Biology subjects

Sarkies, P.

Publications and source records attributed to Sarkies, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Ethanol stress induces siRNA-mediated epimutations and phenotypic variability in fission yeast

Epigenetic information is required for the maintenance of gene expression states through cell division. Some epigenetic information can also persist through generations, raising the possibility that epigenetic differences may arise in populations and contribute to evolutionary processes such as drift and natural selection. Evaluating this requires characterising the properties of such epimutations, so that their rate, stability and genome-wide distribution can be assessed. Recent experiments in many organisms have demonstrated that epimutations occur, but have limited stability relative to DNA sequence mutations. However, the extent to which epimutations contribute to evolution is still unclear, in part because it is not clear whether epimutations respond dynamically to environmental conditions. Here, we use the fission yeast S. pombe, in which evolutionarily relevant epimutations have been observed, but not yet systematically characterised, to test whether a physiologically relevant stress, ethanol exposure, leads to changes in the properties of epimutations. Using laboratory evolution experiments with repeated population bottlenecks to minimise selection, we show that epimutations associated with changes in small interfering (si)RNAs occur at a greater rate in ethanol. Ethanol-induced epimutations are enriched at transposable element loci and correlate with changes in transposon expression. Ethanol exposure also increases gene expression noise genome-wide and variability in growth rate; importantly, both these increases are suppressed in mutants lacking siRNAs. Together, these results demonstrate that stress can alter the generation of heritable epigenetic information, and suggest that siRNAs may have a role in promoting phenotypic variability in response to environmental stress.

genetics↗

bioRxiv2026

Adulthood depletion of Integrator extends lifespan and healthspan via defective pre-mRNA processing

Identifying strategies to mitigate age-related physiological decline remains a central challenge. During ageing, the transcriptome undergoes extensive remodelling, but how this affects organismal health and lifespan is not well understood. The Integrator complex plays a central role in RNA polymerase II transcription and RNA 3' end processing. Surprisingly, we find that depletion of most Integrator subunits specifically in adults extends lifespan and healthspan in the nematode C. elegans. We show that loss of the catalytic subunit INTS-11 disrupts 3' end formation of small nuclear and spliced leader RNAs, impairing trans-splicing and promoting outron retention in a subset of transcripts enriched for spliceosomal and nucleocytoplasmic transport genes. These RNA-processing defects lead to altered levels of endogenous siRNAs, which are required for the longevity and healthspan benefits of INTS-11 depletion. In parallel, outron retention disrupts nuclear-encoded mitochondrial gene expression and protein production, inducing mitochondrial dysfunction and promoting lifespan extension. We also demonstrate that loss of INTS-11 perturbs transcription elongation at genes where Integrator is present at promoters, and that upregulation of enhancer elements within intragenic regions can affect the expression and isoform usage of nearby genes. Together, our findings identify Integrator as a key upstream regulator of non-coding RNA transcription, which in turn impacts protein-coding gene expression and mitochondrial function to shape the ageing process.

cell biology↗

bioRxiv2026

Centromeres are hotspots of cytosine methylation epimutations in a filamentous fungus

Epimutations are changes in chromatin modifications, such as DNA methylation or histone modifications. Some of these epigenetic changes can be inherited for several generations, and they potentially contribute to evolutionary processes. Estimates of epimutation rates now exists in a few species, but the presence and function of epigenetic marks are not conserved across different species. To understand the properties of epimutations in fungi, we performed a mutation accumulation experiment with the filamentous fungus Neurospora crassa and investigated spontaneous changes in DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3) in the mutation accumulation lines. We observed that centromeric regions are hotspots of spontaneous DNA methylation changes in N. crassa. In these hotspot regions, DNA methylation changes were transmitted across mitoses, but changes occurring in euchromatin were not maintained. The rate of DNA methylation changes was around 30 000 fold faster than the genetic mutation rate. We did not observe spontaneous changes in H3K9me3 that were transmitted across mitoses. Our results show that while spontaneous epimutations occur in this species, they occur predominantly in gene poor heterochromatic regions, so their impact for evolutionary adaptation may be limited.

