Telomere-to-telomere Schizosaccharomyces japonicus genome assembly reveals hitherto unknown genome features.

Schizosaccharomyces japonicus belongs to the single-genus class Schizosaccharomycetes, otherwise known as "fission yeasts." As part of a composite model system with its widely studied S. pombe sister species, S. japonicus has provided critical insights into the workings and the evolution of cell biological mechanisms. Furthermore, its divergent biology makes S. japonicus a valuable model organism in its own right. However, the currently available genome assembly contains gaps and has been unable to resolve centromeres and other repeat-rich chromosomal regions. Here we present a telomere-to-telomere long-read genome assembly of the S. japonicus genome. This includes the three megabase-length chromosomes, with centromeres hundreds of kilobases long, rich in 5S ribosomal RNA genes, transfer RNA genes, long terminal repeats, and short repeats. We identify a gene-sparse region on chromosome 2 that resembles a 331 kb centromeric duplication. We revise the genome size of S. japonicus to at least 16.6 Mb and possibly up to 18.12 Mb, at least 30% larger than previous estimates. Our whole genome assembly will support the growing S. japonicus research community and facilitate research in new directions, including centromere and DNA repeat evolution, and yeast comparative genomics.

Schizosaccharomyces versatilis represents a distinct evolutionary lineage of fission yeast.

The fission yeast species Schizosaccharomyces japonicus is currently divided into two varieties-S. japonicus var. japonicus and S. japonicus var. versatilis. Here we examine the var. versatilis isolate CBS5679. The CBS5679 genome shows 88% identity to the reference genome of S. japonicus var. japonicus at the coding sequence level, with phylogenetic analyses suggesting that it has split from the S. japonicus lineage 25 million years ago. The CBS5679 genome contains a reciprocal translocation between chromosomes 1 and 2, together with several large inversions. The products of genes linked to the major translocation are associated with 'metabolism' and 'cellular assembly' ontology terms. We further show that CBS5679 does not generate viable progeny with the reference strain of S. japonicus. Although CBS5679 shares closer similarity to the 'type' strain of var. versatilis as compared to S. japonicus, it is not identical to the type strain, suggesting population structure within var. versatilis. We recommend that the taxonomic status of S. japonicus var. versatilis is raised, with it being treated as a separate species, Schizosaccharomyces versatilis.

Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Systematic Analysis of Copy Number Variations in the Pathogenic Yeast Candida parapsilosis Identifies a Gene Amplification in RTA3 That is Associated with Drug Resistance.

We analyzed the genomes of 170 C. parapsilosis isolates and identified multiple copy number variations (CNVs). We identified two genes, RTA3 (CPAR2_104610) and ARR3 (CPAR2_601050), each of which was the target of multiple independent amplification events. Phylogenetic analysis shows that most of these amplifications originated only once. For ARR3, which encodes a putative arsenate transporter, 8 distinct CNVs were identified, ranging in size from 2.3 kb to 10.5 kb with 3 to 23 copies. For RTA3, 16 distinct CNVs were identified, ranging in size from 0.3 kb to 4.5 kb with 2 to ~50 copies. One unusual amplification resulted in a DUP-TRP/INV-DUP structure similar to some human CNVs. RTA3 encodes a putative phosphatidylcholine (PC) floppase which is known to regulate the inward translocation of PC in Candida albicans. We found that an increased copy number of RTA3 correlated with resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine. Two genes, CPAR2_303950 and CPAR2_102700, coding for putative PC flippases homologous to S. cerevisiae DNF1 gained homozygous protein-disrupting mutations in the evolved strains. Overall, our results show that C. parapsilosis can gain resistance to miltefosine, a drug that has recently been granted orphan drug designation approval by the United States Food and Drug Administration for the treatment of invasive candidiasis, through both CNVs or loss-of-function alleles in one of the flippase genes. IMPORTANCE Copy number variations (CNVs) are an important source of genomic diversity that have been associated with drug resistance. We identify two unusual CNVs in the human fungal pathogen Candida parapsilosis. Both target a single gene (RTA3 or ARR3), and they have occurred multiple times in multiple isolates. The copy number of RTA3, a putative floppase that controls the inward translocation of lipids in the cell membrane, correlates with resistance to miltefosine, a derivative of phosphatidylcholine (PC) that was originally developed as an anticancer drug. In 2021, miltefosine was designated an orphan drug by the United States Food and Drug Administration for the treatment of invasive candidiasis. Importantly, we find that resistance to miltefosine is also caused by mutations in flippases, which control the outward movement of lipids, and that many C. parapsilosis isolates are prone to easily acquiring an increased resistance to miltefosine.

Effectiveness of glass beads for plating cell cultures.

