Extended Data Fig. 8: Compartment profiles of single-Stag GSCs.
From: The mitotic STAG3–cohesin complex shapes male germline nucleome
a, P’(s) curves for WT GSCs. Features analyzed by polymer simulation were annotated. b, Simulation results of P’(s) (top), the separation at slowest decay (representing the average loop size; middle), and the slope between peak and dip (representing the strength of insulation; bottom) in various parameters for the CTCF-capture ratio (left), loop extrusion velocity (middle), and cohesin residence time (right). LEF: loop extrusion factor. c, Diagram of the ATPase assay. Each component of the cohesin complex and DNA was purified (top), and ATP hydrolysis rates were compared based on their sequential addition (bottom) using thin-layer chromatography and autoradiography. d, Coomassie-blue staining of the indicated recombinant proteins on SDS-PAGE. Two biological replicates were analyzed. e, ATPase rates in the presence of the indicated components were determined using thin-layer chromatography/autoradiography. The upper and lower ends of the error bars represent mean + and – SD, respectively. p-values are measured by two-sided Welch’s t-test. Replicates per condition: n = 3 for most groups; n = 5 for Trimer+NM + DNA, and n = 4 for Trimer+STAG3 + NM + DNA. All data were obtained from two biological replicates of protein preparations. f, Scheme for the generation of STAG1-only and STAG3-only Hela cells for iFRAP experiments. OSTIR1: Oryza sativa Transport inhibitor response 1; DHB: DNA helicase B; IAA: indole-3-acetic acid (auxin); Dox: doxycycline. Halo, SNAP, AID, and FLAG are tags for proteins. g, Western blot for STAG1, STAG3, and α-tubulin in the whole cell lysate and for STAG2 and histone H3 in the chromatin fraction in STAG1-only and STAG3-only Hela cells in the presence (+) and absence (−) of IAA and Dox as indicated (one experiment). h, Representative Hi-C contact maps depicting chr5:9–42 mb at 50-kb resolution (a compartment scale) in Stag1-only and Stag3-only GSCs. The heatmap color scale represents the normalized contact frequency. i, Enrichment of homotypic (that is, intra-compartmental) interaction strength as measured using the saddle strength function of cooltools152 in Stag1- and Stag3-only GSCs. The enrichment of interactions between the A-A and B-B compartments over the A-B compartment is represented as higher y values.
