Convergent genes shape budding yeast pericentromeres

Abstract

The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1,2,3,4,5,6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Convergent genes mark pericentromere borders.
Fig. 2: The pericentromere is a multilooped structure in mitosis that extends to an open V shape under tension.
Fig. 3: Gene orientation determines pericentromere size.
Fig. 4: Gene orientation affects biorientation efficiency and cell viability.

Data availability

Sequencing datasets are available at the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/), accession number GSE104135. Source data for Figs. 1, 3, 4 and Extended Data Figs. 2, 5, 9, 10 are provided with the paper.

Code availability

The ImageJ plugin for measuring the fluorescent intensity and distance between GFP foci can be found on the GitHub repository at https://github.com/dkelly604/CellClicker_. Scripts for Hi-C data analysis are available at https://github.com/danrobertson87/Paldi_2019.

References

  1. 1.

    Rowley, M. J. & Corces, V. G. Organizational principles of 3D genome architecture. Nat. Rev. Genet. 19, 789–800 (2018).

    CAS  Article  Google Scholar 

  2. 2.

    Ng, T. M., Waples, W. G., Lavoie, B. D. & Biggins, S. Pericentromeric sister chromatid cohesion promotes kinetochore biorientation. Mol. Biol. Cell 20, 3818–3827 (2009).

    CAS  Article  Google Scholar 

  3. 3.

    Weber, S. A. et al. The kinetochore is an enhancer of pericentric cohesin binding. PLoS Biol. 2, E260 (2004).

    Article  Google Scholar 

  4. 4.

    Fernius, J. et al. Cohesin-dependent association of Scc2/4 with the centromere initiates pericentromeric cohesion establishment. Curr. Biol. 23, 599–606 (2013).

    CAS  Article  Google Scholar 

  5. 5.

    Fernius, J. & Marston, A. L. Establishment of cohesion at the pericentromere by the Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3. PLoS Genet. 5, e1000629 (2009).

    Article  Google Scholar 

  6. 6.

    Eckert, C. A., Gravdahl, D. J. & Megee, P. C. The enhancement of pericentromeric cohesin association by conserved kinetochore components promotes high-fidelity chromosome segregation and is sensitive to microtubule-based tension. Genes Dev. 21, 278–291 (2007).

    CAS  Article  Google Scholar 

  7. 7.

    Ocampo-Hafalla, M. T., Katou, Y., Shirahige, K. & Uhlmann, F. Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting. Chromosoma 116, 531–544 (2007).

    Article  Google Scholar 

  8. 8.

    Nerusheva, O. O., Galander, S., Fernius, J., Kelly, D. & Marston, A. L. Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation. Genes Dev. 28, 1291–1309 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Lopez-Serra, L., Lengronne, A., Borges, V., Kelly, G. & Uhlmann, F. Budding yeast Wapl controls sister chromatid cohesion maintenance and chromosome condensation. Curr. Biol. 23, 64–69 (2013).

    CAS  Article  Google Scholar 

  10. 10.

    Hinshaw, S. M., Makrantoni, V., Kerr, A., Marston, A. L. & Harrison, S. C. Structural evidence for Scc4-dependent localization of cohesin loading. eLife 4, e06057 (2015).

    Article  Google Scholar 

  11. 11.

    Hinshaw, S. M., Makrantoni, V., Harrison, S. C. & Marston, A. L. The kinetochore receptor for the cohesin loading complex. Cell 171, 72–84 (2017).

    CAS  Article  Google Scholar 

  12. 12.

    Lengronne, A. et al. Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430, 573–578 (2004).

    CAS  Article  ADS  Google Scholar 

  13. 13.

    Hu, B. et al. Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq. Nucleic Acids Res. 43, e132 (2015).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Verzijlbergen, K. F. et al. Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere. eLife 3, e01374 (2014).

    Article  Google Scholar 

  15. 15.

    Peplowska, K., Wallek, A. U. & Storchová, Z. Sgo1 regulates both condensin and Ipl1/Aurora B to promote chromosome biorientation. PLoS Genet. 10, e1004411 (2014).

    Article  Google Scholar 

  16. 16.

