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The Scc2–Scc4 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions

Nature Genetics volume 46, pages 1147–1151 (2014)Cite this article

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Abstract

The cohesin complex is at the heart of many chromosomal activities, including sister chromatid cohesion and transcriptional regulation1,2,3. Cohesin loading onto chromosomes depends on the Scc2–Scc4 cohesin loader complex4,5,6, but the chromatin features that form cohesin loading sites remain poorly understood. Here we show that the RSC chromatin remodeling complex recruits budding yeast Scc2–Scc4 to broad nucleosome-free regions, which the cohesin loader helps to maintain. Consequently, inactivation of either the cohesin loader or the RSC complex has similar effects on nucleosome positioning, gene expression and sister chromatid cohesion. These results show an intimate link between local chromatin structure and higher-order chromosome architecture. Our findings pertain to the similarities between two severe human disorders, Cornelia de Lange syndrome, which is caused by alterations in the human cohesin loader, and Coffin-Siris syndrome, which results from alterations in human RSC complex components7,8,9. Both syndromes can arise from gene misregulation due to related changes in the nucleosome landscape.

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Figure 1: Scc2–Scc4 associates with promoters that are characterized by broad nucleosome-free regions.
Figure 2: Scc2–Scc4 recruitment is independent of an active, open promoter.
Figure 3: Scc2–Scc4 is recruited by the RSC chromatin remodeling complex.
Figure 4: Shared roles of Scc2–Scc4 and RSC in chromatin remodeling and transcriptional regulation.

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Acknowledgements

We thank P. Chambers at the Cancer Research UK Genome Variation Laboratory at St. James's University Hospital, Leeds, and N. Matthews from the Advanced Sequencing Facility at the Cancer Research UK London Research Institute for high-throughput sequencing. We thank C. Esnault, A. Lengronne and our laboratory members for discussions and comments on the manuscript. This work was supported by a Beca Postdoctoral del Ministerio de Ciencia y Tecnologia (Spain) and a Marie Curie Intra-European Fellowship (L.L.-S.) and the European Research Council (L.L.-S. and F.U.).

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Authors and Affiliations

  1. Chromosome Segregation Laboratory, Cancer Research UK London Research Institute, London, UK

    Lidia Lopez-Serra & Frank Uhlmann

  2. Bioinformatics and Biostatistics Service, Cancer Research UK London Research Institute, London, UK

    Gavin Kelly, Harshil Patel & Aengus Stewart

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  1. Lidia Lopez-Serra
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Contributions

L.L.-S. conceived, designed and performed the experiments. G.K. performed the statistical analyses. H.P. and A.S. analyzed high-throughput sequencing data sets. F.U. supervised the study. L.L.-S. and F.U. wrote the manuscript.

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Correspondence to Frank Uhlmann.

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Integrated supplementary information

Supplementary Figure 1 Additional characterization of Scc2–Scc4 binding sites.

(a) Scc2–Scc4 prefers intergenic regions. The fraction of assigned cohesin loader peaks that lie in intergenic regions or in ORFs, and the fraction that these features take up in the S. cerevisiae genome, is depicted. (b) The cohesin loader accumulates at RP gene promoters. Dot plot of Scc2–Scc4 peak to TSS distances for cohesin loader-bound RP genes. (c) The cohesin loader associates with highly expressed genes. The gene expression profiles of Scc2–Scc4–bound genes, divided as to whether they are encoded on the Watson or Crick strand, is compared to that of all other genes. ‘Scc2–Scc4–bound genes’ are the genes that lie closest to each of the 423 Scc2–Scc4 peaks, tRNA genes being excluded from this analysis. The y axis represents probability density of expression as estimated by a Gaussian kernel, the proportion of genes within an expression range is given as the area under the curve above that range. On average, Scc2–Scc4–bound genes are 1.5 fold more highly expressed than all other genes (P < 0.00001; Wilcoxon signed-rank test). (d) Validation by ChIP followed by quantitative real time PCR of five Scc2–Scc4–binding sites. Three negative control sites (N1, N2, N3)12, were analyzed as comparison. Scc2–Scc4 was significantly enriched at the RPL19B promoter compared to the negative control. The means and standard error of three independent experiments are shown. (e) Heat maps of ChIP sequence counts across 423 Scc2-Pk binding sites, normalized as detailed in Online Methods, compared to a control ChIP experiment with the same antibody but using a strain lacking a Pk epitope tag.

