Biology subjects

Sarkies, P.

Publications and source records attributed to Sarkies, P..

33 records · Page 2Linked to original sources

bioRxiv2022

Gene editing in the nematode parasite Nippostrongylus brasiliensis using extracellular vesicles to deliver active Cas9/guide RNA complexes

Despite recent advances, animal-parasitic nematodes have thus far been largely refractory to genetic manipulation. We describe here a new approach providing proof of principle that CRISPR/Cas9-mediated gene editing of parasitic nematodes is achievable using vesicular stomatitis virus glycoprotein-pseudotyped extracellular vesicles (EV) for the delivery of Cas9-synthetic guide RNA (RNP) complexes. We demonstrate that EV-delivered RNPs can be used to disrupt a secreted DNase II in Nippostrogylus brasiliensis. Introduction of a repair template encoding multiple stop codons led to measurable reduction in expression of the targeted gene. Altered transcripts corresponding to the edited locus were detected by RT-PCR, demonstrating that vesicles can access cells of tissues actively expressing the gene of interest. These data provide evidence that this technique can be employed for targeted gene editing in N. brasiliensis, making this species genetically tractable for the first time and providing a new platform for genetic analysis of parasitic nematodes. Author SummaryParasitic nematodes have a complex life cycle involving passage through a host organism, which makes them very difficult to manipulate genetically. Recently, a method for deleting, changing or replacing genes (gene editing) has been developed in other organisms which has revolutionised our ability to understand fine details of how these organisms work. It has generally not been possible to adapt this method to parasitic nematodes because delivery of the components is difficult, and this has proved to be a bottleneck in understanding how parasites develop, survive and interact with their host. We show here that the components for gene editing can be introduced into a widely used laboratory model of intestinal nematode infection by encapsulation in membrane-bound vesicles which have been modified to carry a protein which facilitates fusion of the vesicles with parasite cells and delivery of the contents. This resulted in accurate editing of a specific gene by deletion and repair, such that the amount of functional protein produced from that gene was reduced. This system should be applicable to all nematode species, and will facilitate understanding of their complex biology, in addition to defining new targets for control of infection.

molecular biology↗

bioRxiv2022

The slow evolving genome of the xenacoelomorph worm Xenoturbella bocki

The evolutionary origins of Bilateria remain enigmatic. One of the more enduring proposals highlights similarities between a cnidarian-like planula larva and simple acoel-like flatworms. This idea is based in part on the view of the Xenacoelomorpha as an outgroup to all other bilaterians which are themselves designated the Nephrozoa (protostomes and deuterostomes). Genome data can help to elucidate phylogenetic relationships and provide important comparative data. Here we assemble and analyse the genome of the simple, marine xenacoelomorph Xenoturbella bocki, a key species for our understanding of early bilaterian and deuterostome evolution. Our highly contiguous genome assembly of X. bocki has a size of [~]111 Mbp in 18 chromosome like scaffolds, with repeat content and intron, exon and intergenic space comparable to other bilaterian invertebrates. We find X. bocki to have a similar number of genes to other bilaterians and to have retained ancestral metazoan synteny. Key bilaterian signalling pathways are also largely complete and most bilaterian miRNAs are present. We conclude that X. bocki has a complex genome typical of bilaterians, in contrast to the apparent simplicity of its body plan. Overall, our data do not provide evidence supporting the idea that Xenacoelomorpha are a primitively simple outgroup to other bilaterians and gene presence/absence data support a relationship with Ambulacraria.

evolutionary biology↗

bioRxiv2021

Malignancy and NF-kB signalling strengthen coordination between the expression of mitochondrial and nuclear-encoded oxidative phosphorylation genes