    He, X., Asthana, S. & Sorger, P. K. Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast. Cell 101, 763–775 (2000).

    CAS  Article  Google Scholar 

  17. 17.

    Goshima, G. & Yanagida, M. Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell 100, 619–633 (2000).

    CAS  Article  Google Scholar 

  18. 18.

    Tanaka, T., Fuchs, J., Loidl, J. & Nasmyth, K. Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation. Nat. Cell Biol. 2, 492–499 (2000).

    CAS  Article  Google Scholar 

  19. 19.

    Yeh, E. et al. Pericentric chromatin is organized into an intramolecular loop in mitosis. Curr. Biol. 18, 81–90 (2008).

    CAS  Article  Google Scholar 

  20. 20.

    Schalbetter, S. A. et al. SMC complexes differentially compact mitotic chromosomes according to genomic context. Nat. Cell Biol. 19, 1071–1080 (2017).

    CAS  Article  Google Scholar 

  21. 21.

    Lazar-Stefanita, L. et al. Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle. EMBO J. 36, 2684–2697 (2017).

    CAS  Article  Google Scholar 

  22. 22.

    Ganji, M. et al. Real-time imaging of DNA loop extrusion by condensin. Science 360, 102–105 (2018).

    CAS  Article  ADS  Google Scholar 

  23. 23.

    Davidson, I. F. et al. DNA loop extrusion by human cohesin. Science 366, 1338–1345 (2019).

    CAS  Article  ADS  Google Scholar 

  24. 24.

    Kim, Y., Shi, Z., Zhang, H., Finkelstein, I. J. & Yu, H. Human cohesin compacts DNA by loop extrusion. Science 366, 1345–1349 (2019).

    CAS  Article  ADS  Google Scholar 

  25. 25.

    Vian, L. et al. The energetics and physiological impact of cohesin extrusion. Cell 173, 1165–1178 (2018).

    CAS  Article  Google Scholar 

  26. 26.

    Fudenberg, G. et al. Formation of chromosomal domains by loop extrusion. Cell Rep. 15, 2038–2049 (2016).

    CAS  Article  Google Scholar 

  27. 27.

    Bausch, C. et al. Transcription alters chromosomal locations of cohesin in Saccharomyces cerevisiae. Mol. Cell. Biol. 27, 8522–8532 (2007).

    CAS  Article  Google Scholar 

  28. 28.

    Ocampo-Hafalla, M., Muñoz, S., Samora, C. P. & Uhlmann, F. Evidence for cohesin sliding along budding yeast chromosomes. Open Biol. 6, 150178 (2016).

    Article  Google Scholar 

  29. 29.

    Bresson, S., Tuck, A., Staneva, D. & Tollervey, D. Nuclear RNA decay pathways aid rapid remodeling of gene expression in yeast. Mol. Cell 65, 787–800 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Biggins, S. & Murray, A. W. The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint. Genes Dev. 15, 3118–3129 (2001).

    CAS  Article  Google Scholar 

  31. 31.

    Tanaka, T. U. et al. Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections. Cell 108, 317–329 (2002).

    CAS  Article  Google Scholar 

  32. 32.

    Perea-Resa, C. & Blower, M. D. Centromere biology: transcription goes on stage. Mol. Cell. Biol. 38, e00263-18 (2018).

    Article  Google Scholar 

  33. 33.

    Stempor, P. & Ahringer, J. Seqplots—interactive software for exploratory data analyses, pattern discovery and visualization in genomics. Wellcome Open Res. 1, 14 (2014).

  34. 34.

    Belton, J.-M. & Dekker, J. Hi-C in budding yeast. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot085209 (2015).

  35. 35.

    Schalbetter, S. A., Fudenberg, G., Baxter, J., Pollard, K. S. & Neale, M. J. Principles of meiotic chromosome assembly revealed in S. cerevisiae. Nat. Commun. 10, 4795 (2018).

    Article  ADS  Google Scholar 

  36. 36.

    Servant, N. et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol. 16, 259 (2015).

    Article  Google Scholar 

  37. 37.

