Fig 2: Cohesin and Condensin II Occupy Super-Enhancers and Are Required for Proper Expression of the Key Stem Cell Identity Genes(A) ChIP-seq binding profiles (reads per million) for the ESC transcription factors OCT4, SOX2, and NANOG (OSN), the Mediator coactivator (MED1), NIPBL, condensin II (CAPH2), and cohesin (SMC1) at the Gck and miR-290-295 loci in ESCs. Enhancer bars and scale bars are depicted above the binding profiles.(B) Metagene representations of Mediator, NIPBL, condensin II, and cohesin ChIP-seq density (reads per million per base pair) across the 8,563 typical enhancers and the 231 super-enhancers. Metagenes are centered on the enhancer region (703 base pairs for typical enhancers and 8.7 kb for super-enhancers), with 3 kb surrounding each enhancer region. ChIP-seq fold difference for Mediator, NIPBL, condensin II, and cohesin at super-enhancers versus typical enhancers are displayed below the metagenes. Fold difference at enhancers refers to the mean ChIP-seq signal (total reads) at super-enhancers divided by the mean ChIP-seq signal at typical enhancers. Fold difference at enhancer constituents refers to the mean ChIP-seq density (reads per million per base pair) at super-enhancer constituents divided by the mean ChIP-seq density at typical enhancer constituents.(C) Depletion of condensin II and cohesin caused a decrease in expression of super-enhancer associated genes. Box plots of fold change expression in condensin II and cohesin knockdown cells relative to GFP. Box plot whiskers extend to 1.5× the interquartile range. p values (Condensin II = 7.6e-13 and Cohesin = 2.2e-20) were calculated using a two-tailed t test.

Fig 3: Condensin II and Cohesin Depletions Disrupt the ESC Gene Expression Program(A) CAPH2 and SMC1 mRNA levels in ESCs infected with shRNA lentiviral constructs. Transcript levels were normalized to GAPDH. The error bars represent the SD of the average of three to six independent PCRs. p values (Condensin II #1 = 2e-4, Condensin II #2 = 3e-4, and SMC1 = 1.9e-3) were calculated using a one-tailed t test.(B) Gene expression changes following CAPH2 and SMC1 knockdown at co-occupied genes. Gene expression changes were calculated by comparing the RNA-seq data from cells transduced with condensin II shRNA (left axis) or cohesin shRNA (right axis) to cells transduced with control GFP shRNA. For CAPH2, two highly similar RNA-seq data sets from two different shRNA constructs were pooled. The 1,752 co-occupied genes were ranked based on the average fold change for all shRNAs.See also Figure S2 and Tables S2 and S3.

Fig 4: The SWI/SNF complex interacts with the condensin II complex.(A) Validation of the interaction between the SWI/SNF and condensin II complexes by co-IP in RMG1 cells with endogenously FLAG-tagged ARID1A. Nuclear fractions were subjected to IP using an anti-FLAG antibody and immunoblotting using the indicated antibodies. (B) Expression of the indicated SWI/SNF and condensin II subunits in ARID1A wild-type OVCAR429 and RMG1 cells with or without ARID1A knockout determined by immunoblotting. (C) Co-IP analysis between NCAPH2 and BAF155 using an anti-NCAPH2 antibody from nuclear extracts prepared from RMG1 cells treated with or without DNase I. (D) Venn diagram showing the overlap of ChIP-seq peaks among NCAPH2, ARID1A, H3K27ac, and H3K4me3 in RMG1 cells. (E) Heatmap clustering of ChIP-seq profiles of NCAPH2, ARID1A, SNF5, H3K27ac, and H3K4me3 in RMG1 cells. The number of binding sites in each of the four clusters is indicated. (F) Average profiles of the ChIP-seq signal for the indicated antibodies within four distinct clusters found by k-means clustering. Immunoglobulin G, IgG.

Fig 5: 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).