Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

Development of a Comprehensive Genotype-to-Fitness Map of Adaptation-Driving Mutations in Yeast.

Adaptive evolution plays a large role in generating the phenotypic diversity observed in nature, yet current methods are impractical for characterizing the molecular basis and fitness effects of large numbers of individual adaptive mutations. Here, we used a DNA barcoding approach to generate the genotype-to-fitness map for adaptation-driving mutations from a Saccharomyces cerevisiae population experimentally evolved by serial transfer under limiting glucose. We isolated and measured the fitness of thousands of independent adaptive clones and sequenced the genomes of hundreds of clones. We found only two major classes of adaptive mutations: self-diploidization and mutations in the nutrient-responsive Ras/PKA and TOR/Sch9 pathways. Our large sample size and precision of measurement allowed us to determine that there are significant differences in fitness between mutations in different genes, between different paralogs, and even between different classes of mutations within the same gene.

Errors of the Egg: The Establishment and Progression of Human Aneuploidy Research in the Maternal Germline.

Meiosis, a key process in the creation of haploid gametes, is a complex cellular division incorporating unique timing and intricate chromosome dynamics. Abnormalities in this elaborate dance can lead to the production of aneuploid gametes, i.e., eggs containing an incorrect number of chromosomes, many of which cannot generate a viable pregnancy. For many decades, research has been attempting to address why this process is notoriously error prone in humans compared to many other organisms. Rapidly developing technologies, access to new clinical material, and a mounting public infertility crisis have kept the field both active and quickly evolving. In this review, we discuss the history of aneuploidy in humans with a focus on its origins in maternal meiosis. We also gather current working mechanistic hypotheses, as well as up-and-coming areas of interest that point to future scientific avenues and their potential clinical applications.

Gametogenesis: Exploring an Endogenous Rejuvenation Program to Understand Cellular Aging and Quality Control.

Gametogenesis is a conserved developmental program whereby a diploid progenitor cell differentiates into haploid gametes, the precursors for sexually reproducing organisms. In addition to ploidy reduction and extensive organelle remodeling, gametogenesis naturally rejuvenates the ensuing gametes, leading to resetting of life span. Excitingly, ectopic expression of the gametogenesis-specific transcription factor Ndt80 is sufficient to extend life span in mitotically dividing budding yeast, suggesting that meiotic rejuvenation pathways can be repurposed outside of their natural context. In this review, we highlight recent studies of gametogenesis that provide emerging insight into natural quality control, organelle remodeling, and rejuvenation strategies that exist within a cell. These include selective inheritance, programmed degradation, and de novo synthesis, all of which are governed by the meiotic gene expression program entailing many forms of noncanonical gene regulation. Finally, we highlight critical questions that remain in the field and provide perspective on the implications of gametogenesis research on human health span.

Signatures of copy number alterations in human cancer.

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage-fusion-bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3-5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

Haploid mouse embryonic stem cells: rapid genetic screening and germline transmission.

Most animal genomes are diploid, and mammalian development depends on specific adaptations that have evolved secondary to diploidy. Genomic imprinting and dosage compensation restrict haploid development to early embryos. Recently, haploid mammalian development has been reinvestigated since the establishment of haploid embryonic stem cells (ESCs) from mouse embryos. Haploid cells possess one copy of each gene, facilitating the generation of loss-of-function mutations in a single step. Recessive mutations can then be assessed in forward genetic screens. Applications of haploid mammalian cell systems in screens have been illustrated in several recent publications. Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice. Different strategies for introducing genetic modifications from haploid ESCs into the mouse germline have been further developed. Haploid ESCs therefore introduce new possibilities in mammalian genetics and could offer an unprecedented tool for genome exploration in the future.

Interactions between U and V sex chromosomes during the life cycle of Ectocarpus.

