Abstract Centromeres are essential for chromosome segregation, yet in many genomes they are composed entirely of rapidly evolving repetitive DNA, embedded in other repetitive DNA that forms pericentromeric heterochromatin. Due to the difficulties of manipulating these repeat-rich regions, how the relative size of pericentromeric repeat regions influences chromosome segregation remains an open question. Here, we take advantage of the tractable Schizosaccharomyces pombe system by combining population-level analysis, complete long-read assemblies, and engineered near-isogenic strains to test how pericentromeric repeat copy number affects chromosome biology in its native context. We find that pericentromeric dh/dg arrays on chromosome 3 vary almost tenfold in size among natural S. pombe isolates, ranging from 35 to 265 kb. We converted this natural diversity into an experimental system of nearly isogenic strains that primarily differ in pericentromere size (35 to >350 kb). We found that pericentromere size does not alter baseline growth under standard conditions. However, larger pericentromeres alter transcriptional output and sensitize cells to spindle stress. We show that this spindle-stress phenotype depends on heterochromatin: loss of the H3K9 methyltransferase Clr4 abolishes size-dependent differences, whereas artificial targeting of the Chromosomal Passenger Complex to heterochromatin partially rescues the defect. Thus, we find that larger pericentromeres act as sinks for limiting regulatory factors, weakening their effective concentration at centromeres and compromising faithful chromosome segregation under stress. These results establish that naturally occurring copy-number variation within repetitive pericentromeric DNA is not merely noise, but a functional source of variation in chromosome segregation and gene regulation. Our work provides an experimentally tractable framework for understanding how repeat expansion in centromere-proximal heterochromatin influences chromosome behavior across eukaryotes.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the C. neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that insertions of transposons into the 5' upstream region can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5' insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans, particularly in examining the consequences of genotypic diversity.

Most fungi can produce dormant, long-lived cells known as spores (e.g., conidia and ascospores). Spores play a critical role in fungal biology and human health, but much about spores is unknown. Here, we investigate factors affecting spore fitness using ascospores of the fission yeast Schizosaccharomyces pombe as a model. We found that storage conditions affect spore longevity and that spore health declines over time. We identified a delay in dormancy breaking (germination), decreased asymmetry during cell division, and reduced stress tolerance as aging phenotypes. These results support that S. pombe ascospores are affected by both time and experiences during dormancy, highlighting critical features of spore biology and revealing parallels between aging in spores and aging in animal cells. IMPORTANCE Fungi are integral to human health and well-being. They also offer scientists easy experimental systems to study facets of life shared with other eukaryotes, including humans. Most studies of fungal cell biology focus on actively growing cells, but fungi can also produce dormant cells known as spores. Spores promote fungal survival and dispersal and are often the agents of infection in pathogenic fungi. In this work, we characterize the spores produced by an easy-to-study model fungus, Schizosaccharomyces pombe. We find that the longevity and health of S. pombe spores decline over time and in response to heat stress. We characterize several traits associated with stressed and aged spores and identify parallels to aging cells in animals. This study expands the foundation for using S. pombe spores as a model system for fungal spore biology and as a model for the aging of non-dividing cells.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the Cryptococcus neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that 5' insertions of transposons can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5' insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans, particularly in examining the consequences of genotypic diversity.

Numerous genes required for sexual reproduction remain to be identified even in simple model species like Schizosaccharomyces pombe. To address this, we developed an assay in S. pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wild type. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities and can be rescued by conditioned medium. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

Many genes required for sexual reproduction remain to be identified. Moreover, many of the genes that are known have been characterized in distinct experiments using different conditions, which complicates understanding the relative contributions of genes to sex. To address these challenges, we developed an assay in Schizosaccharomyces pomb e that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 ( i mportant f or s ex), that is required for spore viability. In two other hits, plb1 and alg9 , we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wildtype. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 ( s ocial d istancing g ene), is instead required for growth at low cell densities. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans. ### Competing Interest Statement SEZ is an inventor on a patent application based on wtf killers (patent application serial 62/491,107)

Meiotic drivers are genetic elements that break Mendel’s law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing (mating between individuals from distinct lineages) because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe , a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity for cells from distinct clonal lineages to mate varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed levels of preferential mating between cells from the same clonal lineage can slow, but not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs linked to the driver. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

