Two variant cohesin complexes containing SMC1, SMC3, RAD21 and either SA1 (also known as STAG1) or SA2 (also known as STAG2) are present in all cell types. We report here their genomic distribution and specific contributions to genome organization in human cells. Although both variants are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-containing cohesin complexes (hereafter referred to as cohesin-SA2) localize to enhancers lacking CTCF, are linked to tissue-specific transcription and cannot be replaced by the SA1-containing cohesin complex (cohesin-SA1) when SA2 is absent, a condition that has been observed in several tumors. Downregulation of each of these variants has different consequences for gene expression and genome architecture. Our results suggest that cohesin-SA1 preferentially contributes to the stabilization of topologically associating domain boundaries together with CTCF, whereas cohesin-SA2 promotes cell-type-specific contacts between enhancers and promoters independently of CTCF. Loss of cohesin-SA2 rewires local chromatin contacts and alters gene expression. These findings provide insights into how cohesin mediates chromosome folding and establish a novel framework to address the consequences of mutations in cohesin genes in cancer.
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We thank Y. Cuartero and J. Quilez (4D Genome–CRG) for technical help with the Hi-C experiments, D. Rico (Newcastle University), F. X. Real (CNIO) and M. Manzanares (CNIC) for comments on the manuscript, T. Hirano (RIKEN) and H. Yu (UT Southwestern) for reagents, and M. Quintela (CNIO) for MCF10A cells. This work has been supported by the Spanish Ministry of Economy and Competitiveness and FEDER funds (grant no. BFU2013-48481-R (A.L.), BFU2016-79841-R (A.L.) and BFU2013-47736-P (M.A.M.-R.), fellowship no. BES-2014-069166 (M.D.K.), and Centro de Excelencia Severo Ochoa grant no. SEV-2015-0510 (to CNIO) and SEV-2012-0208 (to CRG), the European Research Council (FP7/2010-2015, ERC grant agreement 609989; M.A.M.-R.), the EU Horizon 2020 Research and Innovation Program (agreement 676556; M.A.M.-R.), the CERCA Programme–Generalitat de Catalunya (M.A.M.-R.) and the La Caixa Foundation (PhD fellowship to A.K.).
The authors declare no competing interests.
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Integrated supplementary information
a, Immunoblots of total extracts from human primary cells (NHEK, normal human epithelial keratinocytes; PrEC, prostate epithelial cells; NHBE, normal human bronchial epithelial cells; NHA, normal human astrocytes; HCAEC, human coronary artery endothelial cells; SKMC, skeletal muscle cells; HMEC, human mammary epithelial cells; HUVEC, human umbilical vein endothelial cells; NHOst, normal human osteoblasts) and MCF10A cells. b, Known amounts of C-terminal fragments of SA1 and SA2 (around 50 kDa each) were run alongside the indicated fractions from MCF10A cells to assess relative abundance of cohesin-SA1 and cohesin-SA2 on chromatin (T, total cell extract; Cyt, cytosolic fraction; N, soluble nuclear fraction; Chr, chromatin-bound protein). The dotted line indicates that two parts of the film corresponding to the same exposure but different sizes have been pasted together. This was repeated with all cell lines used in a (data not shown). c, Quantification of the signals in the blots above was used to generate the histograms for total cohesin levels and the SA2:SA1 ratio in the same cell lines.
a, Motif-based analysis of the indicated positions in HMECs. E-values of the significantly enriched motifs and percentage of regions containing each motif for a given condition are indicated. The “TF” column contains the transcription factors with statistically significant binding (blue scale; right) to the identified motifs. b, Distribution of SMC1, SA1, SA2 and CTCF around Zmym2 and activated YAP (YAP-5SA) positions (±2.5 kb) defined by ChIP–seq in MCF10A cells.
Enrichment plots for genes within KEGG pathways specific for the hematopoietic system (“Hematopoietic cell lineage”) and the nervous system (“Neuroactive Ligand-Receptor interaction”) after downregulation of SA1, SA2 or CTCF. Both these pathways appear significantly upregulated in the siSA2 condition (NES > 0, in red; FWER q < 0.01). In the other two conditions, the gene expression changes are either not significant (FWER q value in gray) or in the opposite direction (NES < 0, in blue). The y axis shows the enrichment score (ES).
Supplementary Figure 4 Preferential accumulation of cohesin in SA2-only sites in Wapl-knockout cells.
Read density plots showing cohesin SMC1 distribution in wild-type (wt) and Wapl-knockout (KO) human HAP1 cells from ref. 23. Cohesin positions were classified into common and SA2-only based on their co-occurrence with CTCF in Wapl-knockout cells.
a, MCF10A cells depleted for SA1 or SA2 by RNAi were arrested in G1 by contact inhibition. Left, immunoblot analysis of the remaining levels of cohesin 72 h after transfection. Right, cell cycle profiles of MCF10A proliferating cells and of each of the samples used for Hi-C analysis (two replicas, r1 and r2, per condition). b, Hi-C normalized interaction maps for chromosome 15 at 100-kb resolution. Left, contact maps for r1 and r2 for each condition (siControl, siSA1 and siSA2). Middle, correlation between r1 and r2 of each condition of all data in the normalized interaction map for chromosome 15. Right, correlation between the Eigen values. c, Scatterplot of eigenvectors of the intrachromosomal interaction matrices indicated. Numbers within the plot show the percentage of bins that change compartment between replicates for each condition. d, TAD number, TAD border strength and TAD border conservation analysis as shown in Fig. 6c–e, including replicas. e, Hi-C interactions as a function of genomic distance averaged across the genome for the two replicas in the siSA1 and siSA2 conditions.
Supplementary Figure 6 Chromosomal interactions are differently affected by depletion of SA1 or SA2.
Chromosome 15 differential interaction map at 100-kb resolution of siSA1 (top) or siSA2 (down) versus siControl for the merged dataset. The panel is complemented by tracks of A/B compartment assignment based on PC1 Eigen values as well as gene density across the genome (middle of the interaction matrices). Replicate experiments are also shown on both sides.
Supplementary Figures 1–6
Uncropped blot images
ChIP–seq and RNA-seq datasets
Proteomic analyses of SA1 and SA2 immunoprecipitates
DEGs in siSA1 versus siControl
DEGs in siSA2 versus siControl
DEGs in siCTCF versus siControl
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Kojic, A., Cuadrado, A., De Koninck, M. et al. Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization. Nat Struct Mol Biol 25, 496–504 (2018). https://doi.org/10.1038/s41594-018-0070-4
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