Fig. 1: NDF interacts with FACT and synergistically stimulates transcription.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Proteins identified by mass spectrometry following tandem affinity purification of NDF−Flag−GFP from HeLa cells, listed from three independent experiments. b, Co-immunoprecipitation (IP) of the endogenous NDF interaction with Spt16 in HeLa cells. c, Interaction between recombinant NDF−Flag and the FACT complex analysed by a FLAG pull-down assay. In b and c, representative results from three independent experiments are shown. d, A schematic of Pol II−nucleosome complexes for in vitro transcription elongation assays. The major pausing sites at nucleosome SHL −5 and SHL −1 are labelled as bp1 and bp42, respectively. e, A nucleosome transcription assay shows an increase in the full-length (run-off) product in the presence of 0.1 µM, 0.2 µM, 0.4 µM or 0.8 µM NDF or FACT and shows synergy in the presence of equimolar amounts of both NDF and FACT. Transcription reactions were performed with 1.0 mM NTPs for 10 min at room temperature. RNA length and corresponding nucleosomal base pairs are indicated. f, Quantification of run-off products from the experiment shown in e, based on three biological replicates, normalized to run-off product from 0.1 µM NDF alone. Mean values and s.d. are displayed, with black dots representing individual data points. The reaction rates were calculated using Michaelis−Menten analysis: for NDF alone, Vmax = 1.97, Km = 0.11, goodness of fit (R2) = 0.67; for FACT alone, Vmax = 11.22, Km = 0.40, R2 = 0.92; and for equimolar NDF and FACT together, Vmax = 33.09, Km = 0.10, R2 = 0.94. A P value of 1.02 × 10−9 was obtained from the one-way analysis of variance (ANOVA) test for the 0.8 µM concentration reactions.
