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. 2017 Aug 30;114(38):E7949–E7958. doi: 10.1073/pnas.1711158114

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Fig. 4. — RSF1 represses transcription activation from H2Aub nucleosome-containing chromatin in vitro. (A) Supercoiling assay of reconstituted chromatin template. The purified supercoiled plasmids (lane 1) were relaxed to closed circular templates using topoisomerase I (lane 2). After protease digestion and phenol-chloroform extraction, the relaxed plasmids (lane 2) were mixed with histone/ACF/Nap1 in the presence of topoisomerase I to reconstitute chromatin template that restore DNA supercoiling. DNA was then purified and changes in linking number were demonstrated (lanes 3 and 4). (B) In vitro transcription assay on naked DNA and on chromatin templates reconstituted with H2A or semisynthetic H2Aub. GAL4-VP16 and p300/AcCoA activate transcription. RSF1 inhibits transcription activation on H2Aub chromatin template, but has no effect on transcription activation on H2A chromatin.