NE preparation from HeLa S3 cells, IVT and looping assays, and chromatin reconstitution of the IVT templates were conducted as described (48 (link)). All IVT assays involved 0.2 pmoles of chromatinized template, 50µg NE, and 1 pmole of activator (ERα or GAL4-VP16) or repressor (GAL4-SID) where indicated. All reactions contained 100 nM E2. The template and all reagents as indicated were mixed to a volume of 45 µL at RT to allow the formation of preinitiation complex. Transcription was initiated by addition of 5 µL NTPs mix (5 mM) and shifting the reactions to 30 °C. IVT reactions described in Fig. 3A were carried out slightly differently. The 5′biotinylated CompF chromatin was first immobilized on M280 streptavidin beads. Preinitiation complex was formed with NE without or with ERα, and the unbound proteins were washed with Buffer D. Protein-bound CompF-chromatin on beads were resuspended in 45 µL buffer mix that provided the final composition of 12 mM HEPES-KOH (pH 7.9), 12% glycerol, 60 mM KCl, 12 mM MgCl2, 0.12 mM EDTA, 0.3 mM DTT, 1 mM ATP, 0.9 mM acetyl CoA. control oligos, ASOs, and RNase H were added as indicated, and transcription was initiated with NTPs and shifting the reactions to 30 °C. After 45 min, transcription was terminated with 250 µL TriReagent. RNA was extracted, digested with DNase with the Ambion DNase-free kit as per the manufacturer’s instruction, and the RNA was used in qRT-PCR. Aqueous solutions of 1,6HD, and 2,5HD were added to the reactions prior to NTPs at final concentrations as indicated.