Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast

Yue Shen, Feng Gao, Yun Wang, Ju Zheng, Jianhui Gong, Jintao Zhang, Zhouqing Luo, Daniel Schindler, Yang Deng, Weichao Ding, Tao Lin, Teem Swidah, Hongcui Zhao, Shuangying Jiang, Cheng Zeng, Shihong Chen, Tai Chen, Yong Wang, Yisha Luo, Leslie Mitchell, Joel S Bader, Guojie Zhang, Xia Shen, Jian Wang, Xian Fu, Junbiao Dai, Jef D Boeke, Huanming Yang, Xun Xu, Yizhi Cai

Preprint posted on 2 September 2022 https://www.biorxiv.org/content/10.1101/2022.09.01.506252v1

Article now published in Cell Genomics at http://dx.doi.org/10.1016/j.xgen.2023.100364

Disomic yeast cells find relief from the stress of too much DNA by scrambling up the additional chromosome

Selected by Grace Thomas

Categories: cancer biology, cell biology, genetics, genomics, synthetic biology, systems biology

Background

Aneuploidy, an abnormality in chromosomal copy number, is typically the result of aberrant chromosome segregation during cell division. The resulting genomic instability can result in cell death, or in some organisms, can provide an opportunity for the cell to adapt to environmental conditions ¹. In humans, aneuploidy is bad news; often the cause of embryo inviability, genetic disease, and tumorigenesis ².

The effect of aneuploidy varies widely depending on the cell type and karyotype. This is challenging to study in multicellular model organisms, due to the difficulty in generating stable aneuploid cell populations. With decades worth of sophisticated molecular tools available, the budding yeast Saccharomyces cerevisiae is a widely used model for looking at the molecular mechanisms behind aneuploidy. By forcing mis-segregation, previous studies have been able to produce targeted aneuploid cells, and study the effect of various karyotypic changes ³. This preprint takes this a step further by investigating contributions of specific regions of chromosome VII to the fitness of aneuploid cells.

The Synthetic yeast genome project, or Sc2.0, led by Jef Boeke, is the first ever attempt to design and build a synthetic eukaryotic genome. The designer chromosomes have been streamlined and modified, with unstable repetitive motifs removed, and additional elements – like loxP sites and PCRTags – added in. These loxP sites are part of the SCRaMbLE system (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution), which facilitates the generation of combinatorial genomic diversity ⁴. In the presence of inducible Cre recombinase, DNA sequences flanked by loxP sites recombine with other loxP sites across the chromosome, resulting in inversions, deletions or insertions. By introducing loxP sites across the synthetic chromosomes, massive random chromosomal rearrangements can be induced at will. Using this system, the authors present a compelling new method to systematically investigate the effect of the loss or retention of sub-chromosomal regions on aneuploidy phenotypes.

Key findings

Synthetic chromosome VII can be stably maintained in a disomic yeast strain (n+1) and its presence leads to aneuploidy-specific phenotypes.

Using the standard design principles for producing a synthetic chromosome with SCRaMbLE capabilities, the authors constructed and debugged synthetic chromosome VII (synVII). At each stage of construction of the chromosome, cells containing the construct were assayed for fitness defects in a process called debugging. These growth defects could be caused by modifications to gene flanking sequences. To restore fitness that section of synVII had to be redesigned.

In order to generate an aneuploid strain with synVII, the authors first made an n+chrVII disomic strain. This chrVII-specific aneuploid yeast strain (YSy140 n+chrVII) was generated by inserting a galactose inducible promoter adjacent to chrVII CEN7, resulting in non-disjunction of chrVII. YSy140 and the strain containing synVII (YSy105) were mated and sporulated to finally obtain a disomic strain with one copy of native chrVII and one copy of synVII (YSy142 n+synVII). This strain was verified with whole genome sequencing and exhibited aneuploidy-specific phenotypes, namely increased sensitivity to hydroxyurea (an inhibitor of ribonucleotide reductase), cycloheximide (a protein synthesis inhibitor), and methyl methanesulfonate (a DNA-damaging agent).

969 recombination events were detected in 219 SCRaMbLEd aneuploid yeast strains

To induce SCRaMbLE in synVII, a Cre recombinase expression plasmid was transformed into YSy142. Induction of Cre expression resulted in recombination across the genome between segments flanked by loxP sites. SCRaMbLEd aneuploid derivatives were recovered on selective agar medium and analyzed by genome sequencing.

Overall, 989 recombination events were identified in 291 strains; 62% were deletions, 29% were inversions and 9% were duplications. On average, there were 42 events per strain. The sequence adjacent to the centromere was the most consistently retained sequence likely due to the presence of the selection marker LEU2 by the centromere. The authors speculated that it is possible that genes near the telomeres could be toxic when multiple copies are present, as these regions were frequently absent from the SCRaMbLEd strains.

