Full-spectral genome analysis of natural killer/T cell lymphoma highlights impacts of genome instability in driving its progression.

BACKGROUND: Natural killer/T cell lymphoma (NKTCL) is a clinically and genetically heterogeneous disease with poor prognosis. Genome sequencing and mutation characterization provides a powerful approach for patient stratification, treatment target discovery, and etiology identification. However, previous studies mostly concentrated on base-level mutations in primary NKTCL, whereas the large-scale genomic alterations in NKTCL and the mutational landscapes in relapsed/refractory NKTCL remain largely unexplored. METHODS: Here, we assembled whole-genome sequencing and whole-exome sequencing data from 163 patients with primary or relapsed/refractory NKTCL and compared their somatic mutational landscapes at both nucleotide and structure levels. RESULTS: Our study not only confirmed previously reported common NKTCL mutational targets like STAT3, TP53, and DDX3X but also unveiled several novel high-frequency mutational targets such as PRDM9, DST, and RBMX. In terms of the overall mutational landscape, we observed striking differences between primary and relapsed/refractory NKTCL patient groups, with the latter exhibits higher levels of tumor mutation burden, copy number variants (CNVs), and structural variants (SVs), indicating a strong signal of genomic instability. Complex structural rearrangements such as chromothripsis and focal amplification are also significantly enriched in relapsed/refractory NKTCL patients, exerting a substantial impact on prognosis. Accordingly, we devised a novel molecular subtyping system (i.e., C0-C4) with distinct prognosis by integrating potential driver mutations at both nucleotide and structural levels, which further provides an informative guidance for novel treatments that target these specific driver mutations and genome instability as a whole. CONCLUSIONS: The striking differences underlying the mutational landscapes between the primary and relapsed/refractory NKTCL patients highlight the importance of genomic instability in driving the progression of NKTCL. Our newly proposed molecular subtyping system is valuable in assisting patient stratification and novel treatment design towards a better prognosis in the age of precision medicine.

GetPrimers: A generalized PCR-based genetic targeting primer designer enabling easy and standardized targeted gene modification across multiple systems.

Genetic targeting (e.g., gene knockout and tagging) based on polymerase chain reaction (PCR) is a simple yet powerful approach for studying gene functions. Although originally developed in classic budding and fission yeast models, the same principle applies to other eukaryotic systems with efficient homologous recombination. One-step PCR-based genetic targeting is conventionally used but the sizes of the homologous arms that it generates for recombination-mediated genetic targeting are usually limited. Alternatively, gene targeting can also be performed via fusion PCR, which can create homologous arms that are orders of magnitude larger, therefore substantially increasing the efficiency of recombination-mediated genetic targeting. Here, we present GetPrimers (https://www.evomicslab.org/app/getprimers/), a generalized computational framework and web tool to assist automatic targeting and verification primer design for both one-step PCR-based and fusion PCR-based genetic targeting experiments. Moreover, GetPrimers by design runs for any given genetic background of any species with full genome scalability. Therefore, GetPrimers is capable of empowering high-throughput functional genomic assays at multipopulation and multispecies levels. Comprehensive experimental validations have been performed for targeting and verification primers designed by GetPrimers across multiple organism systems and experimental setups. We anticipate GetPrimers to become a highly useful and popular tool to facilitate easy and standardized gene modification across multiple systems.

Genome instability footprint under rapamycin and hydroxyurea treatments.