In many animals and flowering plants, sex determination occurs in the diploid phase of the life cycle with XX/XY or ZW/ZZ sex chromosomes. However, in early diverging plants and most macroalgae, sex is determined by female (U) or male (V) sex chromosomes in a haploid phase called the gametophyte. Once the U and V chromosomes unite at fertilization to produce a diploid sporophyte, sex determination no longer occurs, raising key questions about the fate of the U and V sex chromosomes in the sporophyte phase. Here, we investigate genetic and molecular interactions of the UV sex chromosomes in both the haploid and diploid phases of the brown alga Ectocarpus. We reveal extensive developmental regulation of sex chromosome genes across its life cycle and implicate the TALE-HD transcription factor OUROBOROS in suppressing sex determination in the diploid phase. Small RNAs may also play a role in the repression of a female sex-linked gene, and transition to the diploid sporophyte coincides with major reconfiguration of histone H3K79me2, suggesting a more intricate role for this histone mark in Ectocarpus development than previously appreciated.

Constructing telomere-to-telomere diploid genome by polishing haploid nanopore-based assembly.

Draft genomes generated from Oxford Nanopore Technologies (ONT) long reads are known to have a higher error rate. Although existing genome polishers can enhance their quality, the error rate (including mismatches, indels and switching errors between paternal and maternal haplotypes) can be significant. Here, we develop two polishers, hypo-short and hypo-hybrid to address this issue. Hypo-short utilizes Illumina short reads to polish an ONT-based draft assembly, resulting in a high-quality assembly with low error rates and switching errors. Expanding on this, hypo-hybrid incorporates ONT long reads to further refine the assembly into a diploid representation. Leveraging on hypo-hybrid, we have created a diploid genome assembly pipeline called hypo-assembler. Hypo-assembler automates the generation of highly accurate, contiguous and nearly complete diploid assemblies using ONT long reads, Illumina short reads and optionally Hi-C reads. Notably, our solution even allows for the production of telomere-to-telomere diploid genomes with additional manual steps. As a proof of concept, we successfully assembled a fully phased telomere-to-telomere diploid genome of HG00733, achieving a quality value exceeding 50.

Ploidy evolution in a wild yeast is linked to an interaction between cell type and metabolism.

Ploidy is an evolutionarily labile trait, and its variation across the tree of life has profound impacts on evolutionary trajectories and life histories. The immediate consequences and molecular causes of ploidy variation on organismal fitness are frequently less clear, although extreme mating type skews in some fungi hint at links between cell type and adaptive traits. Here, we report an unusual recurrent ploidy reduction in replicate populations of the budding yeast Saccharomyces eubayanus experimentally evolved for improvement of a key metabolic trait, the ability to use maltose as a carbon source. We find that haploids have a substantial, but conditional, fitness advantage in the absence of other genetic variation. Using engineered genotypes that decouple the effects of ploidy and cell type, we show that increased fitness is primarily due to the distinct transcriptional program deployed by haploid-like cell types, with a significant but smaller contribution from absolute ploidy. The link between cell-type specification and the carbon metabolism adaptation can be traced to the noncanonical regulation of a maltose transporter by a haploid-specific gene. This study provides novel mechanistic insight into the molecular basis of an environment-cell type fitness interaction and illustrates how selection on traits unexpectedly linked to ploidy states or cell types can drive karyotypic evolution in fungi.

The nature of Neanderthal introgression revealed by 27,566 Icelandic genomes.

Human evolutionary history is rich with the interbreeding of divergent populations. Most humans outside of Africa trace about 2% of their genomes to admixture from Neanderthals, which occurred 50-60 thousand years ago1. Here we examine the effect of this event using 14.4 million putative archaic chromosome fragments that were detected in fully phased whole-genome sequences from 27,566 Icelanders, corresponding to a range of 56,388-112,709 unique archaic fragments that cover 38.0-48.2% of the callable genome. On the basis of the similarity with known archaic genomes, we assign 84.5% of fragments to an Altai or Vindija Neanderthal origin and 3.3% to Denisovan origin; 12.2% of fragments are of unknown origin. We find that Icelanders have more Denisovan-like fragments than expected through incomplete lineage sorting. This is best explained by Denisovan gene flow, either into ancestors of the introgressing Neanderthals or directly into humans. A within-individual, paired comparison of archaic fragments with syntenic non-archaic fragments revealed that, although the overall rate of mutation was similar in humans and Neanderthals during the 500 thousand years that their lineages were separate, there were differences in the relative frequencies of mutation types-perhaps due to different generation intervals for males and females. Finally, we assessed 271 phenotypes, report 5 associations driven by variants in archaic fragments and show that the majority of previously reported associations are better explained by non-archaic variants.

