Extended Data Fig. 1. Chromosome structure is altered upon degradation of factors involved in loop extrusion.
A. Western Blots showing degradation of RAD21, WAPL and CTCF upon 1.5 h, 24 h and 6 h, respectively. Loading control: α-tubulin, n = 1–2 replicates for each cell line. B. Left: Average enrichment in Hi-C read counts at CTCF sites based on Hi-C data in RAD21-AID-eGFP cells either untreated (left), treated for 1.5 h (middle) or 4 h (right) with auxin. Right: Differences in enrichment at CTCF peaks. Peaks were called on Hi-C data from untreated cells. C. Flow cytometry analysis of fixed cells stained with DAPI showing cell-cycle stage distributions of RAD21-AID-eGFP mESC cultured with serum, LIF and 2i, either before (green) or after 1.5 h (blue) and 6 h (red) auxin treatment. D. Integration site numbers in two clones of RAD21-AID-eGFP lines with and without 3xCTCF sites. E. Distribution of integration sites from lines shown in panel D that belong to A and B compartments called on distance-normalized Hi-C map (same as panel B). F. Integration sites distances from the closest endogenous CTCF site. Boxplot: lower and upper quartiles (Q1 and Q3, respectively); whiskers: 1.5x interquartile region (IQR) below Q1 and above Q3. n = 15 and 19 insertions for -3xCTCF-TetO clones 1 and 2, respectively, n = 14 and 19 insertions for +3xCTCF-TetO clones 1 and 2, respectively. G. Example of genotyping PCR upon removal of 3xCTCF sites in a RAD21-AID-eGFP +3xCTCF-TetO clonal line. PCR1 amplifies the entire 3xCTCF cassette and product size changes from 470 bp to 147 bp if the cassettes are successfully removed. PCR2 amplifies half of the 3xCTCF cassette and no product is expected if 3xCTCF cassettes were removed from all insertion sites; otherwise a PCR band of 303 bp is expected. H. Representative 4C-seq profiles from insertions on chromosomes 6 and 9 using TetO as a viewpoint showing that 3xCTCF-TetOs lead to the formation of ectopic contacts (dashed red lines) with nearby endogenous CTCF sites in the presence of RAD21. Contacts are lost upon deletion of 3xCTCF cassette (−3xCTCF-TetO) and upon degradation of RAD21 (−RAD21).