The mutational processes dictating the accumulation of mutations in genomes are shaped by genetic background, environment and their interactions. Accurate quantification of mutation rates and spectra under drugs has important implications in disease treatment. Here, we used whole-genome sequencing and time-resolved growth phenotyping of yeast mutation accumulation lines to give a detailed view of the mutagenic effects of rapamycin and hydroxyurea on the genome and cell growth. Mutation rates depended on the genetic backgrounds but were only marginally affected by rapamycin. As a remarkable exception, rapamycin treatment was associated with frequent chromosome XII amplifications, which compensated for rapamycin induced rDNA repeat contraction on this chromosome and served to maintain rDNA content homeostasis and fitness. In hydroxyurea, a wide range of mutation rates were elevated regardless of the genetic backgrounds, with a particularly high occurrence of aneuploidy that associated with dramatic fitness loss. Hydroxyurea also induced a high T-to-G and low C-to-A transversion rate that reversed the common G/C-to-A/T bias in yeast and gave rise to a broad range of structural variants, including mtDNA deletions. The hydroxyurea mutation footprint was consistent with the activation of error-prone DNA polymerase activities and non-homologues end joining repair pathways. Taken together, our study provides an in-depth view of mutation rates and signatures in rapamycin and hydroxyurea and their impact on cell fitness, which brings insights for assessing their chronic effects on genome integrity.

Telomere-to-telomere assemblies of 142 strains characterize the genome structural landscape in Saccharomyces cerevisiae.

Pangenomes provide access to an accurate representation of the genetic diversity of species, both in terms of sequence polymorphisms and structural variants (SVs). Here we generated the Saccharomyces cerevisiae Reference Assembly Panel (ScRAP) comprising reference-quality genomes for 142 strains representing the species' phylogenetic and ecological diversity. The ScRAP includes phased haplotype assemblies for several heterozygous diploid and polyploid isolates. We identified circa (ca.) 4,800 nonredundant SVs that provide a broad view of the genomic diversity, including the dynamics of telomere length and transposable elements. We uncovered frequent cases of complex aneuploidies where large chromosomes underwent large deletions and translocations. We found that SVs can impact gene expression near the breakpoints and substantially contribute to gene repertoire evolution. We also discovered that horizontally acquired regions insert at chromosome ends and can generate new telomeres. Overall, the ScRAP demonstrates the benefit of a pangenome in understanding genome evolution at population scale.

Gut-specific telomerase expression counteracts systemic aging in telomerase-deficient zebrafish.

Telomere shortening is a hallmark of aging and is counteracted by telomerase. As in humans, the zebrafish gut is one of the organs with the fastest rate of telomere decline, triggering early tissue dysfunction during normal zebrafish aging and in prematurely aged telomerase mutants. However, whether telomere-dependent aging of an individual organ, the gut, causes systemic aging is unknown. Here we show that tissue-specific telomerase expression in the gut can prevent telomere shortening and rescues premature aging of tert-/-. Induction of telomerase rescues gut senescence and low cell proliferation, while restoring tissue integrity, inflammation and age-dependent microbiota dysbiosis. Averting gut aging causes systemic beneficial impacts, rescuing aging of distant organs such as reproductive and hematopoietic systems. Conclusively, we show that gut-specific telomerase expression extends the lifespan of tert-/- by 40%, while ameliorating natural aging. Our work demonstrates that gut-specific rescue of telomerase expression leading to telomere elongation is sufficient to systemically counteract aging in zebrafish.

Spontaneous Mutation Rates and Spectra of Respiratory-Deficient Yeast.

The yeast petite mutant was first discovered in the yeast Saccharomyces cerevisiae, which shows growth stress due to defects in genes encoding the respiratory chain. In a previous study, we described that deletion of the nuclear-encoded gene MRPL25 leads to mitochondrial genome (mtDNA) loss and the petite phenotype, which can be rescued by acquiring ATP3 mutations. The mrpl25Δ strain showed an elevated SNV (single nucleotide variant) rate, suggesting genome instability occurred during the crisis of mtDNA loss. However, the genome-wide mutation landscape and mutational signatures of mitochondrial dysfunction are unknown. In this study we profiled the mutation spectra in yeast strains with the genotype combination of MRPL25 and ATP3 in their wildtype and mutated status, along with the wildtype and cytoplasmic petite rho0 strains as controls. In addition to the previously described elevated SNV rate, we found the INDEL (insertion/deletion) rate also increased in the mrpl25Δ strain, reinforcing the occurrence of genome instability. Notably, although both are petites, the mrpl25Δ and rho0 strains exhibited different INDEL rates and transition/transversion ratios, suggesting differences in the mutational signatures underlying these two types of petites. Interestingly, the petite-related mutagenesis effect disappeared when ATP3 suppressor mutations were acquired, suggesting a cost-effective mechanism for restoring both fitness and genome stability. Taken together, we present an unbiased genome-wide characterization of the mutation rates and spectra of yeast strains with respiratory deficiency, which provides valuable insights into the impact of respiratory deficiency on genome instability.