Meiotic drivers are selfish alleles that can force their transmission into more than 50% of the viable gametes made by heterozygotes. Meiotic drivers are known to cause infertility in a diverse range of eukaryotes and are predicted to affect the evolution of genome structure and meiosis. The wtf gene family of Schizosaccharomyces pombe includes both meiotic drivers and drive suppressors and thus offers a tractable model organism to study drive systems. Currently, only a handful of wtf genes have been functionally characterized and those genes only partially reflect the diversity of the wtf gene family. In this work, we functionally test 22 additional wtf genes for meiotic drive phenotypes. We identify eight new drivers that share between 30-90% amino acid identity with previously characterized drivers. Despite the vast divergence between these genes, they generally drive into >85% of gametes when heterozygous. We also identify three wtf genes that suppress other wtf drivers, including two that also act as autonomous drivers. Additionally, we find that wtf genes do not underlie a weak (64% allele transmission) meiotic driver on chromosome 1. Finally, we find that some Wtf proteins have expression or localization patterns that are distinct from the poison and antidote proteins encoded by drivers and suppressors, suggesting some wtf genes may have non-meiotic drive functions. Overall, this work expands our understanding of the wtf gene family and the burden selfish driver genes impose on S. pombe.

Histone variants expand chromatin functions in eukaryote genomes. H2A.B genes are testis-expressed short histone H2A variants that arose in placental mammals. Their biological functions remain largely unknown. To investigate their function, we generated a knockout (KO) model that disrupts all 3 H2A.B genes in mice. We show that H2A.B KO males have globally altered chromatin structure in postmeiotic germ cells. Yet, they do not show impaired spermatogenesis or testis function. Instead, we find that H2A.B plays a crucial role postfertilization. Crosses between H2A.B KO males and females yield embryos with lower viability and reduced size. Using a series of genetic crosses that separate parental and zygotic contributions, we show that the H2A.B status of both the father and mother, but not of the zygote, affects embryonic viability and growth during gestation. We conclude that H2A.B is a novel parental-effect gene, establishing a role for short H2A histone variants in mammalian development. We posit that parental antagonism over embryonic growth drove the origin and ongoing diversification of short histone H2A variants in placental mammals.

Natural selection works best when the two alleles in a diploid organism are transmitted to offspring at equal frequencies. Despite this, selfish loci known as meiotic drivers that bias their own transmission into gametes are found throughout eukaryotes. Drive is thought to be a powerful evolutionary force, but empirical evolutionary analyses of drive systems are limited by low numbers of identified meiotic drive genes. Here, we analyze the evolution of the wtf gene family of Schizosaccharomyces pombe that contains both killer meiotic drive genes and suppressors of drive. We completed assemblies of all wtf genes for two S. pombe isolates, as well as a subset of wtf genes from over 50 isolates. We find that wtf copy number can vary greatly between isolates and that amino acid substitutions, expansions and contractions of DNA sequence repeats, and nonallelic gene conversion between family members all contribute to dynamic wtf gene evolution. This work demonstrates the power of meiotic drive to foster rapid evolution and identifies a recombination mechanism through which transposons can indirectly mobilize meiotic drivers.

Meiotic drivers are selfish genes that bias their transmission into gametes, defying Mendelian inheritance. Despite the significant impact of these genomic parasites on evolution and infertility, few meiotic drive loci have been identified or mechanistically characterized. Here, we demonstrate a complex landscape of meiotic drive genes on chromosome 3 of the fission yeasts Schizosaccharomyces kambucha and S. pombe. We identify S. kambucha wtf4 as one of these genes that acts to kill gametes (known as spores in yeast) that do not inherit the gene from heterozygotes. wtf4 utilizes dual, overlapping transcripts to encode both a gamete-killing poison and an antidote to the poison. To enact drive, all gametes are poisoned, whereas only those that inherit wtf4 are rescued by the antidote. Our work suggests that the wtf multigene family proliferated due to meiotic drive and highlights the power of selfish genes to shape genomes, even while imposing tremendous costs to fertility.

Hybrid sterility is one of the earliest postzygotic isolating mechanisms to evolve between two recently diverged species. Here we identify causes underlying hybrid infertility of two recently diverged fission yeast species Schizosaccharomyces pombe and S. kambucha, which mate to form viable hybrid diploids that efficiently complete meiosis, but generate few viable gametes. We find that chromosomal rearrangements and related recombination defects are major but not sole causes of hybrid infertility. At least three distinct meiotic drive alleles, one on each S. kambucha chromosome, independently contribute to hybrid infertility by causing nonrandom spore death. Two of these driving loci are linked by a chromosomal translocation and thus constitute a novel type of paired meiotic drive complex. Our study reveals how quickly multiple barriers to fertility can arise. In addition, it provides further support for models in which genetic conflicts, such as those caused by meiotic drive alleles, can drive speciation.

