Fig 2: Condensin II and TFIIIC complexes associate in mammalian cells.(A and B) Subunits of the TFIIIC complex (blue) were pulled down using anti-condensin (SMC2 and SMC4) antibodies (Ab) in nuclear extracts from HEK293T cells for co-IP experiments, followed by immunoblotting. IgG, immunoglobulin G. (C) All subunits of the condensin II complex (magenta) were pulled down using anti–TFIIIC-220 antibodies in co-IP, followed by immunoblotting with the indicated antibody. (D and E) Subunits of the TFIIIC complex were pulled down from nuclear extracts made from cell lines that stably expressed Flag-SMC2 or Flag-NCAPH2, followed by immunoblotting with the indicated antibody. (F) All subunits of the condensin II complex were pulled down with anti-FLAG antibody from a cell line stably expressing Flag-TFIIIC-110, followed by immunoblotting with the indicated antibody. (G) Subunits of the TFIIIC complex were pulled down from nuclear extracts derived from a cell line that stably expressed BirA*-NCAPH2 in a BioID assay. (H) BioID assays with nuclear extracts from a cell line that stably expressed BirA*-TFIIIC-110 had all subunits of the TFIIIC complex. Each experiment was performed in triplicate.

Fig 3: Condensin’s ATPase Site 1 Drives Loop Formation(A) Schematic depiction of the S. cerevisiae condensin complex.(B) Coomassie-stained SDS-PAGE of the condensin holocomplex samples as used in (C).(C) ATP hydrolysis rates measured for condensin holocomplexes (5 μM) in the absence or presence of 10 μg/μL of a relaxed 6.4-kb plasmid DNA. The graph shows mean ± SD of five independent experiments for wild-type, AS1LV (Smc2 L1048V), and AS2LV (Smc4 L1323V) mutants, and three independent experiments for the negative control, the Q-loop mutant AS1QL (Smc4 Q302L)-AS2QL (Smc2 Q147L).(D) Schematic overview of single-molecule fluorescence assay for condensin-mediated loop extrusion. DNA is double-tethered to a streptavidin-containing surface and stained with Sytox Orange. The addition of 1 nM condensin and 5 mM ATP results in a loop as judged by the local increase in intensity of the fluorescently labeled DNA. DNA loops can be visualized by applying a flow perpendicular to the tethered DNA molecule.(E) Rate of DNA loop extrusion by wild-type (n = 27), AS1LV (n = 51), and AS2LV (n = 51) condensin. Each circle represents one DNA molecule. Red line depicts the mean. Only DNA molecules with an extension of <0.65 were included in this graph (for extrusion rates of other DNA extensions check Figure S4A). The experiment was performed using 1 nM condensin and 5 mM ATP.(F) Fraction of DNA molecules that shows a loop using wild-type (n = 109), AS1LV (n = 237), or AS2LV (n = 124) condensin complexes. The experiment was performed using 1 nM condensin and 5 mM ATP. The graph shows the mean ± 95% confidence interval. Statistical significance was measured using unpaired two-tailed Student’s t test, ∗∗∗∗p value < 0.0001. ns, non-significant.

Fig 4: Independent strategies do not support physical interactions between condensin II and SWI/SNF subunits. A. Immunoblots (IB) show enrichment of the condensin subunit Smc4 and the SWI/SNF subunit Smarca4 in immunoprecipitations (IP) conducted from thymic whole cell extract using the B276 antibody compared to IgG control. B. Immunoblots show enrichment of the SWI/SNF subunit Arid1a (~270 KDa), but not the condensin II subunit Ncaph2 (~80 KDa), in immunoprecipitations conducted using an anti-Smarca4 antibody compared to IgG control. C. Schematic illustration of anti-FLAG affinity purification mass spectrometry strategy using Ncaph2 FLAG/FLAG mouse ESCs and untagged control. D. Scatter plot showing protein enrichment in the immunoprecipitation experiments shown in panel C (n = 4, from cultures grown and processed in parallel). The x-axis depicts mean Log2 fold-enrichment in Ncaph2 FLAG/FLAG (Flag) versus untagged control cells (WT). The y-axis shows the Log10 probability of enrichment from Bayes-moderated t-tests. Condensin II and SWI/SNF subunits are labelled in blue and red, respectively. Uncropped blots for panels A and B are available via Figshare ( Wood & Taylor, 2020a).