Telomeres are shorter in wild Saccharomyces cerevisiae isolates than in domesticated ones.

Telomeres are ribonucleoproteins that cap chromosome-ends and their DNA length is controlled by counteracting elongation and shortening processes. The budding yeast Saccharomyces cerevisiae has been a leading model to study telomere DNA length control and dynamics. Its telomeric DNA is maintained at a length that slightly varies between laboratory strains, but little is known about its variation at the species level. The recent publication of the genomes of over 1,000 S. cerevisiae strains enabled us to explore telomere DNA length variation at an unprecedented scale. Here, we developed a bioinformatic pipeline (YeaISTY) to estimate telomere DNA length from whole-genome sequences and applied it to the sequenced S. cerevisiae collection. Our results revealed broad natural telomere DNA length variation among the isolates. Notably, telomere DNA length is shorter in those derived from wild rather than domesticated environments. Moreover, telomere DNA length variation is associated with mitochondrial metabolism, and this association is driven by wild strains. Overall, these findings reveal broad variation in budding yeast's telomere DNA length regulation, which might be shaped by its different ecological life-styles.

Genetically controlled mtDNA deletions prevent ROS damage by arresting oxidative phosphorylation.

Deletion of mitochondrial DNA in eukaryotes is currently attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that this process critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication through Rtg2 and Rtg3. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. This shows that oxidative stress-induced mitochondrial impairment may be under strict regulatory control. If the results extend to human cells, the results may prove to be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.

RecombineX: A generalized computational framework for automatic high-throughput gamete genotyping and tetrad-based recombination analysis.

Meiotic recombination is an essential biological process that ensures faithful chromosome segregation and promotes parental allele shuffling. Tetrad analysis is a powerful approach to quantify the genetic makeups and recombination landscapes of meiotic products. Here we present RecombineX (https://github.com/yjx1217/RecombineX), a generalized computational framework that automates the full workflow of marker identification, gamete genotyping, and tetrad-based recombination profiling based on any organism or genetic background with batch processing capability. Aside from conventional reference-based analysis, RecombineX can also perform analysis based on parental genome assemblies, which facilitates analyzing meiotic recombination landscapes in their native genomic contexts. Additional features such as copy number variation profiling and missing genotype inference further enhance downstream analysis. RecombineX also includes a dedicate module for simulating the genomes and reads of recombinant tetrads, which enables fine-tuned simulation-based hypothesis testing. This simulation module revealed the power and accuracy of RecombineX even when analyzing tetrads with very low sequencing depths (e.g., 1-2X). Tetrad sequencing data from the budding yeast Saccharomyces cerevisiae and green alga Chlamydomonas reinhardtii were further used to demonstrate the accuracy and robustness of RecombineX for organisms with both small and large genomes, manifesting RecombineX as an all-around one stop solution for future tetrad analysis. Interestingly, our re-analysis of the budding yeast tetrad sequencing data with RecombineX and Oxford Nanopore sequencing revealed two unusual structural rearrangement events that were not noticed before, which exemplify the occasional genome instability triggered by meiosis.

Aborting meiosis allows recombination in sterile diploid yeast hybrids.

Hybrids between diverged lineages contain novel genetic combinations but an impaired meiosis often makes them evolutionary dead ends. Here, we explore to what extent an aborted meiosis followed by a return-to-growth (RTG) promotes recombination across a panel of 20 Saccharomyces cerevisiae and S. paradoxus diploid hybrids with different genomic structures and levels of sterility. Genome analyses of 275 clones reveal that RTG promotes recombination and generates extensive regions of loss-of-heterozygosity in sterile hybrids with either a defective meiosis or a heavily rearranged karyotype, whereas RTG recombination is reduced by high sequence divergence between parental subgenomes. The RTG recombination preferentially arises in regions with low local heterozygosity and near meiotic recombination hotspots. The loss-of-heterozygosity has a profound impact on sexual and asexual fitness, and enables genetic mapping of phenotypic differences in sterile lineages where linkage analysis would fail. We propose that RTG gives sterile yeast hybrids access to a natural route for genome recombination and adaptation.

