Fig. 4: NDF−FACT condensate formation, not merely protein interaction, is essential for synergistic transcription enhancement.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Domain scheme of the NDF protein and the focused regions that are required for interaction with Spt16. b, Amino acid sequence alignments around the second Spt16 interaction domain from different species. c,d, Interaction analysis between NDF mutants and Spt16 using Ni-NTA pull-down assays. PWWP-less NDF mutants (K161A and R162A) show abolished interaction with Spt16 (c), while full-length versions of the same mutants retain Spt16 binding due to intact PWWP domain interaction (d). e, Representative fluorescence microscopy images showing condensate formation between NDF−GFP variants (WT, K161A, R162A and KR; 25 nM each) and FACT−mCherry (0.2 μM) in droplet formation buffer at room temperature. f, Droplet formation between NDF-WT−GFP, NDF-KR−GFP and FACT−mCherry at 200 nM for FACT−mCherry and specific concentrations for NDF variants in droplet formation buffer. Scale bar, 10 μm. g, Nucleosome transcription assay with full-length NDF-WT (25 nM and 50 nM), NDF-K161A (25 nM and 50 nM) or NDF-R162A (25 nM and 50 nM) mutants, together with 200 nM FACT. Transcription reactions were performed with 0.25 mM NTPs for 10 min. h, Statistical analysis of the run-off product from g (mean ± s.d., n = 3 biological replicates). Normalized to run-off product from FACT alone (*P < 0.05 with a two-tailed t-test). i, Nucleosome transcription assay with 0.2 µM NDF and NDF_F1 truncates and FACT with 0.3 µM TFIIS. Transcription reactions were performed with 0.25 mM NTPs for 10 min at 30 °C. Representative results from three independent experiments are shown in a−f and i.
