Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Crosland, M. W. & Crozier, R. H. Myrmecia pilosula, an ant with only one pair of chromosomes. Science 231, 1278 (1986).
Green, E. D., Watson, J. D. & Collins, F. S. Human Genome Project: Twenty-five years of big biology. Nature 526, 29–31 (2015).
Lee, J. Y. et al. Simple purification of human chromosomes to homogeneity using muntjac hybrid cells. Nat. Genet. 7, 29–33 (1994).
Goffeau, A. et al. Life with 6000 genes. Science 274, 546–567 (1996).
Wood, V. et al. The genome sequence of Schizosaccharomyces pombe. Nature 415, 871–880 (2002).
McClintock, B. The stability of broken ends of chromosomes in Zea mays. Genetics 26, 234–282 (1941).
DiCarlo, J. E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).
Moretti, P., Freeman, K., Coodly, L. & Shore, D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 8, 2257–2269 (1994).
Gotta, M. et al. The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae. J. Cell Biol. 134, 1349–1363 (1996).
Zhou, J., Zhou, B. O., Lenzmeier, B. A. & Zhou, J. Q. Histone deacetylase Rpd3 antagonizes Sir2-dependent silent chromatin propagation. Nucleic Acids Res. 37, 3699–3713 (2009).
Zhang, W. et al. Engineering the ribosomal DNA in a megabase synthetic chromosome. Science 355, eaaf3981 (2017).
Duan, Z. et al. A three-dimensional model of the yeast genome. Nature 465, 363–367 (2010).
Mercy, G. et al. 3D organization of synthetic and scrambled chromosomes. Science 355, eaaf4597 (2017).
Dekker, J., Rippe, K., Dekker, M. & Kleckner, N. Capturing chromosome conformation. Science 295, 1306–1311 (2002).
van Berkum, N. L. et al. Hi-C: a method to study the three-dimensional architecture of genomes. J. Vis. Exp. 39, 1869 (2010).
Le, T. B., Imakaev, M. V., Mirny, L. A. & Laub, M. T. High-resolution mapping of the spatial organization of a bacterial chromosome. Science 342, 731–734 (2013).
Engel, S. R. et al. The reference genome sequence of Saccharomyces cerevisiae: then and now. G3 (Bethesda) 4, 389–398 (2014).
Wellinger, R. J. & Zakian, V. A. Everything you ever wanted to know about Saccharomyces cerevisiae telomeres: beginning to end. Genetics 191, 1073–1105 (2012).
Mitchell, L. A. & Boeke, J. D. Circular permutation of a synthetic eukaryotic chromosome with the telomerator. Proc. Natl Acad. Sci. USA 111, 17003–17010 (2014).
Xie, Z. X. et al. Rapid and efficient CRISPR/Cas9-based mating-type switching of Saccharomyces cerevisiae. G3 (Bethesda) 8, 173–183 (2018).
Richardson, S. M. et al. Design of a synthetic yeast genome. Science 355, 1040–1044 (2017).
Spilianakis, C. G., Lalioti, M. D., Town, T., Lee, G. R. & Flavell, R. A. Interchromosomal associations between alternatively expressed loci. Nature 435, 637–645 (2005).
Cremer, T. & Cremer, M. Chromosome territories. Cold Spring Harb. Perspect. Biol. 2, a003889 (2010).
Verdaasdonk, J. S. & Bloom, K. Centromeres: unique chromatin structures that drive chromosome segregation. Nat. Rev. Mol. Cell Biol. 12, 320–332 (2011).
Kegel, A. et al. Chromosome length influences replication-induced topological stress. Nature 471, 392–396 (2011).
Yue, J. X. et al. Contrasting evolutionary genome dynamics between domesticated and wild yeasts. Nat. Genet. 49, 913–924 (2017).
Luo, J., Sun, X., Cormack, B. P. & Boeke, J. D. Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast. Nature https://doi.org/10.1038/s41586-018-0374-x (2018).
Zhou, J., Wu, R., Xue, X. & Qin, Z. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA. Nucleic Acids Res. 44, e124 (2016).
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Gietz, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protocols 2, 31–34 (2007).
Wu, Z. et al. Rad6-Bre1-mediated H2B ubiquitination regulates telomere replication by promoting telomere-end resection. Nucleic Acids Res. 45, 3308–3322 (2017).
Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722–736 (2017).
Li, H. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. Bioinformatics 32, 2103–2110 (2016).
Frith, M. C. & Kawaguchi, R. Split-alignment of genomes finds orthologies more accurately. Genome Biol. 16, 106 (2015).
Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).
Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).
Imakaev, M. et al. Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9, 999–1003 (2012).
Crane, E. et al. Condensin-driven remodelling of X chromosome topology during dosage compensation. Nature 523, 240–244 (2015).
Varoquaux, N., Ay, F., Noble, W. S. & Vert, J. P. A statistical approach for inferring the 3D structure of the genome. Bioinformatics 30, i26–i33 (2014).
Sauria, M. E., Phillips-Cremins, J. E., Corces, V. G. & Taylor, J. HiFive: a tool suite for easy and efficient HiC and 5C data analysis. Genome Biol. 16, 237 (2015).