genetics↗

bioRxiv2026

Introduction of CG methylation in E. coli induces mutagenesis at AT base pairs

In many eukaryotic species, DNA is methylated at the 5 position of cytosine to form 5mC, predominantly within CG dinucleotides. Despite being conserved since the dawn of eukaryotic life, 5mC is often lost from individual lineages, suggesting that it may have detrimental effects. One such effect is genotoxicity, through the effect of 5mC on the process of cytosine deamination and its repair. Additionally, enzymes that introduce 5mC (DNA methyltransferases, DNMTs) can also damage DNA through alkylation and oxidative stress, but how these genotoxic effects combine to influence mutagenesis is unclear. To investigate how mutagenesis changes upon methylation of CG dinucleotides we introduced high levels of CG methylation into the bacteria E. coli. 5mC induction increased mutation at CG dinucleotides consistent with increased C to T mutations. We also discovered that 5mC induction led to increased mutations at AT base pairs, specifically in the absence of the alkylation repair enzyme AlkB. This effect was specific to certain E. coli strains and was not dependent on the DNA repair enzyme RecA, so its exact mechanism remains unclear. Together, our work highlights multiple mutagenic consequences of DNMT expression, which might act as selective pressures for organisms to lose 5mC across evolution.

molecular biology↗

bioRxiv2025

An Evolutionarily Conserved N-terminal Domain of RRF-3 Governs GTSF-1 Binding in Nematodes

GTSF1 is an essential activating cofactor for PIWI proteins in many metazoans. In the nematode Caenorhabditis elegans, however, GTSF-1 does not bind PIWI, but associates with the RNA-dependent RNA polymerase RRF-3, supporting endo-siRNA (26G-RNA) biogenesis. Here, we demonstrate that this rewiring is deeply conserved across nematodes. For C. briggsae and Pristionchus pacificus we show that GTSF-1 interacts with RRF-3 and is essential for 26G-RNA production and fertility. We map this interaction to an N-terminal domain of RRF-3, termed the GTSF-1 interacting domain (GID), and show that the GTSF-1 zinc finger region alone is sufficient for binding. Mutagenesis identifies critical residues mediating this interaction and reveals that GTSF-1 stability depends on RRF-3. Other RdRPs possess GID-like domains, which we propose to bind GTSF-1-related proteins. Phylogenomic and structural analyses support GTSF-1-RRF-3 interactions across all major nematode lineages and map the shift in GTSF-1 activity to the last common nematode ancestor. We propose that binding of GTSF-1 induces conformational changes in RRF-3 that facilitate ERI complex assembly and activate RdRP function, paralleling its role as a PIWI activator.

molecular biology↗

bioRxiv2025

ATRX safeguards cellular identity during C. elegans development

ATRX is a member of the SWI/SNF family of ATP-dependent chromatin remodellers. In humans, loss of ATRX function leads to ATRX syndrome, a neurodevelopmental disorder. ATRX mutation in human cell lines is associated with multiple phenotypes including activation of the alternative lengthening of telomere (ALT) pathway, upregulation of retrotransposons and increased sensitivity to replication stress. However, the principal role of ATRX and the reason why its mutation causes such diverse phenotypes is currently unclear. To address this, we studied the role of ATRX in the model organism Caenorhabditis elegans. We find that loss of XNP-1, the C. elegans homologue of ATRX, recapitulates many human phenotypes. Loss of XNP-1 causes ectopic activation of germline genes in somatic cells, indicating a loss of cellular identity control. Strikingly, mutation of the germline transcription factor gsox-1 suppresses both this misexpression and multiple xnp-1 phenotypes, including developmental delay and telomeric defects. These findings suggest that ectopic germline gene expression underlies the majority of XNP-1-dependent phenotypes, consistent with a role for XNP-1 in maintaining cellular identity, offering insights into the functions of ATRX in humans.

molecular biology↗

bioRxiv2024

Identification of SLC45A4 as a pain gene encoding a neuronal polyamine transporter.

Polyamines are regulatory metabolites with key roles in transcription, translation, cell signalling and autophagy1. They are implicated in multiple neurological disorders including stroke, epilepsy and neurodegeneration and can regulate neuronal excitability through interactions with ion channels2. Polyamines have been linked to pain showing altered levels in human persistent pain states and modulation of pain behaviour in animal models3. However, the systems governing polyamine transport within the nervous system remain unclear. In undertaking a Genome Wide Association Study (GWAS) of chronic pain intensity in the UK-Biobank we found significant association with variants mapping to the SLC45A4 gene locus. In the mouse nervous system SLC45A4 expression is enriched in all sensory neuron sub-types within the dorsal root ganglion including nociceptors. Cell-based assays show that SLC45A4 is a selective plasma membrane polyamine transporter, whilst the cryo-EM structure reveals a novel regulatory domain and basis for polyamine recognition. Mice lacking SLC45A4 show normal mechanosensitivity but reduced sensitivity to noxious heat and algogen induced tonic pain that is associated with reduced excitability of peptidergic nociceptors. Our findings thus establish a role for neuronal polyamine transport in pain perception and identify a new target for therapeutic intervention in pain treatment.