    Kerpedjiev, P. et al. HiGlass: web-based visual exploration and analysis of genome interaction maps. Genome Biol. 19, 125 (2018).

    Article  Google Scholar 

Download references

Acknowledgements

We thank B. Baying and V. Benes (Genecore, EMBL) for next-generation sequencing and library preparation; the Wellcome Centre for Cell Biology bioinformatics core for computational support; the Centre Optical Imaging Laboratory for support with microscopy; and W. Borek for help with statistical analysis. We thank R. Allshire, S. Bresson and D. Tollervey for helpful discussions; P. Megee for yeast strains; and W. Borek, S. Hinshaw, V. Makrantoni and P. Romé for comments on the manuscript. This study was funded by a Wellcome Senior Research Fellowship (grant 107827 to A.L.M., B.A. and F.P.); a Wellcome PhD studentship (109091 to F.P.); core funding for the Wellcome Centre for Cell Biology (203149 to A.L.M., F.P., B.A., D.R., A.K. and D.A.K.); a European Research Council (ERC) Consolidator Award (311336 to M.J.N. and S.A.S.); and a Wellcome Trust Investigator Award (200843 to M.J.N. and S.A.S.).

Author information

Affiliations

Authors

Contributions

A.L.M., F.P. and B.A. conceived the study. F.P. performed Hi-C, ChIP-seq, microscopy and viability experiments. B.A. performed ChIP-seq and ChIP–qPCR experiments. F.P., B.A. and A.L.M. generated reagents and analysed data. D.R., A.K. and S.A.S. performed bioinformatics analyses. S.A.S. developed protocols. D.A.K. wrote custom code for image analysis. F.P., A.L.M., S.A.S., J.B. and M.J.N. interpreted Hi-C data. A.L.M. supervised the study. A.L.M. and F.P. wrote the paper, with input from all authors.

Corresponding author

Correspondence to Adele L. Marston.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Zhijun Duan, Katsuhiko Shirahige and Hongtao Yu for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Tension-dependent cohesin removal at metaphase is restricted to the pericentromere and occurs independently of Wpl1/Rad61.

a, Scc1–6HA-calibrated ChIP-seq profiles for the pericentromeric region of chromosome IV are shown for rad61Δ cells arrested in metaphase, in the absence and presence of spindle tension (n = 1). b, Mean calibrated ChIP reads (solid lines), standard errors (dark shading) and 95% confidence intervals (light shading) at a pericentromere-proximal cohesin site on each chromosome arm (n = 32 sites), for wild-type and chl4Δ cells, in either the presence or the absence of tension.

Extended Data Fig. 2 Overview of border gene organization and pericentromere size.

a, Diagram showing the positions of convergent gene pairs flanking centromeres. Grey ovals represent the centromeres; convergent gene pairs at borders are indicated by arrows. b, Genomic distances (thick grey lines) between the 3′ ends of convergent genes (black and orange arrows) at borders, as well as the 3′ and 5′ ends of the two genes transcribed towards centromeres (black arrows) (n = 49). Centre line, median; box limits, second and third quartiles; whiskers, first and fourth quartiles. c, Table of convergent genes identified at pericentromere borders for each chromosome, along with the corresponding pericentromere size. A border was defined as the innermost cohesin peak near the centromere that persisted in the presence of tension. d, Pericentromere sizes determined in c are plotted against chromosome size. e, Percentages of genes essential for growth on rich glucose media among genes at borders and among all genes. f, Scc1 ChIP-seq in asynchronous C. glabrata cells, showing chromosome F from ref. 13. Source Data

Extended Data Fig. 3 An ectopic centromere establishes new borders at convergent genes on a chromosome arm.

Shown are cohesin (Scc1) ChIP-seq profiles for the region surrounding the endogenous centromere on chromosome III (left), and for a roughly 50-kb region of chromosome III surrounding the ectopic centromeric arm site (right) (n = 1). Regions of tension-insensitive cohesin peaks at convergent sites flanking the endogenous and ectopic centromeres are highlighted.