Non-genetic and genetic rewiring underlie adaptation to hypomorphic alleles of an essential gene.

Adaptive evolution to cellular stress is a process implicated in a wide range of biological and clinical phenomena. Two major routes of adaptation have been identified: non-genetic changes, which allow expression of different phenotypes in novel environments, and genetic variation achieved by selection of fitter phenotypes. While these processes are broadly accepted, their temporal and epistatic features in the context of cellular evolution and emerging drug resistance are contentious. In this manuscript, we generated hypomorphic alleles of the essential nuclear pore complex (NPC) gene NUP58. By dissecting early and long-term mechanisms of adaptation in independent clones, we observed that early physiological adaptation correlated with transcriptome rewiring and upregulation of genes known to interact with the NPC; long-term adaptation and fitness recovery instead occurred via focal amplification of NUP58 and restoration of mutant protein expression. These data support the concept that early phenotypic plasticity allows later acquisition of genetic adaptations to a specific impairment. We propose this approach as a genetic model to mimic targeted drug therapy in human cells and to dissect mechanisms of adaptation.

Uncoupling transcription and translation through miRNA-dependent poly(A) length control in haploid male germ cells.

As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for hours or even days before translation. In addition to oocytes and neurons, developing spermatids display significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoprotein (RNP) granules for translational repression and stabilization. In contrast, re-polyadenylation might allow for translocation of the translationally repressed transcripts from RNP granules to polysomes. Overall, our data suggest that miRNA-dependent poly(A) length control represents a previously unreported mechanism underlying uncoupled translation and transcription in haploid male mouse germ cells.

A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.

The molecular pathways that trigger the initiation of embryogenesis after fertilization in flowering plants, and prevent its occurrence without fertilization, are not well understood1. Here we show in rice (Oryza sativa) that BABY BOOM1 (BBM1), a member of the AP2 family2 of transcription factors that is expressed in sperm cells, has a key role in this process. Ectopic expression of BBM1 in the egg cell is sufficient for parthenogenesis, which indicates that a single wild-type gene can bypass the fertilization checkpoint in the female gamete. Zygotic expression of BBM1 is initially specific to the male allele but is subsequently biparental, and this is consistent with its observed auto-activation. Triple knockout of the genes BBM1, BBM2 and BBM3 causes embryo arrest and abortion, which are fully rescued by male-transmitted BBM1. These findings suggest that the requirement for fertilization in embryogenesis is mediated by male-genome transmission of pluripotency factors. When genome editing to substitute mitosis for meiosis (MiMe)3,4 is combined with the expression of BBM1 in the egg cell, clonal progeny can be obtained that retain genome-wide parental heterozygosity. The synthetic asexual-propagation trait is heritable through multiple generations of clones. Hybrid crops provide increased yields that cannot be maintained by their progeny owing to genetic segregation. This work establishes the feasibility of asexual reproduction in crops, and could enable the maintenance of hybrids clonally through seed propagation5,6.

Gene-rich X chromosomes implicate intragenomic conflict in the evolution of bizarre genetic systems.

