Specific Contributions of Cohesin-SA1 and Cohesin-SA2 to TADs and Polycomb Domains in Embryonic Stem Cells

Abstract

Cohesin exists in two variants carrying either STAG/SA1 or SA2. Here we have addressed their specific contributions to the unique spatial organization of the mouse embryonic stem cell genome, which ensures super-enhancer-dependent transcription of pluripotency factors and repression of lineage-specification genes within Polycomb domains. We find that cohesin-SA2 facilitates Polycomb domain compaction through Polycomb repressing complex 1 (PRC1) recruitment and promotes the establishment of long-range interaction networks between distant Polycomb-bound promoters that are important for gene repression. Cohesin-SA1, in contrast, disrupts these networks, while preserving topologically associating domain (TAD) borders. The diverse effects of both complexes on genome topology may reflect two modes of action of cohesin. One, likely involving loop extrusion, establishes overall genome arrangement in TADs together with CTCF and prevents excessive segregation of same-class compartment regions. The other is required for organization of local transcriptional hubs such as Polycomb domains and super-enhancers, which define cell identity.

Keywords: CTCF; Hi-C; Hox network; PRC1; STAG1; STAG2; chromatin loop; cohesin; compartment; pluripotency.

Graphical abstract

Figure 1

Cohesin-SA2 Is Enriched at Polycomb-Repressed Regions and Super-enhancers (A) ChIP-seq read distribution for the chromatin components indicated around common and cohesin-SA2-only positions within a 5 kb window in serum-grown mESCs. OSN, Oct4, Sox2, Nanog. SA2-only positions were divided into two categories that cluster according to their relative enrichment in Polycomb-repressed (2,002 peaks) or active enhancers and promoters chromatin marks (6,853 peaks). The SA2-only positions within super-enhancers (SEs) are indicated with a bracket. Color bars below heatmaps indicate ChIP-seq read number. Datasets used are summarized in Table S1. (B) Pie charts showing the distribution of cohesin positions in chromatin states defined in mESCs. (C) SA1 and SA2 enrichment along the 231 SEs defined in mESCs (Whyte et al., 2013). Med1 and Nipbl enrichments are also shown.

Figure 2

Cohesin-SA2 Facilitates the Establishment of Polycomb-Repressed Regions in mESCs (A) Comparison of protein (left) and mRNA levels (right) of SA1, SA2, and Nanog in mESCs growing in 2i and serum conditions. Tubulin, loading control for immunoblot. For qRT-PCR (right), data represent mean and SD from three independent experiments. Low Dnmt3b expression is a feature of mESCs cultured in 2i (Leitch et al., 2013). (B) Distribution of SA1, SA2, and PRC2 protein Ezh2 assayed by ChIP-seq around bivalent gene promoters defined by Mas et al. (2018) in mESCs grown in the indicated conditions. Data for Ezh2 are from Marks et al. (2012). (C) Cell cycle profiles of mock-depleted (control) and SA1- or SA2-depleted mESCs (top). The presence of cohesin and Polycomb proteins on chromatin after downregulation of SA1 or SA2 was assessed by chromatin fractionation. T, total cell extract; Cyt, cytosol; N, nuclear soluble; Chr, chromatin. (D) Read density plots (top) and read heatmap (bottom) comparing the distribution of Suz12 and Ring1B around the 2,002 SA2-only positions containing Polycomb, described in Figure 1A, in mock-depleted (control), SA1-depleted (siSA1), and SA2-depleted (siSA2) cells. (E) UCSC Genome Browser image of the HoxB and HoxC loci showing ChIP-seq read distribution for SA1 and SA2 in mESCs as well as for Ring1B in control and SA1- or SA2-depleted cells.