Mitochondria are ancient endosymbiotic organelles crucial to eukaryotic growth and metabolism. Mammalian mitochondria carry a small genome containing thirteen protein-coding genes with the remaining mitochondrial proteins encoded by the nuclear genome. Little is known about how coordination between the two sets of genes is achieved. Correlation analysis of RNA-seq expression data from large publicly-available datasets is a common method to leverage genetic diversity to infer gene co-expression modules. Here we use this method to investigate nuclear-mitochondrial gene expression coordination. We identify a pitfall in correlation analysis that results from the large variation in the proportion of transcripts from the mitochondrial genome in RNA-seq data. Commonly used normalization techniques based on total read count (such as FPKM or TPM) produce artefactual negative correlations between mitochondrial- and nuclear-encoded transcripts. This also results in artefactual correlations between pairs of nuclear-encoded genes, thus having important consequences for inferring co-expression modules beyond mitochondria. We show that these effects can be overcome by normalizing using the median-ratio normalization (MRN) or trimmed mean of M values (TMM) methods. Using these normalizations, we find only weak and inconsistent correlations between mitochondrial and nuclear-encoded mitochondrial genes in the majority of healthy human tissues from the GTEx database. However, a subset of healthy tissues with high expression of NF-{kappa}B show significant coordination supporting a role for NF-{kappa}B in retrograde signalling. Contrastingly, most cancer types show robust coordination of nuclear and mitochondrial OXPHOS gene expression, identifying this as a feature of gene regulation in cancer.

genomics↗

bioRxiv2021

Evolved changes in DNA methylation in response to the sustained loss of parental care in the burying beetle

Levels of parental care critically influence the developmental environment with the capacity to impact the growth, survival, physiology, and behaviour of offspring. Plastic changes in DNA methylation have been hypothesised to modulate gene expression responses to parental environments. Moreover, these effects can be inherited and so may affect the process of adaptive evolution. In this study, using experimental evolution, we investigated how plastic changes in DNA methylation induced by the loss of parental care have evolved in a biparental insect (Nicrophorus vespilloides) using experimental evolution. We show that removal of care in a single generation is associated with changes in gene expression in stress-related pathways in 1st instar larvae. However, in larvae that have adapted to the loss of parental care after being deprived of care for 30 generations, gene expression is shifted from stress-related gene expression towards growth and brain development pathways. We found that changes in gene body methylation arose both as a direct response to the loss of parental care and stochastically as populations diverged. Overall, our results suggest that a complex interplay between transcription and DNA methylation shapes the molecular adaptation to environmental change.

evolutionary biology↗

bioRxiv2020

Integrator terminates promoter-proximal Pol II to generate C. elegans piRNA precursors

Piwi-interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C. elegans, piRNAs are transcribed from >15000 discrete genomic loci by RNA polymerase II, resulting in 28 nt short-capped piRNA precursors. Here we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. We show that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter-proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Our results draw new parallels between snRNA and piRNA biogenesis in nematodes, and provide evidence of a role for the Integrator complex as a terminator of promoter-proximal RNA polymerase II. Highlights- Integrator localises to sites of piRNA biogenesis in nematodes - Integrator cleaves nascent RNAs associated with promoter-proximal Pol II at piRNA loci to release short capped piRNA precursors from chromatin - Repression of Pol II elongation at the majority of piRNA loci is independent of Integrator

molecular biology↗

bioRxiv2020

The RNA Polymerase II core subunit RPB-9 directs transcriptional elongation at piRNA loci in Caenorhabditis elegans

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA Polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. rpb-9 mutants fail to initiate heritable piRNA-mediated gene silencing. Furthermore, we show that RPB-9 is required to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis. We demonstrate that RPB-9 acts to promote transcriptional elongation/termination at endogenous piRNA loci. We conclude that as a part of its rapid evolution the piRNA pathway has co-opted another ancient machinery, this time for high-fidelity transcription.

genetics↗

bioRxiv2020

CD8+ T-cell transcription and DNA methylation show age specific differences and lack correlation with clinical outcome in pediatric Inflammatory Bowel Disease