Fig 1- The SCRaMbLE strategy produced strains with rearranged synVII that could be rescued on agar selection media. 291 SCRaMbLEd synVII were sequenced and the sequence was reconstructed. The genomes of these strains had unique deletions, inversions or duplication events.

SCRaMbLEing of the SynVII chromosome rescued aneuploidy phenotypes

Relative fitness of the SCRaMbLEd strains was calculated by comparing average colony size of >200 colonies to the original YSy142 aneuploid colonies. The SCRaMbLEd colonies showed improved fitness in the presence of cycloheximide, varying from 6.6%-80%. The authors identified two types of structural conformation of the SCRaMbLEd synVII (circular and linear). 89% of the 219 strains had the circular form of synVII. Some of these strains had lost most of the chromosomal arms, retaining just 1-19% of synVII. These strains had a higher fitness recovery rate (40%-60%) (figure 2). On the other hand, the authors found that 18/24 of strains that had retained <50% of the synVII had a more modest recovery rate (average of 32.4%). This supports the “mass action of genes hypothesis”, suggesting that aneuploidy phenotypes are the result of copy alterations of many genes that may not individually have observable phenotypes.

Fig 2- General improvement of fitness of the top 18 SCRaMbLEd strains in 5 representative conditions compared to parental disomic strain. Each dot represents one strain’s recovery rate calculated based off the size of >200 single colonies. From left to right: YPD, YDP + hydroyurea (HU 100mM), YPD + cycloheximide (Cyc 0.01µg/mL), YPD + DL-Dithiothreitol (DDT 2.5mM), YPD + methyl methane sulfone (MMS 0.01%).

Removal of a specific 20Kb region led to improved fitness and was linked to up-regulation of translation.

Using a chromosome-wide association analysis, the authors identified a 20kb deletion present in several strains that may play a role in fitness recovery in aneuploidy strains. In addition, a proteomic analysis of 5 strains showed that protein synthesis and ribosome biogenesis were up-regulated. The authors suggested that this result implies that there are specific genes that can cause aneuploidy phenotypes when duplicated. Their findings therefore provide evidence for both the “mass action of genes” theory and the idea that there are key genes that are toxic when present in multiple copies ⁵.

Why I chose this study:

Synthetic biology provides creative approaches to explore molecular mechanisms. I like this study because it attempts to unpick an incredibly complicated phenotype. By producing hundreds of scrambled-up, aneuploid strains the authors have attempted to reach the level of diversity seen in aneuploidy in nature. I appreciate the systematic approach that allows us to focus solely on one chromosome at a time to identify particular genes and regions, and I look forward to seeing the authors apply their SCRaMbLE approach to the other 15 chromosomes in yeast.

I find it curious that practically any rearrangement seems to improve the aneuploidy phenotypes, even when much of the chromosome sequence is retained. I would assume that the rearrangements cause a general disruption to the expression and function of the genes on synVII. It would be very interesting to further investigate the mechanisms behind some of these chromosomal rearrangements, and to see if any equivalent perturbations could be identified in aneuploid mammalian cells.

Questions for the author:

You show that aneuploidy phenotypes can be somewhat rescued by introducing the 20 kb deletion – identified in SCRaMbLEd strains – into synVII and wild type chrVII. Would you also expect to see an associated upregulation of protein synthesis and ribosome biogenesis? Perhaps additional proteomic analysis of these modified diploid strains could confirm the proposed causal relationship between the deletion and the upregulation of these processes.

In the case of the SCRaMbLEd chromosomes that retained most, or all the chromosome content, what mechanism do you propose causes the mitigation of aneuploidy phenotypes? Is it possible that SCRaMbLE causes loss of function of genes across the chromosomes?

References:

Kaya, A., Mariotti, M., Tyshkovskiy, A. et al. Molecular signatures of aneuploidy-driven adaptive evolution. Nat Commun 11, 588 (2020). https://doi.org/10.1038/s41467-019-13669-2

Ben-David, U., Amon, A. Context is everything: aneuploidy in cancer. Nat Rev Genet 21, 44–62 (2020). https://doi.org/10.1038/s41576-019-0171-x

Beach R R., Ricci-Tam C., Brennan CM., Moomau CA., Hsu P., Hua B., Silberman RE., Springer M., Amon A., Aneuploidy Causes Non-genetic Individuality., Cell 169, (2), (2017) http://dx.doi.org/10.1016/j.cell.2017.03.021

Jia, B., Wu, Y., Li, BZ. et al. Precise control of SCRaMbLE in synthetic haploid and diploid yeast. Nat Commun 9, 1933 (2018). https://doi.org/10.1038/s41467-018-03084-4

Bonney ME, Moriya H, Amon A., Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes.,Genes & Dev, 29, (2015) https://doi.org/10.1101/gad.261743.115

Posted on: 17 October 2022

doi: https://doi.org/10.1242/prelights.32928

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