription in cells.

A. Scheme depicting the reporter system (left) where local RNA expression near a luciferase reporter gene can be induced by doxycycline.

B. Experimental design to test the effect of increasing local RNA levels on condensate formation and on reporter gene expression.

C. Live-cell imaging showing localization of Mediator condensates and MS2-tagged RNA expressed near the reporter gene at indicated dox stimulations. Med1-GFP mESCs have an integrated reporter system and 2xMCP-mCherry to visualize MS2-tagged RNA (2456 nt). Representative images are maximum projections that have been subtracted by a median filter and smoothed (STAR Methods).

D. Average density of MED1 signal centered at RNA signal at indicated dox stimulations (p=0.066 10 ng/mL vs 100 ng/mL Dox;p=0.013 10 ng/mL vs 1000 ng/mL Dox, p=0.315 100 ng/mL vs 1000 ng/mL, 2-way Kolmongorov-Smirnoff test).

E. Simulations predict the variation of condensate size with increasing effective rates of RNA synthesis (abscissa) (2D simulation grid). The condensate radius is normalized by value at rate=1 and RNA synthesis rates are normalized to k_p = 0.02 (STAR Methods)

F. qRT-PCR of various “feedback RNAs” with increasing dox concentrations. Markers show the mean of at least 3 replicates and error bars depict the S.D.

G. Luciferase luminescence with increasing dox concentrations. Markers show the mean of at least 3 replicates and error bars depict the S.D.

See also Figure S7