Background & AimsCD8+ T-cell gene expression has been implicated in the pathogenesis of Inflammatory Bowel Diseases (IBD) and has been shown to correlate with disease outcome in adult patients. Moreover, CD8+ T-cell exhaustion was identified as a contributing mechanism that impacts on disease behaviour. We aimed to explore CD8+ T-cell gene expression and DNA methylation in children newly diagnosed with IBD and test their correlation with disease outcome. MethodsWe prospectively recruited 112 children with IBD at the point of diagnosis and 19 non-IBD controls. Follow-up samples were obtained from a subset of patients at 3-month intervals (n=62). CD8+ T-cells were purified from peripheral blood samples using magnetic bead sorting and genome-wide transcriptional (n=192) and DNA methylation (n=66) profiles were generated using Affymetrix and Illumina arrays respectively. Publicly available adult CD8+ T-cell transcriptomes and DNA methylomes were included in data analyses to investigate age dependant differences. ResultsVariation amongst CD8+ T-cell transcriptomes obtained from children showed association with disease, systemic inflammation, age and gender but lacked correlation with disease outcome in pediatric IBD. In contrast to CD8+ T-cell transcriptomes in adult Crohns Disease (CD), samples from pediatric patients did not show variation within genes forming part of the previously reported prognostic expression or T-cell exhaustion signatures. Pediatric CD patient derived DNA methylation profiles also lacked correlation with disease outcome but in comparison to adult CD8+ methylomes showed a higher predicted proportion of CD8+ naive T-cells. ConclusionsOur findings indicate age-related differences in IBD pathogenesis and highlight the importance of validating adult clinical biomarkers in pediatric cohorts.

immunology↗

bioRxiv2020

Trichinella spiralis secretes abundant unencapsulated small RNAs with potential effects on host gene expression

Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment largely encapsulated within small vesicles. Parasite secreted material often contains microRNAs (miRNAs), raising the possibility that they might contribute to pathology by regulating host genes in target cells. Here we characterise material from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis, which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly however, T. spiralis muscle stage larvae (MSL), which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests a new potential mechanism of RNA secretion with implications for the pathology of T. spiralis infection.

molecular biology↗

bioRxiv2020

Widespread conservation and lineage-specific diversification of genome-wide DNA methylation patterns across arthropods

Cytosine methylation is an ancient epigenetic modification yet its function and extent within genomes is highly variable across eukaryotes. In mammals, methylation controls transposable elements and regulates the promoters of genes. In insects, DNA methylation is generally restricted to a small subset of transcribed genes, with both intergenic regions and transposable elements (TEs) depleted of methylation. The evolutionary origin and the function of these methylation patterns are poorly understood. Here we characterise the evolution of DNA methylation across the arthropod phylum. While the common ancestor of the arthropods had low levels of TE methylation and did not methylate promoters, both of these functions have evolved independently in centipedes and mealybugs. In contrast, methylation of the exons of a subset of transcribed genes is ancestral and widely conserved across the phylum, but has been lost in specific lineages. Remarkably the same set of genes are likely to be methylated in all species that retained exon-enriched methylation. We show that these genes have characteristic patterns of expression correlating to broad transcription initiation sites and well-positioned nucleosomes, providing new insights into potential mechanisms driving methylation patterns over hundreds of millions of years. Author SummaryAnimals develop from a single cell to form a complex organism with many specialised cells. Almost all of the fantastic variety of cells must have the same sequence of DNA, and yet they have distinct identities that are preserved even when they divide. This remarkable process is achieved by turning different sets of genes on or off in different types of cell using molecular mechanisms known as "epigenetic gene regulation". Surprisingly, though all animals need epigenetic gene regulation, there is a huge diversity in the mechanisms that they use. Characterising and explaining this diversity is crucial in understanding the functions of epigenetic pathways, many of which have key roles in human disease. We studied how one particular type of epigenetic regulation, known as DNA methylation, has evolved within arthropods. Arthropods are an extraordinarily diverse group of animals ranging from horseshoe crabs to fruit flies. We discovered that the levels of DNA methylation and where it is found within the genome changes rapidly throughout arthropod evolution. Nevertheless, there are some features of DNA methylation that seem to be the same across most arthropods-in particular we found that there is a tendency for a similar set of genes to acquire methylation of DNA in most arthropods, and that this is conserved over 350 million years. We discovered that these genes have distinct features that might explain how methylation gets targeted. Our work provides important new insights into the evolution of DNA methylation and gives some new hints to its essential functions.