Natural variants suppress mutations in hundreds of essential genes.

The consequence of a mutation can be influenced by the context in which it operates. For example, loss of gene function may be tolerated in one genetic background, and lethal in another. The extent to which mutant phenotypes are malleable, the architecture of modifiers and the identities of causal genes remain largely unknown. Here, we measure the fitness effects of ~ 1,100 temperature-sensitive alleles of yeast essential genes in the context of variation from ten different natural genetic backgrounds and map the modifiers for 19 combinations. Altogether, fitness defects for 149 of the 580 tested genes (26%) could be suppressed by genetic variation in at least one yeast strain. Suppression was generally driven by gain-of-function of a single, strong modifier gene, and involved both genes encoding complex or pathway partners suppressing specific temperature-sensitive alleles, as well as general modifiers altering the effect of many alleles. The emerging frequency of suppression and range of possible mechanisms suggest that a substantial fraction of monogenic diseases could be managed by modulating other gene products.

The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin.

Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.

Lager Yeast Design Through Meiotic Segregation of a Saccharomyces cerevisiae × Saccharomyces eubayanus Hybrid.

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae × Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15°C, 15 °Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

Meiotic Cells Counteract Programmed Retrotransposon Activation via RNA-Binding Translational Repressor Assemblies.

Retrotransposon proliferation poses a threat to germline integrity. While retrotransposons must be activated in developing germ cells in order to survive and propagate, how they are selectively activated in the context of meiosis is unclear. We demonstrate that the transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that budding yeast Ty3/Gypsy co-opts binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking its transcriptional activation to meiotic progression. We also elucidate how yeast cells thwart Ty3/Gypsy proliferation by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, several inactive Ty3/Gypsy elements are undergoing domestication. We show that mammals utilize equivalent master meiotic regulators (Stra8, Mybl1, Dazl) to regulate Ty3/Gypsy-derived genes in developing gametes. Our findings inform how genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication.

Molecular asymmetry in the cephalochordate embryo revealed by single-blastomere transcriptome profiling.

Studies in various animals have shown that asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification in early embryos. However, comprehensive analyses of the maternal transcriptomes with spatial information are scarce and limited to a handful of model organisms. In cephalochordates (amphioxus), an early branching chordate group, maternal transcripts of germline determinants form a compact granule that is inherited by a single blastomere during cleavage stages. Further blastomere separation experiments suggest that other transcripts associated with the granule are likely responsible for organizing the posterior structure in amphioxus; however, the identities of these determinants remain unknown. In this study, we used high-throughput RNA sequencing of separated blastomeres to examine asymmetrically localized transcripts in two-cell and eight-cell stage embryos of the amphioxus Branchiostoma floridae. We identified 111 and 391 differentially enriched transcripts at the 2-cell stage and the 8-cell stage, respectively, and used in situ hybridization to validate the spatial distribution patterns for a subset of these transcripts. The identified transcripts could be categorized into two major groups: (1) vegetal tier/germ granule-enriched and (2) animal tier/anterior-enriched transcripts. Using zebrafish as a surrogate model system, we showed that overexpression of one animal tier/anterior-localized amphioxus transcript, zfp665, causes a dorsalization/anteriorization phenotype in zebrafish embryos by downregulating the expression of the ventral gene, eve1, suggesting a potential function of zfp665 in early axial patterning. Our results provide a global transcriptomic blueprint for early-stage amphioxus embryos. This dataset represents a rich platform to guide future characterization of molecular players in early amphioxus development and to elucidate conservation and divergence of developmental programs during chordate evolution.

