Extended Data Fig. 5: Dependence of transcription levels and insulation on enhancer strength.
From: Nonlinear control of transcription through enhancer–promoter interactions
a. Top: UCSC genome browser snapshot of the endogenous Sox2 locus and Sox2 control region (SCR). Bottom: close-up view showing the SCR (black) identified in ref. 29 and the enhancer regions used in the transient reporter assays shown in panel b. Full-length enhancer is in red (same as in Fig. 1); truncated versions are in brown (Ei) and orange (Eii). Experiments in Fig. 5 were performed with Ei. b. Flow cytometry analysis of mES cells transiently transfected with PBase-RFP and different versions of split eGFP plasmids carry either no enhancer, or the full-length SCR (red, see panel a), or the first (brown-Ei) or second (orange-Eii) SCR subregions in front of the Sox2 promoter. Transcription levels generated upon co-transfection with PBase are higher in the presence of the full-length SCR compared to truncated versions. Numbers in each quadrant represent the % of cells either negative or RFP, GFP and RFP-GFP positive. c. Top: distribution of contact probabilities between all active promoters in mES cells and the nearest inner TAD boundaries, calculated as in Extended Data Fig. 2b. Bottom panel: Model prediction for the mean eGFP mRNA numbers per cell plotted against contact probabilities shown as a comparison (same as Fig. 2e). Shaded areas correspond to promoters with contact probability with the closest TAD boundary below 0.2. d. Left panel: scheme of how the probabilities of interaction between promoter and the region before (Pin) and after the TAD boundary (Pout) are calculated, same criteria as in Extended Data Fig. 2b. Central panel: promoters with higher contact probabilities with TAD boundaries experience stronger drops of contact probability across boundaries. Right panel: promoters closer to TAD boundaries experience a stronger drop of contact probability across boundaries.