R2 retrotransposable elements exclusively insert into the tandemly repeated rRNA genes, the rDNA loci, of their animal hosts. R2 elements form stable long-term associations with their host, in which all individuals in a population contain many potentially active copies, but only a fraction of these individuals show active R2 retrotransposition. Previous studies have found that R2 RNA transcripts are processed from a 28S co-transcript and that the likelihood of R2-inserted units being transcribed is dependent upon their distribution within the rDNA locus. Here we analyze the rDNA locus and R2 elements from nearly 100 R2-active and R2-inactive individuals from natural populations of Drosophila simulans. Along with previous findings concerning the structure and expression of the rDNA loci, these data were incorporated into computer simulations to model the crossover events that give rise to the concerted evolution of the rRNA genes. The simulations that best reproduce the population data assume that only about 40 rDNA units out of the over 200 total units are actively transcribed and that these transcribed units are clustered in a single region of the locus. In the model, the host establishes this transcription domain at each generation in the region with the fewest R2 insertions. Only if the host cannot avoid R2 insertions within this 40-unit domain are R2 elements active in that generation. The simulations also require that most crossover events in the locus occur in the transcription domain in order to explain the empirical observation that R2 elements are seldom duplicated by crossover events. Thus the key to the long-term stability of R2 elements is the stochastic nature of the crossover events within the rDNA locus, and the inevitable expansions and contractions that introduce and remove R2-inserted units from the transcriptionally active domain.

In contrast to RNA viruses, double-stranded DNA viruses have low mutation rates yet must still adapt rapidly in response to changing host defenses. To determine mechanisms of adaptation, we subjected the model poxvirus vaccinia to serial propagation in human cells, where its antihost factor K3L is malad-apted against the antiviral protein kinase R (PKR). Viruses rapidly acquired higher fitness via recurrent K3L gene amplifications, incurring up to 7%-10% increases in genome size. These transient gene expansions were necessary and sufficient to counteract human PKR and facilitated the gain of an adaptive amino acid substitution in K3L that also defeats PKR. Subsequent reductions in gene amplifications offset the costs associated with larger genome size while retaining adaptive substitutions. Our discovery of viral "gene-accordions" explains how poxviruses can rapidly adapt to defeat different host defenses despite low mutation rates and reveals how classical Red Queen conflicts can progress through unrecognized intermediates.

BACKGROUND:R2 retrotransposable elements exclusively insert in the 28S rRNA genes of their host. Their RNA transcripts are produced by self-processing from a 28S R2 cotranscript. Because full-length R2 transcripts are found in most tissues of R2-active animals, we tested whether new R2 insertions occurred in somatic tissues even though such events would be an evolutionary dead end.FINDINGS:PCR assays were used to identify somatic R2 insertions in isolated adult tissues and larval imaginal discs of Drosophila simulans. R2 somatic mosaics were detected encompassing cells from individual tissues as well as tissues from multiple body segments. The somatic insertions had 5' junction sequences characteristic of germline insertions suggesting they represented authentic retrotransposition events.CONCLUSIONS:Body segments are specified early in Drosophila development, thus the detection of the same somatic insertion in cells from multiple tissues suggested that the R2 retrotransposition events had occurred before the blastoderm stage of Drosophila development. R2 activity at this stage, when embryonic nuclei are rapidly dividing in a common cytoplasm, suggests that some retrotransposition events appearing as germline events may correspond to germline mosaicism.

Multiple theoretical studies have focused on the concerted evolution of the tandemly repeated rRNA genes of eukaryotes; however, these studies did not consider the transposable elements that interrupt the rRNA genes in many organisms. For example, in insects, R1 and R2 have been stable components of the rDNA locus for hundreds of millions of years, suggesting either that they have minimal effects on fitness or that they are unable to be eliminated. We constructed a simulation model of recombination and retrotransposition within the rDNA locus that addresses the population dynamics and fitness consequences associated with R1 and R2 insertions. The simulations suggest that even without R1 and R2 retrotransposition the frequent sister chromatid exchanges postulated from various empirical studies will, in combination with selection, generate rDNA loci that are much larger than those needed for transcription. These large loci enable the host to tolerate high levels of R1 and R2 insertions with little fitness consequences. Changes in retrotransposition rates are likely to be accommodated by adjustments in sister chromatid exchange (SCE) rate, rather than by direct selection on the number of uninserted rDNA units. These simulations suggest that the rDNA locus serves as an ideal niche for the long-term survival of transposable elements.