SYNTHETIC BIOLOGY 3 D organization of synthetic and scrambled chromosomes

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 averagemultiscale organization of chromosomes in the nuclear space. We exploited Hi-C to investigate the trajectories of synthetic chromosomes within the Saccharomyces cerevisiae nucleus and compare them with their native counterparts. 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 andmodification by loxP-mediated evolution) aimed at accelerating genomic plasticity. Whether these changes affect chromosome organization, DNA metabolism, and fitness is a critical question for completion of the Sc2.0 project. To address these questions, we used Hi-C to characterize the organization of synthetic chromosomes. 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 HML and HMR silent mating-type cassettes on chromosome III led to a loss of their specific interaction. Second, repositioning the large array of rDNA repeats nearer to the centromere cluster forced substantial genome-wide conformational changes— for instance, inserting the array in the middle of the small right arm of chromosome III split the arm into two noninteracting regions. The nucleolus structure was then trapped in the middle between small and large chromosome arms, imposing a physical barrier between them. 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.