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. 2018 Jun;25(6):496-504.
doi: 10.1038/s41594-018-0070-4. Epub 2018 Jun 4.

Distinct Roles of cohesin-SA1 and cohesin-SA2 in 3D Chromosome Organization

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Free PMC article

Distinct Roles of cohesin-SA1 and cohesin-SA2 in 3D Chromosome Organization

Aleksandar Kojic et al. Nat Struct Mol Biol. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. A large fraction of cohesin-SA2 localizes to enhancers independently of CTCF.
a, Analysis of ChIP-seq read distribution for SA1, SA2, SMC1 and CTCF around common, cohesin SA1-only and cohesin SA2-only positions within a 5-kb window in HMECs. b, Average read density plots for SA1 (red) and SA2 (blue) distribution in common, SA1-only and SA2-only positions as well as for CTCF (separate plot on the right). c, Pie charts showing the distribution of cohesin positions in chromatin states defined in HMECs. d-f, same as a-c in MCF10A cells. g-i, same as a-c in HCAECs. CTCF datasets are from ENCODE (Supplementary Dataset 2).
Fig. 2
Fig. 2. Cohesin SA2-only positions are enriched in cell-type specific super-enhancers.
a, Venn diagrams showing overlap of cohesin binding sites between HMECs and HCAECs. Common positions are clearly more conserved. b, Cohesin enrichment in super-enhancers (SE) defined in HMECs. c, Example of cohesin distribution in HMEC and HCAEC cells within a HMEC-specific SE. d, Plot shows SA1 and SA2 enrichment in HMEC and HCAEC cells along HMEC SE. e, Boxplot comparing changes in expression between random genes and genes associated with HMEC-specific SE. Boxes represent interquartile range (IQR), midline represent the median, whiskers are 1.5xIQR and individual points are outliers. Statistical significance was calculated with a Wilcoxon signed-rank test.
Fig. 3
Fig. 3. SA2-specific changes in transcription are related to cell identity.
a, Levels of cohesin and CTCF after siRNA transfection in MCF10A cells (uncropped blot images are shown in Supplementary Dataset 1). b, Venn diagram showing the overlap between genes deregulated after downregulation of SA1, SA2 or CTCF compared to mock transfected cells (FDR<0.05, log2fold change<-0.5 or >0.5 and FPKM>3 in at least one condition). b, UCSC browser image of the S100A gene cluster showing genes (orange dots indicate those deregulated in SA2 depleted cells), CTCF peaks (and motif orientation), and genomic distribution of SMC1, SA1 and SA2 in MCF10A cells. Positions corresponding to common (c) and SA2-only (o) cohesin binding sites used in ChIP-qPCR analyses are shadowed in red and blue, respectively. d, ChIP-qPCR validation of SA1, SA2 and CTCF binding to (c) and (o) positions. e-f, Gene expression levels of S100A genes (e) and cell-type specific transcription factors (f) in control, siSA1, siSA2 and siCTCF conditions (mean and SD from three independent experiments). Student's T test was used to assess statistical significance.
Fig. 4
Fig. 4. Different behaviour of cohesin in common and SA2-only positions.
a, Cohesin SMC1 distribution in HMECs, MCF10A and HCAECs. Maximum mean tag density is indicated on the Y axis. b, SA2 and WAPL binding to the indicated cohesin positions from the S100 locus. Bars represent the mean of at least three independent experiments performed in triplicates; error bar=SD. c, Chromatin bound cohesin was determined upon 0.25M (mid panel) and 0.5M (lower panel) NaCl treatment at different time points. Quantification is shown at the bottom. Uncropped blot images are shown in Supplementary Dataset 1. d-e, The simultaneous presence of at least two cohesin complexes at a given position within the same cell was assayed by Re-ChIP-qPCR (d), and confirmed by Re-ChIP-seq (e). Chromatin eluted from the first ChIP with SA1 or SA2 was incubated with SA2 and SA1 antibodies, respectively, as well as SMC1 and IgG as positive and negative controls. Lower panel, Re-ChIP of chromatin eluted from IgG beads with SA1 and SA2. Positions c3-c8 are "common" cohesin binding sites; n1 and n2, are negative regions. All the positions captured by Re-ChIP-seq (e) correspond to common sites in MCF10A cells.
Fig. 5
Fig. 5. Cohesin-SA1 cannot occupy SA2-only sites
Read heatmap (up) and read density plots (down) showing SA1 and SA2 distribution around cohesin positions defined in Fig. 1d in control cells and cells treated with siRNA against SA1 or SA2. Two independent replicates were performed per condition. Read density plots were built merging the reads from the two replicates.
Fig. 6
Fig. 6. Distinct contribution of cohesin-SA1 and cohesin-SA2 to genome architecture.
a, Vanilla normalized Hi-C matrices for chr13 at 100Kb resolution in MCF10A cells. b, Scatter plot of eigenvectors of the intra-chromosomal interaction matrices indicated. Numbers within the plot show the % of bins that change compartment. First eigenvector for chr13 at 100Kb resolution is shown below. c-e, Boxplots showing number of TADs per chromosome (c), TAD border strength (d) and TAD border conservation (e). The box extends from the lower to upper quartile values of the data, with a line at the median. Notches represent the confidence interval around the median. f, Hi-C interactions as a function of genomic distance averaged across the genome for a maximum distance of 50 Mb. g, Overall genome-wide increased and decreased interactions between siControl and siSA1 or siSA2 in chr15. h, Effect of the SA1 or SA2 depletion in differential inter- and intra-TAD interactions in different compartments. Boxplots are for the chromosome average values. i, Enrichment of SA1-only, SA2-only and common sites in AB compartments, A/B borders and TAD borders. Squares with numbers are significant (Fisher exact test, p-values < 0.001).

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