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Supplementary Figure 2 DNA sequence motif analysis at Scc2–Scc4 binding sites.

The DNA sequence surrounding Scc2–Scc4 binding sites was searched for common sequence motifs using the AlignACE algorithm, as described in Online Methods. Shown are six of the most frequently encountered motifs together with the number of occurrences and the mean offset from the Scc2–Scc4 peak summit ± its variance. Sequence motifs 1 and 2 are oligo(A)-containing motifs, motif 5 is the B-box bound by TFIIIC at tRNA genes, while the origin of motifs 3, 4 and 6 remains unknown.

Supplementary Figure 3 Scc2–Scc4 is recruited to ribosomal gene promoters independently of the transcription factor Fhl1.

(a) RPL34A and RPL19B mRNA levels, relative to total mRNA as quantified by real time PCR, were reduced to less than half in an fhl1Δ strain, as compared to a wild type strain, consistent with results obtained at other ribosomal protein genes26. (b) Scc2 ChIP was performed in parallel in wild type and fhl1Δ strains. Scc2 enrichment at two ribosomal protein gene promoters, and at a tRNA gene as a control, was analyzed by quantitative real time PCR relative to three negative control regions. The means and standard error from three independent experiments are shown.

Source data

Supplementary Figure 4 Additional characterization of Scc2 and Sth1 colocalization.

(a) Colocalization of Scc2 and Sth1 is shown using heatmaps as in Figure 3c, but ChIP-Seq counts are depicted centered on the 830 Sth1 peaks. This confirms colocalization of Scc2 with Sth1 at many of the Sth1 binding sites. It suggests that, in addition to the 423 Scc2 binding sites identified by our peak picking algorithm, Scc2–Scc4 peaks that did not meet the peak threshold requirements exist at additional Sth1 binding sites. Strong Sth1 binding often coincides with strong Scc2 binding (and vice versa, compare Figure 3c), so we retained the original selection of 423 strong Scc2–Scc4 binding sites for further analyses. (b) The degree of co-incidence of Scc2 and Sth1 binding within gene promoters, defined as occurrence of a peak’s summit within the 500 bp window immediately prior to a TSS, based on the sacCer3 genome, was tested using a hypergeometric test across all genes, with a null hypothesis that there be no association between the two factors’ binding patterns. The numbers of upstream regions that contain neither, either or both Scc2 and Sth1 peaks is tabulated, and the P value is shown.

Supplementary Figure 5 Nucleosome-free regions at cohesin loading sites are jointly maintained by the Scc2–Scc4 and RSC complexes.

A region around the RPL19B promoter was chosen to display nucleosome and Scc2-ChIP profiles in the indicated strains at 25 °C and 37 °C. The analysis confirms that maintenance of the RPL19B promoter nucleosome-free region, like that at the RPS8B promoter shown in Figure 4a, requires both Scc2–Scc4 and RSC complex function.

Supplementary Figure 6 Genes regulated by Scc2–Scc4 and Sth1 are often bound by Scc2–Scc4.

Venn diagram representing genes either up- or down-regulated greater than 1.5-fold after inactivation of Scc2 or Sth1, respectively, and those that contain an Scc2–Scc4 binding site within 500 bp upstream of the TSS (Scc2–Scc4–promoter). The significance of co-occurrence of genes in the indicated intersections was determined using a hypergeometric test.

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Lopez-Serra, L., Kelly, G., Patel, H. et al. The Scc2–Scc4 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions. Nat Genet 46, 1147–1151 (2014). https://doi.org/10.1038/ng.3080

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