Cell plating, the spreading out of a liquid suspension of cells on a surface followed by colony growth, is a common laboratory procedure in microbiology. Despite this, the exact impact of its parameters on colony growth has not been extensively studied. A common protocol involves the shaking of glass beads within a Petri dish containing solid growth media. We investigated the effects of multiple parameters in this protocol: the number of beads, the shape of movement, and the number of movements. Standard suspensions of Escherichia coli were spread while varying these parameters to assess their impact on colony growth. Results were assessed by a variety of metrics: the number of colonies, the mean distance between closest colonies, and the variability and uniformity of their spatial distribution. Finally, we devised a mathematical model of shifting billiard to explain the heterogeneities in the observed spatial patterns. Exploring the parameters that affect the most fundamental techniques in microbiology allows us to better understand their function, giving us the ability to precisely control their outputs for our exact needs.

Tos4 mediates gene expression homeostasis through interaction with HDAC complexes independently of H3K56 acetylation.

Saccharomyces cerevisiae exhibits gene expression homeostasis, which is defined as the buffering of transcription levels against changes in DNA copy number during the S phase of the cell cycle. It has been suggested that S. cerevisiae employs an active mechanism to maintain gene expression homeostasis through Rtt109-Asf1-dependent acetylation of histone H3 on lysine 56 (H3K56). Here, we show that gene expression homeostasis can be achieved independently of H3K56 acetylation by Tos4 (Target of Swi6-4). Using Nanostring technology, we establish that Tos4-dependent gene expression homeostasis depends on its forkhead-associated (FHA) domain, which is a phosphopeptide recognition domain required to bind histone deacetylases (HDACs). We demonstrate that the mechanism of Tos4-dependent gene expression homeostasis requires its interaction with the Rpd3L HDAC complex. However, this is independent of Rpd3's well-established roles in both histone deacetylation and controlling the DNA replication timing program, as established by deep sequencing of Fluorescence-Activated Cell Sorted (FACS) S and G2 phase populations. Overall, our data reveals that Tos4 mediates gene expression homeostasis through its FHA domain-dependent interaction with the Rpd3L complex, which is independent of H3K56ac.

Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early- and late-replicating euchromatin.

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.

The Beacon Calculus: A formal method for the flexible and concise modelling of biological systems.

Biological systems are made up of components that change their actions (and interactions) over time and coordinate with other components nearby. Together with a large state space, the complexity of this behaviour can make it difficult to create concise mathematical models that can be easily extended or modified. This paper introduces the Beacon Calculus, a process algebra designed to simplify the task of modelling interacting biological components. Its breadth is demonstrated by creating models of DNA replication dynamics, the gene expression dynamics in response to DNA methylation damage, and a multisite phosphorylation switch. The flexibility of these models is shown by adapting the DNA replication model to further include two topics of interest from the literature: cooperative origin firing and replication fork barriers. The Beacon Calculus is supported with the open-source simulator bcs (https://github.com/MBoemo/bcs.git) to allow users to develop and simulate their own models.

DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family.

Genome replication follows a defined temporal programme that can change during cellular differentiation and disease onset. DNA replication results in an increase in DNA copy number that can be measured by high-throughput sequencing. Here we present a protocol to determine genome replication dynamics using DNA copy-number measurements. Cell populations can be obtained in three variants of the method. First, sort-seq reveals the average replication dynamics across S phase in an unperturbed cell population; FACS is used to isolate replicating and non-replicating subpopulations from asynchronous cells. Second, sync-seq measures absolute replication time at specific points during S phase using a synchronized cell population. Third, marker frequency analysis can be used to reveal the average replication dynamics using copy-number analysis in any proliferating asynchronous cell culture. These approaches have been used to reveal genome replication dynamics in prokaryotes, archaea and a wide range of eukaryotes, including yeasts and mammalian cells. We have found this approach straightforward to apply to other organisms and highlight example studies from across the three domains of life. Here we present a Saccharomyces cerevisiae version of the protocol that can be performed in 7-10 d. It requires basic molecular and cellular biology skills, as well as a basic understanding of Unix and R.

Genome-wide analysis of DNA replication timing in single cells: Yes! We're all individuals.

Recent studies have accomplished the extraordinary feat of measuring the exact status of DNA replication in individual cells. We outline how these studies have revealed surprising uniformity in how cells replicate their DNA, and consider the implications of this remarkable technological advance.

Interspecies conservation of organisation and function between nonhomologous regional centromeres.

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres, here we take an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Interestingly, nonhomologous centromere central-core sequences from S. octosporus and S. cryophilus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin.

Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads.