Extended Data Fig. 4 Shugoshin and condensin localize to pericentromere borders.

a, Representative cohesin (Scc1), shugoshin (Sgo1) and condensin (Brn1) enrichment in metaphase-arrested cells in the presence of nocodazole in the pericentromeric region of chromosome IV (n = 2; immunoprecipitation was performed using proteins tagged with different epitopes in the two biological replicates). b, Plots show mean calibrated ChIP reads (solid lines), standard errors (dark shading) and 95% confidence intervals (light shading) at all 32 borders and 16 centromeres. For comparison, similar plots for the next convergent gene site on each chromosome arm are shown. c, d, Condensin associates with pericentromere borders in a Sgo1-dependent manner in cells arrested in metaphase in the absence of tension. c, These ChIP-seq data have been published previously14, and show the median condensin (Brn1) signal across a 50-kb region surrounding all 16 centromeres. d, The mean Brn1 signal centred around all 32 borders (left) or 16 centromeres (right). Sgo1 is removed from the borders, but not from core centromeres, in response to spindle tension (solid lines show medians; dark shading shows standard errors; light shading shows 95% confidence intervals). e, Median Sgo1 enrichment by ChIP-seq, plotted over a 50-kb region surrounding all 16 centromeres in metaphase-arrested cells in the presence or absence of tension. f, Mean Sgo1 signal centred around all 32 borders (left) or 16 centromeres (right) (solid lines, medians; dark shading, standard errors; light shading, 95% confidence intervals).

Extended Data Fig. 5 Pericentromere borders resist sister-chromatid separation under tension.

a, Assay to measure separation of loci on sister chromatids in metaphase-arrested cells. Cells carry tetO/TetR–GFP foci (green) integrated at various positions and Spc42–tdTomato foci (pink) to mark spindle-pole bodies (SPBs); the cells are arrested in metaphase by Cdc20 depletion. The diagram at the left shows the expected separation of GFP foci inside and outside pericentromere loci; this distance is measured as described in the Methods. A representative image is shown on the right (n = 3 biological replicates). Filled and open arrowheads mark cells with a single GFP focus or split foci, respectively. b, Positions of GFP foci and corresponding calibrated Scc1–6HA ChIP-seq profiles (n = 3) for chromosomes I and III. c, Cells carrying tetO/TetR–GFP foci integrated at various positions were arrested in metaphase and the distance between GFP dots was measured in 100 cells. Horizontal lines indicate means. Source Data

Extended Data Fig. 6 Cis-looping and alternative model for pericentromeric chromosome conformation.

a, Pile-ups (bin size 1 kb) of cis contacts located in the 100 kb around all 16 centromeres for wild-type and chl4Δ cells in the absence or presence of spindle tension. b, Pile-ups of cis contacts in the 100 kb around centromeres (left); ratio of expected/observed signals (second panel); pericentromere pile-up (third panel, 10 kb surrounding centromeres); and its ratio of expected/observed signals (right), for wild-type cells in the absence of spindle tension (n = 16). c, Model proposed previously19 showing the formation of intramolecular loops by centromere-flanking pericentromeric chromatin, with cohesin bridging the two sides of the pericentromere flanking the centromere; meanwhile chromosome arms are paired intermolecularly between sister chromatids, resulting in a cruciform chromosome conformation.

Extended Data Fig. 7 Changes in pericentromere structure on individual chromosomes in response to tension and in the absence of pericentromeric cohesin.

Shown are Hi-C contact maps (1-kb bin) over a 50-kb region surrounding all centromeres in wild-type cells (WT; left panels for each chromosome) without tension (bottom half of heatmap) and with tension (top half of heatmap), and in chl4Δ cells (middle panels) without tension (bottom half) and with tension (top half) (n = 1). Right, log2 ratios between wild-type and chl4Δ cells, without tension (bottom half) and with tension (top half). The extent of the pericentromere for each chromosome is marked by the black bars on top of the contact maps.

Extended Data Fig. 8 Pericentromere structure depends on pericentromeric cohesin rather than condensin.

