Extended Data Fig. 2: Analysis of chromosome structure around the transgenic locus and genome-wide in mES cells.
From: Nonlinear control of transcription through enhancer–promoter interactions
a. Top: capture-C maps (6.4 kb resolution) of four cell lines where the SCR (black arrow) has been reinserted at different distances from the promoter (blue arrow). Bottom: differential contact map between individual cell lines and the founder line. Grey pixels: correspond to ‘noisy’ interactions that did not satisfy quality control filters (see Methods). Right: barplot showing the change in average interaction probabilities between the SCR reinsertion and the cassette, calculated using a square of 5 bins (6.4 kb resolution) centred at the cassette SCR reinsertion interaction. b. Left: example of Hi-C heatmap in mES cells at 6.4 kb resolution. Centre: scheme depicting how the probability of interaction between a promoter and the region immediately before the nearest TAD boundary (Pin, 12.8 kb i.e. two 6.4 kb bins before the boundary called using CaTCH66) and after the nearest TAD boundary (Pout) are calculated. Right: distribution of contact probability between all active promoters in mES cellss and the closest inner TAD boundary (Pin) (n = 9655). Box plot description as in Extended Data Fig. 1k. c. Box plots showing the distribution of contact probability changes within the TAD and across the closest TADs boundary for all active promoters in mES cells (n = 9655) whose contact probability outside the TAD is higher than 0.001 (n = 834). Box plot description as in Extended Data Fig. 1k; outliers not shown. d. Contact probabilities of the founder line from the location of the ectopic Sox2 transgene (black line) and normalized averaged mean number of mRNAs per cell (highest value = 1) generated in individual eGFP+ lines by the SCR mobilization are plotted as a function of its genomic position (dashed red line). The average is calculated within equally spaced 20 kb bins as in Fig. 1h (black dots). e. Coefficients of variation (CV) of eGFP levels measured by flow cytometry plotted against contact probabilities between the ectopic Sox2 promoter and the locations of SCR insertions. Data are presented as mean values +/- standard deviation (n = 3measurements in different days). Shaded light blue area indicates the interval between mean +/- standard deviation of eGFP level CVs in three promoter-only cell lines. f. Coefficients of variation (CV) of mRNA number per cell measured by smRNA-FISH plotted against contact probabilities between the ectopic Sox2 promoter and the locations of SCR in the cell the lines shown in Fig. 2c, d. Data are presented as mean values +/- standard deviation (n = 3 technical replicates).
