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3D organization of synthetic and scrambled chromosomes
Authors
Guillaume Mercy, Julien Mozziconacci, Vittore F. Scolari, Kun Yang, Guanghou Zhao, Agnès Thierry, Yisha Luo, Leslie A. Mitchell, Michael Shen, Yue Shen, Roy Walker, Weimin Zhang, Yi Wu, Ze-xiong Xie, Zhouqing Luo, Yizhi Cai, Junbiao Dai, Huanming Yang, Ying-Jin Yuan, Jef D. Boeke, Joel S. Bader, Héloïse Muller, Romain Koszul
Abstract
INTRODUCTION
The overall organization of budding yeast chromosomes is driven and regulated by four factors: (i) the tethering and clustering of centromeres at the spindle pole body; (ii) the loose tethering of telomeres at the nuclear envelope, where they form small, dynamic clusters; (iii) a single nucleolus in which the ribosomal DNA (rDNA) cluster is sequestered from other chromosomes; and (iv) chromosomal arm lengths. Hi-C, a genomic derivative of the chromosome conformation capture approach, quantifies the proximity of all DNA segments present in the nuclei of a cell population, unveiling the average multiscale organization of chromosomes in the nuclear space. We exploited Hi-C to investigate the trajectories of synthetic chromosomes within the
RATIONALE
The Sc2.0 genome design specifies strong conservation of gene content and arrangement with respect to the native chromosomal sequence. However, synthetic chromosomes incorporate thousands of designer changes, notably the removal of transfer RNA genes and repeated sequences such as transposons and subtelomeric repeats to enhance stability. They also carry loxPsym sites, allowing for inducible genome SCRaMbLE (synthetic chromosome rearrangement and modification by
RESULTS
Comparison of synthetic chromosomes with native counterparts revealed no substantial changes, showing that the redesigned sequences, and especially the removal of repeated sequences, had little or no effect on average chromosome trajectories. Sc2.0 synthetic chromosomes have Hi-C contact maps with much smoother contact patterns than those of native chromosomes, especially in subtelomeric regions. This improved “mappability” results directly from the removal of repeated elements all along the length of the synthetic chromosomes. These observations highlight a conceptual advance enabled by bottom-up chromosome synthesis, which allows refinement of experimental systems to make complex questions easier to address. Despite the overall similarity, differences were observed in two instances. First, deletion of the
In addition to describing the Sc2.0 chromosome organization, we also used Hi-C to identify chromosomal rearrangements resulting from SCRaMbLE experiments. Inducible recombination between the hundreds of loxPsym sites introduced into Sc2.0 chromosomes enables combinatorial rearrangements of the genome structure. Hi-C contact maps of two SCRaMbLE strains carrying synIII and synIXR chromosomes revealed a variety of cis events, including simple deletions, inversions, and duplications, as well as translocations, the latter event representing a class of trans SCRaMbLE rearrangements not previously observed.
CONCLUSION
This large data set is a resource that will be exploited in future studies exploring the power of the SCRaMbLE system. By investigating the trajectories of Sc2.0 chromosomes in the nuclear space, this work paves the way for future studies addressing the influence of genome-wide engineering approaches on essential features of living systems.
Synthetic chromosome organization.
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