Extended Data Fig. 1: Enhancer action is modulated by genomic distance from the target promoter and constrained by TAD boundaries.

a. Top: capture-C contact map at 6.4 kb resolution in wild-type (WT) mES cells in a 2.6 Mb region centred around the neutral TAD on chromosome 15 we used for the experiments. Vertical grey lines: TAD boundaries. Bottom: genomic datasets and ChromHMM analysis showing that the chosen TAD is devoid of active and repressive chromatin states, with the exception of 80 kb at the 3b at t which is enriched in repressive chromatin states. b. Close-up view of panel a, highlighting the presence of CTCF-mediated chromatin loops (dotted boxes) in WT mES cells. c. capture-C contact map at 6.4 kb resolution for the same region as panel b in the cell line with double CTCF site deletions. CTCF deletions lead to loss of CTCF-mediated chromatin loops (dotted boxes). d. Top: UCSC snapshot of the endogenous Sox2 locus and Sox2 control region (SCR). Bottom: close-up views showing the regions of the Sox2 promoter, the SCR region found in ref. ²⁹ and the SCR used in the transgene construct. e. IGV snapshot showing nanopore sequencing reads mapped to a modified mouse genome including the transgene integration. Reads spanning from genomic DNA upstream the left homology arm to genomic DNA downstream the right homology arm confirmed single insertion of the transgene. f. capture-C maps at 6.4 kb resolution of the mES cell line with double CTCF sites deletion (left) and the founder mES cell line with transgene insertion (centre). Right: differential contact map. Grey pixels correspond to ‘noisy’ interactions that did not satisfy our quality control filters (see Methods). Transgene insertion induces new mild interactions with CTCF sites at the 3. and 5a extremities of the TAD (arrows). g. Barplot showing the fraction of piggyBac-SCR reinsertions genome-wide determined by Illumina sequencing of splinkerette PCR products from a pool of cells after PBase expression. See Methods for a detailed description of the protocol. h. Top: Representative smRNA-FISH image and flow cytometry profiles over different passages in a cell line where the SCR was mobilized in the immediate vicinity of the ectopic Sox2 promoter. Scale bar, 10 \(\mu m.\) Bottom: Linear relationship between the mean eGFP intensity and the average number of eGFP mRNAs measured using smRNA-FISH for seven single cell lines (\({R}^{2}=0.9749\), \(p < 0.0001,\) t-test). Error bars on the x-axis: standard deviation of three measurements performed on different days, as in Fig. 1h. Error bars on the y-axis: standard deviation of three technical replicates. i. Normalized mean eGFP intensities levels in individual eGFP+ cell lines are plotted as a function of the genomic position of the SCR in individual eGFP+ lines. Data from 127 individual cell lines (light red dots) from a single experiment are presented as mean +\- standard deviation (n=3 measurements performed in different days, as in Fig. 1g). Average eGFP values calculated within equally spaced 20 kb bins (black dots) are shown. Mean mRNA numbers per cell were inferred from eGFP counts using calibration with smRNA-FISH, see Extended Data Fig. 1h. Shaded light blue area indicates the interval between mean +/- standard deviation of eGFP levels in three promoter-only cell lines. j. Same plot as Fig. 1h showing the only two SCR insertions we detected outside the TAD boundaries (brown dot) and on another chromosome (yellow dot). k. Left: Log10 average eGFP expression (from Fig. 1h) as a function of log10 absolute genomic distance between transgene position and SCR reinsertion. Points are colour-coded as in panel A (chromHMM active, neutral, and repressive states). Black line denotes linear regression. Black circles denote SCR reinsertions within the Npr3 gene body. Right: deviations of eGFP expression levels from the linear regression correlate with chromatin states called using ChromHMM (n: active = 16; neutral = 83; Npr3 = 17; repressive = 7). Reinsertion of SCR within active or repressive regions respectively increases or decreases enhancer activity compared to neutral regions. Box plot: centre line denotes the median; boxes denote lower and upper quartiles (Q1 and Q3, respectively); whiskers denote 1.5x the interquartile region (IQR) below Q1 and above Q3; points denote outliers. l. Coefficients of variation (CV) of eGFP levels measured by flow cytometry plotted against SCR insertion locations in eGFP+ cell lines (light red dots). Data are presented as mean +/- standard deviation (n = 3 measurements in different days). Shaded light blue area indicates the interval between mean +/- standard deviation of eGFP level CVs in three promoter-only cell lines. m. Representative eGFP distributions (normalized to mean eGFP level) in clones with increasing absolute genomic distance (1.7 kb, 42.4 kb, 112.5 kb, and 259.43 kb) between the mobilized enhancer and the ectopic Sox2 promoter. Vertical line indicates normalized mean eGFP levels. n. FACS plot showing standard (top) and less stringent (bottom) gates on eGFP levels used for single cells sort and insertion analysis of corresponding clonal cell lines. o. Left: FACS plot showing the gates used to sort pools of cells for tagmentation-based mapping of PiggyBac-enhancer insertions. For gates “low 1” and “low 2”, six pools of 10000 cells were sorted while for gate “high”, six pools of 337 cells were sorted. Gate “high” corresponds to the standard gate used to isolate eGFP positive cell lines for the mobilization experiments. Centre: Barplot showing the fraction of sequencing reads mapping to non-mobilized enhancer cassette determined by tagmentation-based mapping from the different pools sorted in gates “low 1”, “low 2” and “high”. See Methods for a detailed description of the protocol. Right: Numbers and genomic locations of confident insertion sites (identified as those with at least one read for both 5′oth 5 mapping from the different pools sorted in gates “low 1”, “low 2” and “higeGFP gates.