Haplodiploidy and paternal genome elimination (HD/PGE) are common in invertebrates, having evolved at least two dozen times, all from male heterogamety (i.e., systems with X chromosomes). However, why X chromosomes are important for the evolution of HD/PGE remains debated. The Haploid Viability Hypothesis posits that X-linked genes promote the evolution of male haploidy by facilitating purging recessive deleterious mutations. The Intragenomic Conflict Hypothesis holds that conflict between genes drives genetic system turnover; under this model, X-linked genes could promote the evolution of male haploidy due to conflicts with autosomes over sex ratios and genetic transmission. We studied lineages where we can distinguish these hypotheses: species with germline PGE that retain an XX/X0 sex determination system (gPGE+X). Because evolving PGE in these cases involves changes in transmission without increases in male hemizygosity, a high degree of X linkage in these systems is predicted by the Intragenomic Conflict Hypothesis but not the Haploid Viability Hypothesis. To quantify the degree of X linkage, we sequenced and compared 7 gPGE+X species' genomes with 11 related species with typical XX/XY or XX/X0 genetic systems, representing three transitions to gPGE. We find highly increased X linkage in both modern and ancestral genomes of gPGE+X species compared to non-gPGE relatives and recover a significant positive correlation between percent X linkage and the evolution of gPGE. These empirical results substantiate longstanding proposals for a role for intragenomic conflict in the evolution of genetic systems such as HD/PGE.

Life cycle and functional genomics of the unicellular red alga Galdieria for elucidating algal and plant evolution and industrial use.

Sexual reproduction is widespread in eukaryotes; however, only asexual reproduction has been observed in unicellular red algae, including Galdieria, which branched early in Archaeplastida. Galdieria possesses a small genome; it is polyextremophile, grows either photoautotrophically, mixotrophically, or heterotrophically, and is being developed as an industrial source of vitamins and pigments because of its high biomass productivity. Here, we show that Galdieria exhibits a sexual life cycle, alternating between cell-walled diploid and cell wall-less haploid, and that both phases can proliferate asexually. The haploid can move over surfaces and undergo self-diploidization or generate heterozygous diploids through mating. Further, we prepared the whole genome and a comparative transcriptome dataset between the diploid and haploid and developed genetic tools for the stable gene expression, gene disruption, and selectable marker recycling system using the cell wall-less haploid. The BELL/KNOX and MADS-box transcription factors, which function in haploid-to-diploid transition and development in plants, are specifically expressed in the haploid and diploid, respectively, and are involved in the haploid-to-diploid transition in Galdieria, providing information on the missing link of the sexual life cycle evolution in Archaeplastida. Four actin genes are differently involved in motility of the haploid and cytokinesis in the diploid, both of which are myosin independent and likely reflect ancestral roles of actin. We have also generated photosynthesis-deficient mutants, such as blue-colored cells, which were depleted in chlorophyll and carotenoids, for industrial pigment production. These features of Galdieria facilitate the understanding of the evolution of algae and plants and the industrial use of microalgae.

Establishment and inheritance of minichromosomes from Arabidopsis haploid induction.

Minichromosomes are small, sometimes circular, rearranged chromosomes consisting of one centromere and short chromosomal arms formed by treatments that break DNA, including plant transformation. Minichromosomes have the potential to serve as vectors to quickly move valuable genes across a wide range of germplasm, including into adapted crop varieties. To realize this potential, minichromosomes must be reliably generated, easily manipulated, and stably inherited. Here we show a reliable method for minichromosome formation in haploids resulting from CENH3-mediated genome elimination, a process that generates genome instability and karyotypic novelty specifically on one parental genome. First, we identified 2 out of 260 haploids, each containing a single-copy minichromosome originating from centromeric regions of chromosomes 1 and 3, respectively. The chromosome 1 minichromosome we characterized did not pair at meiosis but displayed consistent transmission over nine selfing generations. Next, we demonstrated that CENH3-based haploid induction can produce minichromosomes in a targeted manner. Haploid inducers carrying a selectable pericentromeric marker were used to isolate additional chromosome-specific minichromosomes, which occurred in 3 out of 163 haploids. Our findings document the formation of heritable, rearranged chromosomes, and we provide a method for convenient minichromosome production.

Strong Purifying Selection in Haploid Tissue-Specific Genes of Scots Pine Supports the Masking Theory.

