Fig. 4: The recruitment of RNA polymerase by FET fusion protein condensates promotes gene transcription activity in vitro.
From: Loci-specific phase separation of FET fusion oncoproteins promotes gene transcription
a Schematic of in vitro single-molecule biomolecular condensate-induced transcription assay for FUS-Gal4 (i). Wide-field TIRFM images of nascent RNA transcripts (iii) colocalized with FUS-Gal4 condensates (ii) after incubation. White arrows in (iii) confirmed the labeled punctum was on DNA. The insert was representative colocalized puncta. DNA substrates were Lambda DNA containing 7× Gal4DBD binding sites. The working buffer was 40 mM Tris-HCl (pH 7.5), 25 mM KCl, and 2 mM MgCl2, 1 mM DTT, and 0.2 mg/ml BSA. b The control experiment of a: the working buffer containing 150 mM KCl in this transcription assay. c The control experiment of a: no FUS-Gal4. d The control experiment of a: DNA substrates were wild-type Lambda DNA (no 7× Gal4DBD binding sites). e The control experiment of a: no Pol II CTDN26-T7 RNAP. f Transcription efficiency for different experimental conditions for FUS-Gal4 from a to d. Independent DNA Curtains experiments were repeated: n = 3 for a, n = 4 for b to d. Error bars, mean ± s.d. g Schematic of in vitro single-molecule biomolecular condensate-induced transcription assay for EWS-FLI1 (i). Wide-field TIRFM images of nascent RNA transcripts (iii) colocalized with EWS-FLI1 condensates (ii) after incubation. DNA substrates were Lambda DNA containing 25× GGAA repeats. White arrows in (iii) confirmed the labeled punctum was on DNA. The working buffer contained 25 mM KCl in a, c, d, e, and g.
