Fig. 1: Enhancer action depends on the genomic distance from the promoter and is constrained by TAD boundaries.

a, Mobilization of an enhancer around its target promoter to measure transcription as a function of their genomic distance. b, Schematic of the transgene: a promoter drives transcription of an eGFP gene split by a piggyBac-enhancer cassette. ITR, inverted terminal repeats. c, After expression of PBase, the piggyBac-enhancer cassette is excised and randomly reinserted, occasionally leading to eGFP expression. d, Sorting of single eGFP⁺ cells results in cell lines in which the enhancer drives transcription from a single position. Splinkerette PCR and flow cytometry analysis are used to determine the enhancer position and promoter expression levels. e, Capture-C (6.4 kb resolution) analysis and genomic datasets in mES cells across 2.6 Mb centred around the selected TAD with both of the internal CTCF motifs deleted (dashed squares; ΔΔCTCF, double CTCF site deletion ). The dashed line indicates the position of the future insertion of the transgene carrying the Sox2 promoter and SCR. A, active; N, neutral; R, repressive; Chr, chromosome. f, Representative flow cytometry profiles from founder mES cells, a promoter-only control cell line and eGFP⁺ cell lines with mobilized SCR. The light blue line indicates the mean eGFP levels in the promoter-only line. The numbers show the median eGFP intensities. AU, arbitrary units. g, eGFP levels in individual eGFP⁺ cell lines over cell passages. The numbers show the median eGFP values. h, Normalized mean eGFP intensities in individual eGFP⁺ cell lines as a function of SCR genomic position. The red dots are data from 135 individual cell lines; data are mean ± s.d. n = 3 measurements on different days. The black dots show the average values within equally spaced 20 kb bins. The dashed red line shows the spline interpolation of average values. Mean mRNA numbers were inferred using smRNA-FISH calibration (Extended Data Fig. 1h). The light blue area shows the interval between the mean ± s.d. of eGFP levels in three promoter-only cell lines. i, Data as in h, colour-coded according to SCR genomic orientation.