Extended Data Fig. 4: Molecular mapping of NDF-FACT interaction domains and their impact on condensate formation and transcription.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Schematic of NDF and its truncated variants. b, Interactions between His6-tagged truncated NDF variants (d) and Spt16-FLAG using a Ni-NTA pull-down assay (4-20% SDS-PAGE, Silver staining). c, Interaction study between His6-tagged truncated variants of F1 and Spt16-FLAG using a Ni-NTA pull-down assay. An asterisk indicates a non-specific band. The F1C construct includes a linker region between NDF and His-tag, resulting in a larger apparent size on the gel. d, Droplet formation visualized with various NDF proteins and Spt16-mCherry at 2 μM in droplet formation buffer (100 mM NaCl, no PEG). Scale bar=10 μm. Note: While the N-terminal PWWP domain interacts with FACT in vitro, proteins containing only the PWWP domain (R4 and R5) do not form condensates. e-f, Amino acid sequences of various F1 mutants (e) and their interaction with Spt16, showing disruption of interactions by mutations at specific residues, as analyzed by a Ni-NTA pull-down assay (f). g, SDS-PAGE analysis of purified full-length NDF and its mutants. h, Nucleosome transcription assay evaluating the impact of full-length NDF and its mutants, with transcription terminated 10 min at 30˚C after addition of NTP (0.25 mM). i, Bar graph comparing the production of full-length transcription products following the addition of full-length NDF or its mutants to the Pol II-nucleosome complexes over 10 min. Results are based on three independent experiments (mean ± SD, two-tailed t-test). j, Assessment of full-length NDF and its KR mutant (K161 and R162 mutated to alanine, AA) on Pol II transcription through nucleosomes, particularly noting decreased activity at the SHL -5 pausing site (near 40nt/bp1). Transcription was terminated 10 min after addition of NTP (0.25 mM). k, Microscopic images show the incorporation of Cy2-labeled nucleosomes into pre-formed (NDF_F1 + Spt16-mCherry) condensates at room temperature. Protein concentrations were 1 µM. Scale bar = 10 µm.