genetics↗

bioRxiv2019

Epimutations driven by small RNAs arise frequently but have limited duration in a metazoan organism

Epigenetic regulation involves changes in gene expression independent of DNA sequence variation that are inherited through cell division (Holliday, 2006). In addition to a fundamental role in cell differentiation, some epigenetic changes can also be transmitted transgenerationally through meiosis (Heard and Martienssen, 2014). Epigenetic alterations ("epimutations") could thus contribute to heritable variation within populations and be subject to evolutionary processes such as natural selection and drift (Burggren, 2016). However, this suggestion is controversial, partly because unlike classical mutations involving DNA sequence changes, key parameters such as the rate at which epimutations arise and their persistence are unknown. Here, we perform the first genome-wide study of epimutations in a metazoan organism. We use experimental evolution to characterise the rate, spectrum and stability of epimutations driven by small silencing RNAs in the model nematode C. elegans. We show that epimutations arise spontaneously at a rate [~]25 times greater than DNA sequence changes and typically have short half-lives of 2-3 generations. Nevertheless, some epimutations last at least 10 generations. Epimutations thus may contribute to evolutionary processes over a short timescale but are unlikely to bring about long-term divergence without further DNA sequence changes.

genetics↗

bioRxiv2019

Long-term experimental evolution reveals purifying selection on piRNA-mediated control of transposable element expression

Transposable elements (TEs) are an almost universal constituent of eukaryotic genomes. In animals, Piwi-interacting small RNAs (piRNAs) and repressive chromatin often play crucial roles in preventing TE transcription and thus restricting TE activity. Nevertheless, TE content varies widely across eukaryotes and the dynamics of TE activity and TE silencing across evolutionary time is poorly understood. Here we used experimentally evolved populations of C. elegans to study the dynamics of TE expression over 400 generations. The experimental populations were evolved at three different population sizes to manipulate the efficiency of natural selection versus genetic drift. We demonstrate increased TE expression relative to the ancestral population, with the largest increases occurring in the smallest populations. We show that the transcriptional activation of TEs within active regions of the genome is associated with failure of piRNA-mediated silencing, whilst desilenced TEs in repressed chromatin domains retain small RNAs. Additionally, we find that the sequence context of the surrounding region influences the propensity of TEs to lose silencing through failure of small RNA-mediated silencing. Together, our results show that natural selection in C. elegans is responsible for maintaining low levels of TE expression, and provide new insights into the epigenomic features responsible.

molecular biology↗

bioRxiv2018

Evolutionary analysis implicates RNA polymerase II pausing and chromatin structure in nematode piRNA biogenesis

Piwi-interacting RNAs (piRNAs) control transposable elements widely across metazoans but have rapidly evolving biogenesis pathways. In Caenorhabditis elegans, almost all piRNA loci are found within two 3Mb clusters on Chromosome IV. Each piRNA locus possesses an upstream motif that recruits RNA polymerase II to produce a [~]28 nt precursor transcript. Here, we use comparative epigenomics across nematodes to gain insight into piRNA biogenesis. We show that the piRNA upstream motif is derived from core promoter elements controlling snRNA biogenesis. We describe two alternative modes of piRNA organisation in nematodes: in C. elegans and closely related nematodes, piRNAs are clustered within repressive H3K27me3 chromatin, whilst in other species, typified by Pristionchus pacificus, piRNAs are distributed genome-wide within introns of actively transcribed genes. In both groups, piRNA production depends on downstream sequence signals associated with RNA polymerase II pausing, which synergise with the chromatin environment to control piRNA precursor transcription.

genomics↗

bioRxiv2018

The conserved phosphatase GSP-2/PP1 promotes germline immortality via small RNA-mediated genome silencing during meiosis