A yeast living ancestor reveals the origin of genomic introgressions.

Genome introgressions drive evolution across the animal1, plant2 and fungal3 kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast4, has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage5, which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.

Deeply conserved synteny resolves early events in vertebrate evolution.

Although it is widely believed that early vertebrate evolution was shaped by ancient whole-genome duplications, the number, timing and mechanism of these events remain elusive. Here, we infer the history of vertebrates through genomic comparisons with a new chromosome-scale sequence of the invertebrate chordate amphioxus. We show how the karyotypes of amphioxus and diverse vertebrates are derived from 17 ancestral chordate linkage groups (and 19 ancestral bilaterian groups) by fusion, rearrangement and duplication. We resolve two distinct ancient duplications based on patterns of chromosomal conserved synteny. All extant vertebrates share the first duplication, which occurred in the mid/late Cambrian by autotetraploidization (that is, direct genome doubling). In contrast, the second duplication is found only in jawed vertebrates and occurred in the mid-late Ordovician by allotetraploidization (that is, genome duplication following interspecific hybridization) from two now-extinct progenitors. This complex genomic history parallels the diversification of vertebrate lineages in the fossil record.

Intragenic repeat expansion in the cell wall protein gene HPF1 controls yeast chronological aging.

Aging varies among individuals due to both genetics and environment, but the underlying molecular mechanisms remain largely unknown. Using a highly recombined Saccharomyces cerevisiae population, we found 30 distinct quantitative trait loci (QTLs) that control chronological life span (CLS) in calorie-rich and calorie-restricted environments and under rapamycin exposure. Calorie restriction and rapamycin extended life span in virtually all genotypes but through different genetic variants. We tracked the two major QTLs to the cell wall glycoprotein genes FLO11 and HPF1 We found that massive expansion of intragenic tandem repeats within the N-terminal domain of HPF1 was sufficient to cause pronounced life span shortening. Life span impairment by HPF1 was buffered by rapamycin but not by calorie restriction. The HPF1 repeat expansion shifted yeast cells from a sedentary to a buoyant state, thereby increasing their exposure to surrounding oxygen. The higher oxygenation altered methionine, lipid, and purine metabolism, and inhibited quiescence, which explains the life span shortening. We conclude that fast-evolving intragenic repeat expansions can fundamentally change the relationship between cells and their environment with profound effects on cellular lifestyle and longevity.

Human RAP1 specifically protects telomeres of senescent cells from DNA damage.

Repressor/activator protein 1 (RAP1) is a highly evolutionarily conserved protein found at telomeres. Although yeast Rap1 is a key telomere capping protein preventing non-homologous end joining (NHEJ) and consequently telomere fusions, its role at mammalian telomeres in vivo is still controversial. Here, we demonstrate that RAP1 is required to protect telomeres in replicative senescent human cells. Downregulation of RAP1 in these cells, but not in young or dividing pre-senescent cells, leads to telomere uncapping and fusions. The anti-fusion effect of RAP1 was further explored in a HeLa cell line where RAP1 expression was depleted through an inducible CRISPR/Cas9 strategy. Depletion of RAP1 in these cells gives rise to telomere fusions only when telomerase is inhibited. We further show that the fusions triggered by RAP1 loss are dependent upon DNA ligase IV. We conclude that human RAP1 is specifically involved in protecting critically short telomeres. This has important implications for the functions of telomeres in senescent cells.

simuG: a general-purpose genome simulator.

SUMMARY: Simulated genomes with pre-defined and random genomic variants can be very useful for benchmarking genomic and bioinformatics analyses. Here we introduce simuG, a lightweight tool for simulating the full-spectrum of genomic variants (single nucleotide polymorphisms, Insertions/Deletions, copy number variants, inversions and translocations) for any organisms (including human). The simplicity and versatility of simuG make it a unique general-purpose genome simulator for a wide-range of simulation-based applications. AVAILABILITY AND IMPLEMENTATION: Code in Perl along with user manual and testing data is available at https://github.com/yjx1217/simuG. This software is free for use under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.