Hi-C analysis of sgo1-3A and sgo1Δ metaphase-arrested cells in the absence of tension reveals similar patterns to wild type. Data for wild type are reproduced from Extended Data Fig. 6 for comparison. a, Pile-ups (bin size 1 kb) of cis contacts surrounding all 16 centromeres in the absence of spindle tension for the indicated strains (left three panels), and log2 ratio maps between wild type and sgo1-3A or sgo1Δ (right two panels), detect little change. b, Pile-ups (bin size 1 kb) and log2 ratio maps of cis contacts surrounding all 32 borders. c, The log2 ratio between 25-kb pile-ups centred on the centromere in wild-type and chl4Δ cells, in the absence (left) and presence (right) of tension (n = 16).

Extended Data Fig. 9 Transcription at pericentromere borders influences cohesin position.

a, Genes at pericentromere borders are moderately transcribed on average. Shown is the relative RNA density for convergent border gene pairs compared with all genes under conditions of no tension or tension (n = 1). RNA-seq analysis of wild-type cells arrested in metaphase in the presence or absence of tension. Dashed lines indicate means; dotted lines mark 95% confidence intervals. b, Box plot showing transcription levels of genes at pericentromere borders based on RNA polymerase II (Rpo21) cross-linking and analysis of cDNA (CRAC) from ref. 29. Rpo21 CRAC sense read counts of genes at borders were normalized to the protein-coding-gene average, and genes at pericentromere borders were grouped by their relative orientation to centromeres. Data points correspond to means of three biological repeats. Centre lines, medians; box limits, second and third quartiles; whiskers, first and fourth quartiles (non-normal distribution, Shapiro–Wilk; *P < 0.05, two-sided Mann–Whitney test). c, Box plot showing relative transcription levels of genes transcribed towards and away from centromeres, at pericentromere borders and at non-border convergent genes inside pericentromeres, as in b. d, e, Insertion of a URA3 cassette between a convergent gene pair shifts the localization of cohesin in the direction of transcription. URA3 was integrated in either orientation between the convergent gene pairs at the left pericentromere border on chromosome IV, and cohesin (Scc1) ChIP–qPCR (n = 3; bars show means ± s.e.m.) using primers at the indicated positions (d) and ChIP-seq (n = 1) (e) were performed. Source Data

Extended Data Fig. 10 Gene reorientation at borders affects pericentromeric protein localization, sister-chromatid separation and chromosome conformation.

a, Cohesin (Scc1), shugoshin (Sgo1) and condensin (Brn1) enrichment in metaphase-arrested cells in the presence of nocodazole in the pericentromeric region of wild-type and reoriented chromosome IV is shown (n = 2; immunoprecipitation was performed using proteins tagged with different epitopes in the two replicates). Asterisks indicate new peaks in reoriented strain. b, The distance between GFP foci does not change following gene reorientation. We measured 100 cells; horizontal lines indicate means. c, The region of separation upon gene reorientation does not extend beyond the next convergent gene pair. Strains with tetO arrays integrated at the indicated positions were arrested in metaphase, and the percentage of cells with two GFP foci was determined. For each biological replicate (data points; n = 3), 200 cells were scored; data are means ± s.e.m. d, Pile-up of cis contacts across all 16 pericentromeres in the reoriented strain, in the presence of spindle tension. e, log2 ratio map of Hi-C signal in pericentromere IV in wild-type and reoriented strains (n = 1). f, Model for pericentromere expansion and disorganization in the absence of convergent genes. g, Diagram showing biorientation of sister kinetochores following spindle repolymerization. Cells carrying chromosomal GFP labels, Spc42–tdTomato and pMET-CDC20 were released from G1 phase into a metaphase arrest by depleting Cdc20 in the presence of nocodazole. Nocadazole was washed out while maintaining metaphase arrest and the percentage of cells with two GFP foci was scored over time. Source Data

Supplementary information

Supplementary Tables

The file contains 5 supplementary tables relevant to the methods section, comprising the lists of yeast strains, primers and plasmids used in this study, reference genome coordinates used to generate plots showing ChIP-seq averages, together with the read counts for the Hi-C sequencing libraries generated during this study.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Paldi, F., Alver, B., Robertson, D. et al. Convergent genes shape budding yeast pericentromeres. Nature 582, 119–123 (2020). https://doi.org/10.1038/s41586-020-2244-6

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.