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Abstract The Sc2.0 global consortium to design and construct a synthetic genome based on theSaccharomyces cerevisiaegenome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp syntheticSaccharomyces cerevisiaechromosomesynXVIof the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects andsynXVI, anin-silicoredesign of thesynXVIchromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. Thein-silicoredesign ofsynXVIincludes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms. 

Summary The evolutionary origins of the genetic point centromere in the brewer’s yeastSaccharomyces cerevisiae,a member of the order Saccharomycetales, are still unknown. Competing hypotheses suggest that the point centromere tripartite genetic centromere DNA elements (CDEs) either evolved from ancestral epigenetic centromeres by descent with modification or were gained through horizontal transfer from selfish DNA plasmids.^1,2Here, we identified centromeres in the sister order Saccharomycodales and termed them “proto-point centromeres” due to sequence features that bridge the evolutionary gap between point centromeres and ancestral centromeres types. Comparative genomic analyses across multiple yeast orders showed an unexpected evolutionary link between point and proto-point centromeres to the long terminal repeats (LTRs) of Ty5 retrotransposons. Strikingly, one Saccharomycodales species,Saccharomycodes ludwigii, harbors compact Ty5-based centromeres, where its CDEII elements are divergent AT-rich Ty5 LTRs. These living fossil centromeres show how retrotransposon cis-regulation was likely co-opted for genetic centromere specification. These insights show that point centromeres are direct descendants of retrotransposons and have evolved by descent with modification. Ultimately, the many diverse centromere types across the yeast subphylum may share a common ancestry rooted in retrotransposon activity. 

Abstract Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering inSaccharomyces cerevisiae. 

Summary Eukaryotic DNA wraps around histone octamers forming nucleosomes, which modulate genome function by defining chromatin environments with distinct accessibility. These well-conserved properties allowed “humanization” of the nucleosome core particle (NCP) inSaccharomyces cerevisiaeat high fitness costs. Here we studied nucleosome-humanized yeast-genomes to understand how species-specific chromatin affects nuclear organization and function. We found a size increase in human-NCP, linked to shorter free linker DNA, supporting decreased chromatin accessibility. 3-D humanized-genome maps showed increased chromatin compaction and defective centromere clustering, correlated with high chromosomal aneuploidy rate. Site-specific chromatin alterations were associated with lack of initiation of early origins of replication and dysregulation of the ribosomal (rDNA and rRNA) metabolism. This latter led to nucleolar fragmentation and rDNA-array instability, through a non-coding RNA dependent mechanism, leading to its extraordinary, but entirely reversible, intra-chromosomal expansion. Overall, our results reveal species-specific properties of the NCP that define epigenome function across vast evolutionary distances. HighlightsHumanized nucleosomes wrap 10 additional nucleotides, shortening free linker lengthHistone-humanized nucleosomes have increased occupancy for DNAHumanized nucleosomes potentially decrease chromatin accessibility by blocking-out free linker DNANucleosome humanization impedes DNA replication by affecting chromatin structure at originsHumanized nucleosomes reversibly destabilize the ribosomal DNA array and leads to massive intrachromosomal rDNA locus expansionHistone humanization disrupts rDNA silencing and leads to nucleolar fragmentation 

Abstract Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here, we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation—mediated by the trans-regulator Spt10—is ancient, likely emerging between 320 and 380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage, which coincided with the loss of 1 copy of its paralogous core histone genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora fast-evolving lineage. 

Abstract The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity ofSaccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a “one-amino-acid-one-codon” strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitutechrXIILfor viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences. 

Abstract Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4–CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation. 

Abstract Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here, we show that aneuploidy in histone‐humanized yeasts is specific to a subset of chromosomes that are defined by their centromeric evolutionary origins but that these aneuploidies are not adaptive. Instead, we find that a set of missense mutations in outer kinetochore proteins drives adaptation to human histones. Furthermore, we characterize the molecular mechanism underlying adaptation in two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. We propose a model through which weakened microtubule attachments promote increased kinetochore‐microtubule turnover and thus suppress chromosome instability. In sum, our data show how a set of point mutations evolved in histone‐humanized yeasts to counterbalance human histone‐induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.