Fig. 2: NDF and FACT form phase-separated condensates.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Microscopic images show that neither NDF−GFP (monomeric GFP) nor FACT−mCherry (Spt16−mCherry + SSRP1) forms droplets at various concentrations in a 110 mM NaCl buffer at room temperature. b, Representative images of droplet formation when equimolar concentrations of NDF−GFP and FACT−mCherry are mixed at room temperature in a 110 mM NaCl buffer, without PEG (LLPS, liquid–liquid phase separation). Images taken with a Zeiss epifluorescence microscope. c, SDS−PAGE image from a sedimentation experiment illustrates the distribution of FACT and NDF in the aqueous (supernatant (S)) and condensed (pellet (P)) phases, mixed at specified concentrations in a 100 mM NaCl buffer without PEG. d, Normalized densitometry data from a reversibility assay where absorbance at 395 nm was measured for droplets formed with 3 µM FACT and 6 µM NDF, and after sequential 1:1 dilutions with and without added NaCl to 430 mM. Data represent mean ± s.d. from three independent experiments in technical triplicate. e, FRAP experiment images of NDF−GFP and FACT−mCherry condensates (equimolar at 0.8 µM) captured at specified times post-photobleaching (1 s). The panel labeled ‘0 s’ corresponds to the pre-bleaching image; note that for quantitative FRAP analysis, time zero is defined as immediately after bleaching. f, Statistical FRAP recovery analysis shows the relative intensity of FACT−mCherry and NDF−GFP signals over time in in vitro formed droplets. Data are presented as average relative intensity ± s.d. (n = 15 puncta, technical replicates). g, Partially overlapping but distinct NDF−FACT puncta do not merge or mix. Mixed droplets of (NDF−GFP + FACT) with (NDF + FACT-mCherry) were incubated at room temperature for 10 min before imaging. Scale bar = 1.5 µm. h, Microscopic images display the incorporation of Cy2-labelled nucleosomes into pre-formed (NDF + Spt16−mCherry) condensates at room temperature. Protein concentrations were 1.2 µM. i, Distribution of transcription products in condensate phases. The transcription reactions were subjected to centrifugation to separate condensates (C) from the total reaction (T), and 0.2 µM NDF or FACT proteins were used for the reaction. RNA gel showing nucleosome-specific pausing at SHL −4/−5 and SHL −2 is reduced in the condensate fraction (lane 4). All lanes cropped from the same gel under identical conditions. j, Experimental scheme for an RNase A (ng µl−1) protection assay with condensate-associated (C) and non-condensate transcripts (S) in the same reaction. In a−c and g−j, representative results from three independent experiments are shown. k, Overlay example image of STORM super-resolution microscopy of 6-FAM-labelled RNA positions (green/yellow) relative to condensate boundaries (red, captured by epifluorescence microscope, condensates were formed with purified NDF and FACT−mCherry) from 169 image acquisitions. Representative of two independent experiments. l, Quantitative analysis of RNA localization within condensates. The left axis shows the ratio of condensate radius to centre-to-centre distance (distance between condensate centre and RNA centre). Values >1 indicate RNA is located inside condensates, while values <1 would indicate RNA outside condensates (n = 26 technical replicates).
