Extended Data Fig. 10. The loop extrusion rate does not change after encounter of cohesin with CTCF.
a, Loop extrusion (LE) rate before and after encounter with N-terminally oriented CTCF when cohesin was blocked at CTCF and then switched extrusion direction to extrude away from it (see e.g. Fig. 3a and Supplementary Video 4). Statistical significance was assessed by a 2-sided Wilcoxon rank-sum test (mean SEM; N = 22). b, As for (a) but for events where cohesin passed over N-terminally oriented CTCF (mean SEM; N = 9). For events where the time between onset of LE and encounter with CTCF was too short to measure the LE rate, the LE rate was determined after passage and compared to the LE rate in the absence of CTCF (split violin plot on the right). For the latter, statistical significance was assessed by a 2-sided 2-sample Kolmogorov-Smirnoff test. c, as for (b) for events where cohesin passed over C-terminally oriented CTCF (mean SEM; N = 38). Error bars on individual data points denote the standard deviations of determined loop extrusion rates in moving 11-frame windows (4.4 s) during the duration of loop extrusion before encounter. Thick horizontal lines on boxplots denote median values, the box extends from the lower to upper quartile values and whisker limits denote the range of data within 1.5 times the interquartile range from the median. Sample sizes (N) refer to biological replicates from 13 independent experiments for N-terminal and 3 independent experiments for C-terminal encounters. d–j, Illustrations of loop size and DNA tension determination, and DNA tension error estimation (see Supplementary Note). (d) In the absence of loop extrusion, a DNA molecule of length L bp is tethered to a surface with end-to-end distance R. The relative DNA extension is thus computed as the ratio of the end-to-end distance R and the contour length of the entire DNA molecule. In the presence of an extruded DNA loop, the non-extruded part of the DNA is effectively shortened by the loop size Lloop, while the DNA inside the loop does not experience any tension in the absence of buffer flow. The relative extension is now computed as ratio of the (constant) end-to-end length R and the contour length of the non-extruded DNA, which has the size Lnonloop = L − Lloop. An increasing loop progressively shortens the non-extruded part of the DNA, giving rise to an increasing tension on the non-extruded DNA due to a fixed end-to-end length R. (e) Illustration of the DNA intensity profile along its long axis. The DNA intensity appears slightly larger than its real end-to-end length due to convolution of the DNA intensity with the microscope point spread function, which is corrected for by the peak peeling algorithm to determine the DNA end-to-end length. The lead intensity is determined as the integrated intensity between one of the DNA ends and the loop position (which corresponds to the peak position of the looped DNA intensity. A 7-pixel window around the loop position is summed and corrected by the intensity contributing in this window from the non-extruded DNA. However, the loop intensity is underestimated due to truncation of the integration outside the range [xloop − w/2, xloop + w/2] (yellow area). (f) At low end-to-end length, the flexibility of DNA might yield a DNA intensity beyond the DNA’s tether points. (g) Cross-sections around point-emitters (grey; N = 15) centred at their maximum value and mean trace (red). (h) Gaussian fits the single traces (grey) and mean fit (red) centred at their maximum value. The average Gaussian width was found to be σ = 180 ± 13 nm. (i) DNA tension with absolute (black line and blue area correspond to mean and error of the DNA tension) and relative (red) error of the DNA tension over loop sizes from Lloop = 0 to 10 kb at fixed end-to-end length R = 3.5 μm. Error bars denote the estimated error, also represented as a red line on the right y-axis. (j) Analogous to (i) for a fixed loop size of Lloop = 5 kb and varying end-to-end length R. Error bars denote the estimated error, also represented as a red line on the right y-axis.