Yardimci, G. et al. Measuring the reproducibility and quality of Hi-C data. Preprint at https://www.biorxiv.org/content/early/2018/02/05/188755 (2017).
Ay, F., Bailey, T. L. & Noble, W. S. Statistical confidence estimation for Hi-C data reveals regulatory chromatin contacts. Genome Res. 24, 999–1011 (2014).
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995).
Xie, T., Yang, Q.-Y., Wang, X.-T., McLysaght, A. & Zhang, H.-Y. Spatial colocalization of human ohnolog pairs acts to maintain dosage-balance. Mol. Biol. Evol. 33, 2368–2375 (2016).
Gayral, P. et al. Next-generation sequencing of transcriptomes: a guide to RNA isolation in nonmodel animals. Mol. Ecol. Resour. 11, 650–661 (2011).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Bennis, S., Chami, F., Chami, N., Bouchikhi, T. & Remmal, A. Surface alteration of Saccharomyces cerevisiae induced by thymol and eugenol. Lett. Appl. Microbiol. 38, 454–458 (2004).
Manukyan, A., Abraham, L., Dungrawala, H. & Schneider, B. L. Synchronization of yeast. Methods Mol. Biol. 761, 173–200 (2011).
We thank X. Gao, W. Zhao, and J. Li for technical help. This research was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000), the National Natural Science Foundation of China (31421061, 31770099, 31370120, 31230040), the National Key Basic Research Program of China (973 Program) (2011CBA00801, 2012CB721102), and the National Key Research and Development Program of China (2016YFA0500701).
Nature thanks G. Liti, K. Wolfe and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
The X, STR, and Y′ elements in each subtelomeric regions are marked with black, grey and white boxes, respectively. The TG1–3 telomeric sequences are marked with arrow tips. The XhoI digestion sites in telomere regions are indicated, and the numbers in kb in parenthesis indicate the sizes of DNA fragments recognized by the TG1–3 probe in the Southern blot analysis.
Extended Data Fig. 2 De novo sequence comparison of BY4742 (light grey) and SY14 (dark grey) genomes.
The chromosomes are labelled with Roman numerals of the yeast reference genome. The telomeres (blue), centromeres (red) and telomere-associated repeats (green) that were cut by experimental design are shown in BY4742 chromosomes. Sequence deletions and insertions identified by genomic comparison between BY4742 and SY14 are highlighted in purple and black, respectively, in SY14 chromosomes.
Extended Data Fig. 3 Comparison of the chromosomal interactions of SY6, SY13 and SY14 cells with those of BY4742 cells.
a, Z-score difference heatmaps. Bin length, 10 kb; red and blue show increased and decreased chromatin interactions, respectively. Green box highlights the interactions of the chromosome XV centromere with other chromosomes. b, Venn diagram of the number of significant (P < 0.01, q < 0.01) ‘inter’- and intra-chromosomal interactions (referring to their locations in the BY4742 genome). c, Strong chromosomal interactions of chromosome XV centromere regions in the BY4742, SY6, SY13 and SY14 genomes. The red bars indicate the centromeres and their flanking regions of 50 MboI restriction sites. Each arc throughout the chromosome XV centromere area represents one strong interaction. In SY6, SY13 and SY14, the reserved interactions are marked with black arcs and new interactions are marked with orange arcs. The green arrowheads mark the ten residual interactions near the centromere regions found in all four strains. d, 3D structures of chromosomes XV and II in SY6, SY13 and SY14 cells compared to those in BY4742 cells. The locations of the 10 residual interactions on Chr. XV and II are marked green.
Chromosome structures in SY6, SY13 and SY14 cells are compared to those in BY4742 cells.
Extended Data Fig. 5 Directional preference plots of SY6, SY13, and SY14 cells compared to BY4742 cells.
Red, BY4742; moss green, SY6; purple, SY13; bright green, SY14. The y-axis denotes the t-test value between the upstream and downstream interactions of each bin. A positive t-value indicates that a bin has more downstream interactions, as described previously16.
a, Blue circles represent BY4742/BY4742a (with HIS3 marker) cells that could grow only on SC-His plates; pink triangles represent SY14/SY14a (with URA3 marker) cells that could grow only on SC-Ura plates; green diamonds represent ‘fusion’ cells of BY4742/BY4742a and SY14/SY14a that could grow on both SC-His and SC-Ura plates. Data from three biological replicates are presented. b, FACS analysis of DNA content of BY4742/BY4742a and SY14/SY14a diploid cells before and after co-culture. Data are representative of two independent experiments. c, PCR verification of genomes from BY4742/BY4742a and SY14/SY14a diploid cells. H1–H3: colonies grown only on SC-His plates; HU1–3: colonies grown on both SC-His and SC-Ura plates. The BY4742/BY4742a and SY14/SY14a diploid cells before co-cultivation were used as control. Two pairs of primers, specific for genomes of BY4742/BY4742a and SY14/SY14a, were used. Data shown are representative images of two independent experiments.
About this article
Moulding the mould: understanding and reprogramming filamentous fungal growth and morphogenesis for next generation cell factories
Biotechnology for Biofuels (2019)
Scientific Reports (2019)
Cell Biology and Toxicology (2019)
Current Genetics (2019)
Science China Life Sciences (2019)