Regulation of pericentromeric DNA loop size via Scc2-cohesin interaction

Abstract

Cohesin exhibits DNA loop extrusion when bound to the ATPase activator Scc2 (NIPBL in humans), which has been proposed to organize higher-order chromosome folding. In budding yeast, most chromosome-bound cohesins lack Scc2. How the Scc2-cohesin interaction is regulated on the chromosome and its physiological consequences remain unclear. Here, we show that the deletion of both ECO1 and WPL1, two known cohesin regulators, but not either alone, caused Scc2-cohesin co-localization in metaphase, particularly around centromeres, using calibrated chromatin immunoprecipitation sequencing (ChIP-seq). Eco1's mitotic activity was required to prevent this co-localization in Δwpl1. We also demonstrate that Scc2-cohesin co-localization enlarged pericentromeric DNA loops, linking centromeres to genome sites hundreds of kilobases away, and delayed mitotic chromosome segregation. These findings suggest that Wpl1 and Eco1 cooperatively regulate Scc2-cohesin interaction, restrict pericentromeric DNA loop size, and facilitate chromosome segregation.

Keywords: Natural sciences; biological sciences; cell biology; functional aspects of cell biology.

Graphical abstract

Figure 1

Simultaneous depletion of Wpl1 and Eco1 promotes Scc2 co-localization at cohesin binding sites (A) Calibrated ChIP-seq profiles of Scc1-PK in wild-type (WT, strain SN75) and Scc2-PK in WT (SN40), Δwpl1 Δeco1 (SN54), Δwpl1 (SN53), GAL-^RECO1 (SN41) in galactose-free medium, and PDS5-AID (SN80) treated with IAA. Cells were arrested at metaphase by benomyl treatment. See Figure S1 for details on the culture conditions of GAL-^RECO1 and PDS5-AID. The y axis indicates normalized fold enrichment (nFE), or ChIP/input ratio normalized by spike-in control, with the range in brackets. CEN, centromere. The identified peaks are indicated by red horizontal bars, and the number of the peaks across the genome is shown in parentheses. (B) Heatmap of Scc1 and Scc2 ChIP-seq nFE in the indicated strains. 10kb-surrounding regions of the cohesin binding sites in WT (excluding those less than 5 kb to the centromeres) are depicted. Regions are sorted in descending order of Scc2 nFE in Δwpl1 Δeco1. In addition to the aforementioned strains, Δwpl1 Δeco1 with SCC1-PK gene (SN74) was used. (C) (Top) The scatterplot showing Scc2 nFE and the distance from the centromeres at each non-centromeric cohesin binding site in WT. Overlay representation of all chromosomes. The cohesin sites are divided into four groups according to their Scc2 FE and depicted by different colors. (Bottom) The kernel density estimation plot for the cohesin sites in each group. (D) Aggregated ChIP-seq profiles of Scc1 and Scc2 in WT and Δwpl1 Δeco1 for each group of the cohesin sites. The profiles are centered at the summit of the Scc1 peaks. Bold line, mean; shaded area, 95% confidence interval. See also Figures S1–S5.

Figure 2

The deletion of Wpl1 and Eco1 synergistically enhances the interaction between Scc2 and cohesin during metaphase (A) Boxplots to compare Scc2, Pds5, and Scc1 ChIP-seq nFE values at the cohesin binding sites in indicated conditions. (B) Boxplots to compare the ratio of Scc2's nFE to Scc1’s nFE, or Pds5’s nFE to Scc1’s nFE at the cohesin sites in indicated conditions (in log₂ scale). The ratios were normalized by setting the median for WT to 1. The numbers at the top of the plots represent the median in linear scale. (C) (Top) Schematic representation of the experimental protocol to deplete Eco1 after metaphase arrest and cell-cycle arrest monitored by flow cytometry (numbers indicate the proportion of cells with 2C DNA content). WT (SN40), Δwpl1 GAL-^RECO1 (SN722), and Δwpl1 Δeco1 (SN54) strains with SCC2-PK gene were used. Asyn, asynchronous; M, metaphase; Gal, galactose. (Bottom left) Western blot assessing Smc3 acetylation level (Smc3-Ac). PS, Ponceau S staining as a loading control. (Bottom right) ChIP-qPCR of Scc2 in the indicated conditions. Analyzed loci are four representative cohesin binding sites and two non-binding sites. The mean of two technical replications was shown. Error bar, standard deviation. See also Figures S6–S10.