Figure 3

Cohesin Variants Make Different Contributions to Genome Architecture in mESCs (A) Vanilla-normalized Hi-C matrices for chromosome 17 at 100 kb resolution in mESCs treated as indicated. All analyses in this figure use data merged from two replicates. (B) Scatterplot of eigenvectors (EVs) of the intrachromosomal interaction matrices indicated in the axis. Numbers within the plot show the percentage of bins that changed compartment. The first eigenvector for chromosome 17 at 100 kb resolution is shown below the plots. Blue and red signals correspond to B and A compartments, respectively. (C) Matrices showing increased (red) and decreased (blue) interactions in chromosome 17 when comparing siSA1 (top), siSA2 (middle), or 2i-growing cells with serum-growing mock-depleted (control) cells. Similar results were obtained in the analysis of individual replicates and additional chromosomes (Figure S2). (D) Three-dimensional interaction meta-plots showing contact strength in long (>500 kb) cohesin-mediated loops previously defined by Hi-ChIP (Mumbach et al., 2016) in the different conditions. For comparison, the effect of CTCF depletion was also analyzed using data previously generated in mESCs carrying auxin-inducible degron (AID)-CTCF (Nora et al., 2017). (E) Meta-analysis of loop strength as in (D) but using borders of high-resolution TADs defined by Bonev et al. (2017). Replicates were also analyzed separately (Figure S3).

Figure 4

Preferential Interaction of Cohesin Dissociating Factor Wapl with Cohesin-SA2 Immunoprecipitation experiments from mESC extracts with non-immune IgG, anti-SA1, or anti-SA2 showing the preferential interaction of cohesin-SA2 with Wapl by immunoblot (left) and mass spectrometry (MS) counts (right). FT, flowthrough; IP, immunoprecipitate. See also Table S2.

Figure 5

Different Roles of Cohesin Variants in the Local Architecture of Polycomb Domains and Super-enhancers (A and B) Meta-plots represent all the interactions identified by Hi-C analyses of cells growing in the indicated conditions in (A) 231 super-enhancers and (B) Ring1B positions that are located within Polycomb-repressed domains. (C) Meta-plots showing genomic interactions identified between promoters within the HoxB (upper panels) or HoxC (lower panels) loci and any promoter from other Hox loci. Plots are centered at TSS. In all cases, each bin corresponds to 5 kb.

Figure 6

Distinct Effects of Cohesin Variants on Transcription (A) Heatmaps showing differentially expressed genes in the indicated conditions (two independent replicates). Eight different clusters of genes were revealed according to their response to SA1 or SA2 depletion (a–h). Numbers on the left indicate the number of genes in each cluster. See also Tables S3 and S4. (B) Boxplots showing changes in expression for upregulated and downregulated genes in serum-growing mESCs depleted from SA1 or SA2 compared with control. Boxes in (B) and (E) represent interquartile range (IQR); the midline represents the median; whiskers are 1.5 × IQR; and individual points are outliers. (C) Some of the most significantly enriched GO terms in each cluster defined in (A) are shown. p values were calculated with a Fisher’s exact test and corrected by FDR (< 0.05). (D) Venn diagram showing the overlap between the KEGG pathways obtained by GSEA of genes deregulated after SA1 or SA2 depletion. See also Table S5. (E) Boxplots comparing changes in expression after SA1 or SA2 depletion for Polycomb-repressed (left) and super-enhancer-dependent (right) genes. Polycomb-repressed genes (n = 1,008) were defined as those having Ring1B at their promoter and fragments per kilobase of transcript per million mapped reads (FPKM) < 1 in the control condition. Super-enhancer-dependent genes (n = 278) were those defined by Novo et al. (2018). Statistical significance was calculated using a Wilcoxon signed rank test. (F) Changes in expression of some Polycomb-repressed and super-enhancer-dependent genes were assessed using qRTPCR and normalized to levels of the housekeeping gene GAPDH. Data are mean and SD from at least three independent experiments. A Student’s t test was used to assess statistical significance. (G) Plot showing the relative distribution of genes deregulated only in the siSA1 condition (up or down) as well as the 4,781 mouse housekeeping genes defined by Li et al. (2017) along TADs in mESCs. Black line represents the distribution of genes not affected by SA1 depletion. (H) Histogram showing the observed and expected distribution of the housekeeping genes among the eight groups of cohesin regulated genes defined in (A). Statistical significance was assessed using a Fisher’s exact test.