Replication of eukaryotic genomes is highly stochastic, making it difficult to determine the replication dynamics of individual molecules with existing methods. We report a sequencing method for the measurement of replication fork movement on single molecules by detecting nucleotide analog signal currents on extremely long nanopore traces (D-NAscent). Using this method, we detect 5-bromodeoxyuridine (BrdU) incorporated by Saccharomyces cerevisiae to reveal, at a genomic scale and on single molecules, the DNA sequences replicated during a pulse-labeling period. Under conditions of limiting BrdU concentration, D-NAscent detects the differences in BrdU incorporation frequency across individual molecules to reveal the location of active replication origins, fork direction, termination sites, and fork pausing/stalling events. We used sequencing reads of 20-160 kilobases to generate a whole-genome single-molecule map of DNA replication dynamics and discover a class of low-frequency stochastic origins in budding yeast. The D-NAscent software is available at https://github.com/MBoemo/DNAscent.git .

Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains.

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

Bayesian inference of origin firing time distributions, origin interference and licencing probabilities from Next Generation Sequencing data.

DNA replication is a stochastic process with replication forks emanating from multiple replication origins. The origins must be licenced in G1, and the replisome activated at licenced origins in order to generate bi-directional replication forks in S-phase. Differential firing times lead to origin interference, where a replication fork from an origin can replicate through and inactivate neighbouring origins (origin obscuring). We developed a Bayesian algorithm to characterize origin firing statistics from Okazaki fragment (OF) sequencing data. Our algorithm infers the distributions of firing times and the licencing probabilities for three consecutive origins. We demonstrate that our algorithm can distinguish partial origin licencing and origin obscuring in OF sequencing data from Saccharomyces cerevisiae and human cell types. We used our method to analyse the decreased origin efficiency under loss of Rat1 activity in S. cerevisiae, demonstrating that both reduced licencing and increased obscuring contribute. Moreover, we show that robust analysis is possible using only local data (across three neighbouring origins), and analysis of the whole chromosome is not required. Our algorithm utilizes an approximate likelihood and a reversible jump sampling technique, a methodology that can be extended to analysis of other mechanistic processes measurable through Next Generation Sequencing data.

Rapid high-resolution measurement of DNA replication timing by droplet digital PCR.

Genomes are replicated in a reproducible temporal pattern. Current methods for assaying allele replication timing are time consuming and/or expensive. These include high-throughput sequencing which can be used to measure DNA copy number as a proxy for allele replication timing. Here, we use droplet digital PCR to study DNA replication timing at multiple loci in budding yeast and human cells. We establish that the method has temporal and spatial resolutions comparable to the high-throughput sequencing approaches, while being faster than alternative locus-specific methods. Furthermore, the approach is capable of allele discrimination. We apply this method to determine relative replication timing across timing transition zones in cultured human cells. Finally, multiple samples can be analysed in parallel, allowing us to rapidly screen kinetochore mutants for perturbation to centromere replication timing. Therefore, this approach is well suited to the study of locus-specific replication and the screening of cis- and trans-acting mutants to identify mechanisms that regulate local genome replication timing.

Investigating the role of Rts1 in DNA replication initiation.

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2.  Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains.   Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

Evolution of Genome Architecture in Archaea: Spontaneous Generation of a New Chromosome in Haloferax volcanii.

The common ancestry of archaea and eukaryotes is evident in their genome architecture. All eukaryotic and several archaeal genomes consist of multiple chromosomes, each replicated from multiple origins. Three scenarios have been proposed for the evolution of this genome architecture: 1) mutational diversification of a multi-copy chromosome; 2) capture of a new chromosome by horizontal transfer; 3) acquisition of new origins and splitting into two replication-competent chromosomes. We report an example of the third scenario: the multi-origin chromosome of the archaeon Haloferax volcanii has split into two elements via homologous recombination. The newly generated elements are bona fide chromosomes, because each bears "chromosomal" replication origins, rRNA loci, and essential genes. The new chromosomes were stable during routine growth but additional genetic manipulation, which involves selective bottlenecks, provoked further rearrangements. To the best of our knowledge, rearrangement of a naturally evolved prokaryotic genome to generate two new chromosomes has not been described previously.

Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

DNA replication timing influences gene expression level.

Eukaryotic genomes are replicated in a reproducible temporal order; however, the physiological significance is poorly understood. We compared replication timing in divergent yeast species and identified genomic features with conserved replication times. Histone genes were among the earliest replicating loci in all species. We specifically delayed the replication of HTA1-HTB1 and discovered that this halved the expression of these histone genes. Finally, we showed that histone and cell cycle genes in general are exempt from Rtt109-dependent dosage compensation, suggesting the existence of pathways excluding specific loci from dosage compensation mechanisms. Thus, we have uncovered one of the first physiological requirements for regulated replication time and demonstrated a direct link between replication timing and gene expression.

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome.

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.