The masking theory states that genes expressed in a haploid stage will be under more efficient selection. In contrast, selection will be less efficient in genes expressed in a diploid stage, where the fitness effects of recessive deleterious or beneficial mutations can be hidden from selection in heterozygous form. This difference can influence several evolutionary processes such as the maintenance of genetic variation, adaptation rate, and genetic load. Masking theory expectations have been confirmed in single-cell haploid and diploid organisms. However, in multicellular organisms, such as plants, the effects of haploid selection are not clear-cut. In plants, the great majority of studies indicating haploid selection have been carried out using male haploid tissues in angiosperms. Hence, evidence in these systems is confounded with the effects of sexual selection and intraspecific competition. Evidence from other plant groups is scarce, and results show no support for the masking theory. Here, we have used a gymnosperm Scots pine megagametophyte, a maternally derived seed haploid tissue, and four diploid tissues to test the strength of purifying selection on a set of genes with tissue-specific expression. By using targeted resequencing data of those genes, we obtained estimates of genetic diversity, the site frequency spectrum of 0-fold and 4-fold sites, and inferred the distribution of fitness effects of new mutations in haploid and diploid tissue-specific genes. Our results show that purifying selection is stronger for tissue-specific genes expressed in the haploid megagametophyte tissue and that this signal of strong selection is not an artifact driven by high expression levels.

Low Spontaneous Mutation Rate in Complex Multicellular Eukaryotes with a Haploid-Diploid Life Cycle.

The spontaneous mutation rate µ is a crucial parameter to understand evolution and biodiversity. Mutation rates are highly variable across species, suggesting that µ is susceptible to selection and drift and that species life cycle and life history may impact its evolution. In particular, asexual reproduction and haploid selection are expected to affect the mutation rate, but very little empirical data are available to test this expectation. Here, we sequence 30 genomes of a parent-offspring pedigree in the model brown alga Ectocarpus sp.7, and 137 genomes of an interspecific cross of the closely related brown alga Scytosiphon to have access to the spontaneous mutation rate of representative organisms of a complex multicellular eukaryotic lineage outside animals and plants, and to evaluate the potential impact of life cycle on the mutation rate. Brown algae alternate between a haploid and a diploid stage, both multicellular and free living, and utilize both sexual and asexual reproduction. They are, therefore, excellent models to empirically test expectations of the effect of asexual reproduction and haploid selection on mutation rate evolution. We estimate that Ectocarpus has a base substitution rate of µbs = 4.07 × 10-10 per site per generation, whereas the Scytosiphon interspecific cross had µbs = 1.22 × 10-9. Overall, our estimations suggest that these brown algae, despite being multicellular complex eukaryotes, have unusually low mutation rates. In Ectocarpus, effective population size (Ne) could not entirely explain the low µbs. We propose that the haploid-diploid life cycle, combined with extensive asexual reproduction, may be additional key drivers of the mutation rate in these organisms.

KATNB1 is a master regulator of multiple katanin enzymes in male meiosis and haploid germ cell development.

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits.

Evolution in the Cycles of Life.

The life cycles of eukaryotes alternate between haploid and diploid phases, which are initiated by meiosis and gamete fusion, respectively. In both ascomycete and basidiomycete fungi and chlorophyte algae, the haploid-to-diploid transition is regulated by a pair of paralogous homeodomain protein encoding genes. That a common genetic program controls the haploid-to-diploid transition in phylogenetically disparate eukaryotic lineages suggests this may be the ancestral function for homeodomain proteins. Multicellularity has evolved independently in many eukaryotic lineages in either one or both phases of the life cycle. Organisms, such as land plants, exhibiting a life cycle whereby multicellular bodies develop in both the haploid and diploid phases are often referred to as possessing an alternation of generations. We review recent progress on understanding the genetic basis for the land plant alternation of generations and highlight the roles that homeodomain-encoding genes may have played in the evolution of complex multicellularity in this lineage.