Figure 3

Δwpl1 Δeco1 promotes extension of pericentromeric DNA loops (A) Micro-C contact maps in WT (SKY001), Δwpl1 Δeco1 (KT110), and log2-ratio between Δwpl1 Δeco1 and WT. Cells were arrested at metaphase by benomyl treatment. Bin size is 500 bp. Squares in the contact maps indicated loops called by HICCUPs. Dashed lines mark the chromosomal domains adjacent to centromeres. Two representative regions in chromosomes XI and XIII are shown. The number of valid pairs in each sample was normalized to 115 million. CEN, centromere. (B) Proportion of the detected loops in each indicated category. Cen, centromere; cohesin BS, cohesin binding site. (C) Length distribution of cohesin loops (loops connecting between cohesin BSs) in WT and Δwpl1 Δeco1. (D) Contact-versus-distance decaying curves of the Micro-C contact matrix in WT and Δwpl1 Δeco1, and their first derivatives (slope). (E) Average contact frequency between cohesin loop anchors in WT and Δwpl1 Δeco1. Observed/expected ratio of the contact frequency around the off-diagonal peaks connecting two cohesin loop anchors found in WT or Δwpl1 Δeco1 were averaged and plotted. The number in the top-left corner of each plot indicates the average enrichment score of the 3 × 3 central pixels. (F) (Left) Average contact frequency around the centromeres in WT and Δwpl1 Δeco1. Black triangles indicate the centromere position. (Right) Averaged insulation score around the centromeres in WT and Δwpl1 Δeco1. Bold line, mean; shaded area, 95% confidence interval. (G) Heatmaps of Scc1 and Scc2 ChIP-seq nFE in WT and Δwpl1 Δeco1. 10-kb surrounding regions of the cohesin-bound loop anchors specific to WT, specific to Δwpl1 Δeco1, and shared between WT and Δwpl1 Δeco1 are depicted. The number of sites for each group is shown in parentheses. See also Figures S11 and S12.

Figure 4

DNA loop expansion in Δwpl1 Δeco1 is impeded by long centromere-oriented genes (A) ChIP-seq profile of Scc1 and Scc2 alongside loops called by HICCUP in WT and Δwpl1 Δeco1 arrested at metaphase. The cohesin sites are categorized into anchor or non-anchor sites depending on their overlapping with anchors of centromere-originated loops in Δwpl1 Δeco1. (B) Aggregated plots of Scc1 and Scc2 ChIP-seq nFE in WT and Δwpl1 Δeco1. 10-kb surrounding regions around the centromeres, anchor cohesin sites, and non-anchor cohesin sites are depicted. Bold line, mean; shaded area, 95% confidence interval. (C) Labeling of the genes adjacent to the cohesin binding sites. CEN-oriented genes, genes distal to the centromere (CEN) and transcribed toward it. TEL-oriented genes, genes proximal to CEN and transcribed away from it. Note that most of the cohesin sites are located in convergent intergenic regions. The number of genes in each category is shown in the table. (D) Boxplot comparison of gene length and Rpo21 ChIP-seq FE between CEN- and TEL-oriented genes. The numbers above the plots are p values (Mann-Whitney U test, two-tailed). NS, non-significant difference (p > 0.05). See also Figures S13–S15.

