Cohesin recruits the Esco1 acetyltransferase genome wide to repress transcription and promote cohesion in somatic cells

Abstract

The cohesin complex links DNA molecules and plays key roles in the organization, expression, repair, and segregation of eukaryotic genomes. In vertebrates the Esco1 and Esco2 acetyltransferases both modify cohesin's Smc3 subunit to establish sister chromatid cohesion during S phase, but differ in their N-terminal domains and expression during development and across the cell cycle. Here we show that Esco1 and Esco2 also differ dramatically in their interaction with chromatin, as Esco1 is recruited by cohesin to over 11,000 sites, whereas Esco2 is infrequently enriched at REST/NRSF target genes. Esco1's colocalization with cohesin occurs throughout the cell cycle and depends on two short motifs (the A-box and B-box) present in and unique to all Esco1 orthologs. Deleting either motif led to the derepression of Esco1-proximal genes and functional uncoupling of cohesion from Smc3 acetylation. In contrast, other mutations that preserved Esco1's recruitment separated its roles in cohesion establishment and gene silencing. We conclude that Esco1 uses cohesin as both a substrate and a scaffold for coordinating multiple chromatin-based transactions in somatic cells.

Keywords: acetylation; chromosomes; gene expression; mitosis; sister chromatids.

Fig. 1.

Human cohesin acetyltransferases (CoATs) differ significantly in their sites of interaction with chromatin. (A) Sequence coverage of Esco1 and Esco2 over a 1.5-Mb region of chromosome 5. Bars below coverage tracks indicate peaks called in both biological replicates. Input used for FLAG-Esco1 ChIP is shown. RPM, reads per million. (B) Scaled Venn diagram comparing replicate-consensus peaks for Esco1 and Esco2 (see also Fig. S1). (C) Scatter plot comparing enrichment and confidence metrics for Esco1 and Esco2 consensus peaks. (D) Esco1 and Esco2 binding sites are enriched for CTCF and REST/NRSF transcription factor motifs. (E) Plots of CTCF and REST motif density around Esco1 and Esco2 peaks.

Fig. 2.

Esco1 is recruited to its binding sites by cohesin. (A) Sequence coverage of K105-acetylated Smc3 (AcSmc3), Rad21, Esco1, and Esco2 over a 1-Mb region of chromosome 11. Input used for AcSmc3 ChIP is shown. (B) Venn diagram comparing replicate-consensus peaks for AcSmc3, Rad21, and Esco1. (C) Plot of Esco1 read density around Rad21 consensus peaks. (D) FLAG-Esco1 cells were transfected with control (siGL2) or Rad21-specific siRNAs, then synchronized in S phase with thymidine. Soluble and chromatin fractions were prepared (24), resolved by SDS/PAGE, and blotted with the indicated antibodies. (E) Cells in D were subjected to FLAG ChIP and quantitative real-time PCR (for primers, see Table S1). Error bars indicate SEMs from triplicate measurements in three independent experiments. Familywise and individual P values were computed with two-way ANOVA and Holm–Sidak multiple comparison tests (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (F) Native chromatin from FLAG-Esco1 cells was digested with benzonase or left intact, then immunoprecipitated with FLAG antibodies in the presence or absence of competitor peptide. Input, flowthrough, and bead-eluted fractions were resolved by SDS/PAGE and blotted as shown.

Fig. 3.

Esco1 colocalizes with cohesin throughout the cell cycle and down-regulates expression of neighboring genes. (A) FLAG-Esco1 cells were synchronized using a double-thymidine block and collected 5 h (G2 phase) or 13 h (G1 phase) after release. DNA content was determined by propidium iodide staining and flow cytometry. (B and C) Cells in A were subjected to FLAG ChIP (B) or AcSmc3 ChIP (C). Error bars reflect SEM of triplicate measurements from two independent experiments. P values were computed using two-way ANOVA and Holm–Sidak multiple comparison tests. (D) Heatmap of genes differentially expressed after Esco1 or Rad21 depletion. A set of 548 Esco1-regulated genes (FDR ≤ 0.05; see Table 1) was used for hierarchical clustering of control, Esco1-, and Rad21-depleted HeLa cells (three biological replicates per condition). Scale displays standardized probe intensities. The final two columns indicate proximity (≤5 kb) to Rad21 or Esco1 binding sites. (E) Validation of Esco1-regulated gene expression. Transcript levels in control and Esco1-depleted HeLa cells were determined by reverse transcription and quantitative PCR (RT-qPCR). Primer sequences are given in Table S2. Fold change is reported relative to the siGL2 control, using GAPDH as an internal reference. Error bars indicate SEMs from triplicate measurements in two experiments. P values were computed using two-way ANOVA and Holm–Sidak multiple comparison tests. (F) Esco1 ChIP-Seq reads at three genes (TAF12, DNER, and CLDN11) validated by RT-qPCR.

Fig. 4.

Two motifs in Esco1 mediate its association with cohesin and gene-silencing activity. (A) Twenty-two Esco1 orthologs were aligned and organized into a phylogenetic tree (Fig. S4A). The A-box, B-box, and a third motif present in terrestrial vertebrates (T) are indicated (see also Fig. S4 B–D). (B) Soluble and chromatin fractions from HeLa cells expressing wild-type (WT) or mutant (ΔA, ΔT, ΔB) FLAG-Esco1 or no transgene (ø) were resolved by SDS/PAGE and blotted as shown. (C) Site-specific Esco1 binding was assessed by FLAG ChIP and quantitative PCR. Data are from two independent experiments. (D and E) N-terminal Esco1 mutants are defective in gene silencing and interfere with endogenous Esco1. Transcript levels were quantified before (D) or after (E) Esco1 knockdown as in Fig. 3E. Error bars indicate SEMs from triplicate RT-qPCR measurements of three to four biological replicates per condition. P values were computed as above.

Fig. 5.

Esco1’s sustained association with cohesin-DNA complexes is required to convert Smc3 acetylation into functional cohesion. (A) HeLa cells expressing RNAi-resistant FLAG-Esco1 (WT, ΔA, ΔT, ΔB) or no transgene (ø) were transfected with control or Esco1-specific siRNAs. Whole-cell lysates were resolved by SDS/PAGE and blotted as shown. Quantitation of total and acetylated Smc3 was performed using infrared dye-coupled antibodies and two-channel imaging system. (B and C) Cells in A were treated with nocodazole for two hours and analyzed by chromosome spreading. The incidence of sister chromatid separation (SCS) was determined from 80 to 200 cells per condition in two separate experiments. P values were computed using one-way ANOVA and Holm–Sidak multiple comparison tests. Error bars indicate SEM. (D) A model for the role of Esco1’s N-terminal domain in gene silencing and cohesion establishment. Cohesin recruits Esco1 during or shortly after its ATPase-dependent loading, via the latter’s A-box and B-box motifs. In addition to acetylating Smc3′s ATPase domain, Esco1 remains bound to the cohesin-DNA complex. Through this interaction, Esco1 is able to silence the expression of nearby genes, likely through stabilization of intrachromosomal loops organized by cohesin and CTCF (Top) or engagement of a transcriptional corepressor (CR, Middle). During DNA replication, both Smc3 acetylation and sustained Esco1 binding are required to establish cohesion between sister chromatids (Bottom). These observations suggest that interactions between Esco1 and the cohesin-DNA complex are required for essential DNA-tethering reaction(s) (for example, opening the cohesin ring to allow entry of a second chromatin fiber without dissociation from the first) or to stabilize the reaction products until the arrival of sororin.