Search for: All records

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. 

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 

The nucleolus is the most prominent membraneless compartment within the nucleus—dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations—where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location—the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This “megablock” rDNA engineering was performed in a fused-karyotype strain ofSaccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to theCEN, in that the locus that isCEN-distal undergoes size reduction at a higher frequency compared to theCEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogouscis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability. 

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.