Genomic silencing can promote germ cell immortality, or transgenerational maintenance of the germ line, via mechanisms that may occur during mitosis or meiosis. Here we report that the gsp-2 PP1/Glc7 phosphatase promotes germ cell immortality. We identified a separation-of-function allele of C. elegans GSP-2 that caused a meiosis-specific chromosome segregation defect and defects in transgenerational small RNA-induced genome silencing. GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. Sterile gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents and histone phosphorylation defects in oocytes, similar to small RNA genome silencing mutants. Epistasis and RNA analysis suggested that GSP-2 functions downstream of small RNAs. We conclude that a meiosis-specific function of GSP-2/LAB-1 ties small RNA-mediated silencing of the epigenome to germ cell immortality. Given that hemizygous genetic elements can drive transgenerational epigenomic silencing, and given that LAB-1 promotes pairing of homologous chromosomes and localizes to the interface between homologous chromosomes during pachytene, we suggest that discontinuities at this interface could promote nuclear silencing in a manner that depends on GSP-2.\n\nAuthor SummaryThe germ line of an organism is considered immortal in its capacity to give rise to an unlimited number of future generations. To protect the integrity of the germ line, mechanisms act to suppress the accumulation of transgenerational damage to the genome or epigenome. Loss of germ cell immortality can result from mutations that disrupt the small RNA-mediated silencing pathway that helps to protect the integrity of the epigenome. Here we report for the first time that the C. elegans protein phosphatase GSP-2 that promotes core chromosome biology functions during meiosis is also required for germ cell immortality. Specifically, we identified a partial loss of function allele of gsp-2 that exhibits defects in meiotic chromosome segregation and is also dysfunctional for transgenerational small RNA-mediated genome silencing. Our results are consistent with a known role of Drosophila Protein Phosphatase 1 in heterochromatin silencing, and point to a meiotic phosphatase function that is relevant to germ cell immortality, conceivably related to its roles in chromosome pairing or sister chromatid cohesion.

genetics↗

bioRxiv2017

Pan-arthropod analysis reveals somatic piRNAs as an ancestral TE defence

In animals, small RNA molecules termed PIWI-interacting RNAs (piRNAs) silence transposable elements (TEs), protecting the germline from genomic instability and mutation. piRNAs have been detected in the soma in a few animals, but these are believed to be specific adaptations of individual species. Here, we report that somatic piRNAs were likely present in the ancestral arthropod more than 500 million years ago. Analysis of 20 species across the arthropod phylum suggests that somatic piRNAs targeting TEs and mRNAs are common among arthropods. The presence of an RNA-dependent RNA polymerase in chelicerates (horseshoe crabs, spiders, scorpions) suggests that arthropods originally used a plant-like RNA interference mechanism to silence TEs. Our results call into question the view that the ancestral role of the piRNA pathway was to protect the germline and demonstrate that small RNA silencing pathways have been repurposed for both somatic and germline functions throughout arthropod evolution.

evolutionary biology↗

bioRxiv2017

An alternative STAT signaling pathway acts in antiviral immunity in Caenorhabditis elegans

Across metazoans, innate immunity is vital in defending organisms against viral infection. In mammals, antiviral innate immunity is orchestrated by interferon signaling, activating the STAT transcription factors downstream of the JAK kinases to induce expression of antiviral effector genes. In the nematode C. elegans, which lacks the interferon system, the major antiviral response so far described is RNA interference but whether additional gene expression responses are employed is not known. Here we show that, despite the absence of both interferon and JAK, the C. elegans STAT homologue STA-1 orchestrates antiviral immunity. Intriguingly, mutants lacking STA-1 show increased resistance to antiviral infection. Using gene expression analysis and chromatin immunoprecipitation we show that, in contrast to the mammalian pathway, STA-1 acts as a transcriptional repressor. Thus STA-1 might act to suppress a constitutive antiviral response in the absence of infection. Using a reverse genetic screen we identify the SID-3 as a kinase upstream of STA-1 in the response to infection. Together, our work identifies a novel STAT regulatory cascade controlling its activity in antiviral resistance, illustrating the complex evolutionary trajectory displayed by innate immune signaling pathways across metazoan organisms.

immunology↗