Figure 5

Synergetic effect of Wpl1 and Eco1 depletion on loop extension (A) Micro-C contact maps in PDS5-AID (SN80) treated with vehicle (+EtOH) or IAA, Δwpl1 (KT127), and Δwpl1 Δeco1 (SN54) cells arrested at metaphase. Vehicle-treated PDS5-AID serves as a control. PDS5-AID cells were cultures as in Figure S1B. Contact maps were computed on 500 bp-resolution data. The number of valid pairs in each sample was normalized to 42 million. (B) Average contact frequency (represented as observed/expected ratio) between a centromere and a cohesin site on the same chromosome in the indicated strains arrested at metaphase. The centromere-cohesin site pairs were grouped by the distance between the two sites. (C) Contact-versus-distance decaying curves of the contact matrix in the indicated strains, and their first derivatives (slope). (D) Comparison of the centromere-originated loops identified in the indicated samples. (Left) The number of the loops. (Middle) Length distribution. (Right) Scc2 ChIP-seq nFE at the non-centromeric anchor sites. Outliers were not depicted in the boxplots.

Figure 6

Acute depletion of Scc2 eliminates cohesin-mediated extended loops in Δwpl1 Δeco1 (A) (Left) Schematic representation of the experimental protocol used to arrest cells in metaphase and induce rapid degradation of Scc2-AID, and cell cycle monitoring by flow cytometry (numbers indicate the proportion of cells with 2C DNA content). (Right) Western blot to verify Scc2-AID depletion. (B) Micro-C contact maps in SCC2-AID (SN75) and Δwpl1 Δeco1 SCC2-AID (SN74) strains treated with vehicle (EtOH) or IAA at the resolution of 1 kb. The number of valid reads in each sample was normalized to 28 million. (C) Average contact frequency for the centromere-originated loops detected in vehicle-treated SCC2-AID and Δwpl1 Δeco1 SCC2-AID strains (+EtOH). The averaged contact frequency for the same locus-pairs in IAA-treated condition (+IAA) was shown side by side for comparison. (D) The number and length of centromere-originated loops detected in the indicated conditions. Outliers were omitted from the boxplot. (E) (Left) Average contact frequency around the centromeres. Black triangles indicate the centromere position. (Right) Averaged insulation score around the centromeres in the indicated condition. Bold line, mean; shaded area, 95% confidence interval. See also Figures S16 and S17.

Figure 7

Expanded pericentromeric loops impeded the smooth progression of chromosome segregation (A) (Left) Schematic representation of the experimental protocol to make cells proceed synchronously through mitosis in the absence or presence of Scc2. SCC2-AID (SN75), Δwpl1 SCC2-AID (ST726), and Δwpl1 Δeco1 SCC2-AID (SN74) were analyzed. Ben, benomyl; αF, α factor. (Right) Proportion of budded cells with one nucleus, budded cells with two nuclei, and unbudded cells with one nucleus are depicted. The mean of three or more biological replicates, in each of which >150 cells were counted, are shown. Error bar, SEM. Two-tailed t test was conducted to evaluate the statistical significance of the difference in the fraction of budded cells with one nucleus (orange). ∗∗∗, p < 0.001; ∗, p < 0.05. (B) (Upper left) Schematic representation of the experimental protocol to make cells proceed synchronously through mitosis in the absence or presence of Eco1. Δwpl1 SCC2-AID (ST730) and Δwpl1 GAL-^RECO1 SCC2-AID (ST731) were analyzed. Gal, galactose. (Lower left) Categorization of cells based on morphology and URA3-GFP foci. (Right) The proportion of cells in each category. Cells were collected at 0, 40, or 60 min after the release from benomyl-arrest. The means of three biological replicates, in each of which >150 cells were counted, are shown. Error bar, SEM. Two-tailed t test was conducted to evaluate the statistical significance of the difference in the fraction of budded cells with a single GFP dot (orange). ∗∗∗, p < 0.001; ∗∗, p < 0.01.