neuroscience↗

bioRxiv2024

Polycomb-mediated transgenerational epigenetic inheritance of Drosophila eye colour is independent of small RNAs

Transgenerational epigenetic inheritance (TEI) describes the process where distinct epigenetic states may be transmitted between generations, resulting in stable gene expression and phenotypic differences between individuals that persist independently of DNA sequence variation. Chromatin modifications have been demonstrated as important in TEI, however, the extent to which they require other signals to establish and maintain epigenetic states is still unclear. Here we investigate whether small non-coding RNAs contribute to different epigenetic states of the Fab2L transgene in Drosophila triggered by transient chromatin contacts, which requires Polycomb complex activity to deposit the H3K27me3 modification for long-term TEI. Using mutants deficient in known small non-coding RNAs, high throughput sequencing, investigation of chromatin conformation and gene expression analysis we demonstrate that small non-coding RNAs do not contribute directly to initiation or maintenance of silencing. However, we uncover an indirect role for microRNA expression in transgene silencing through effects on Polycomb complex expression. Additionally, we show that a commonly used marker gene, Stubble (Sb), affects Polycomb complex expression, which may be important in interpreting experiments assaying Polycomb function in Drosophila development. By ruling out a plausible candidate for TEI at the Fab2L transgene our work highlights the variability in different modes of TEI across species.

biochemistry↗

bioRxiv2024

Introduction of cytosine-5 DNA methylation sensitizes cells to oxidative damage

DNA methylation at the 5 position of cytosine (5mC) is an ancient epigenetic mark in eukaryotes. The levels of total 5mC vary enormously between different species, and the DNA methyltransferases that introduce 5mC have been repeatedly lost in several independent lineages. DNA methyltransferases are a threat to genomic stability due to the increased mutagenicity of 5mC bases and the propensity of DNA methyltransferases themselves to introduce DNA alkylation damage as an off-target effect. However, whether alkylation damage explains why 5mC is frequently lost in evolution is unclear. Here we tested the fitness consequences of DNA methyltransferase-induced alkylation damage by introducing a eukaryotic-like 5mC system into E. coli. We showed that introducing 5mC genome-wide leads to increased sensitivity to alkylating agents, which is strongly enhanced by removal of the 3mC repair enzyme AlkB. Unexpectedly, we discovered that 5mC introduction led to increased sensitivity to oxidative stress. We showed that this is due to increased formation of reactive oxygen in the presence of 5mC. We determined that reactive oxygen species led to non-enzymatic oxidation of 5mC, producing modified cytosines such as 5fC that are recognised as DNA base damage in E. coli. Overall, our work identifies increased sensitivity to oxidative stress, as well as alkylating agents, as a negative consequence of genome-wide 5mC. Oxidative stress is frequently encountered by organisms in their environment, thus offering a plausible reason for total loss of 5mC in some species.

molecular biology↗

bioRxiv2024

Substantial hierarchical reductions of genetic and morphological traits in the evolution of rotiferan parasites

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/605096v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@469ecborg.highwire.dtl.DTLVardef@6330f4org.highwire.dtl.DTLVardef@22faf6org.highwire.dtl.DTLVardef@8a6704_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG During the last 800 million years of evolution animals radiated into a vast range of diversity of species and disparity of forms and lifestyles. The process involved a near hierarchical increase in complexity from life forms with few cell types to organisms with many hundreds of cell-types. However, neither genome size nor number of protein-coding genes can explain these differences and their biological basis remains elusive. Yet, recent studies have suggested that the evolution of complexity is closely linked to the acquisition of a class of protein coding gene-regulators called microRNAs. In a regressive approach, to investigate the association between loss of organismal complexity and microRNAs, we here studied Syndermata, an invertebrate group including free-living rotifers (Monogononta, Bdelloidea), the epibiotic Seisonidea and the endoparasitic Acanthocephala. Genomic, transcriptomic and morphological characterization and comparisons across 25 syndermatan species revealed a strong correlation between loss of microRNAs, loss of protein-coding genes and decreasing morphological complexity. The near hierarchical loss extends to [~]85% loss of microRNAs and a [~]50% loss of BUSCO genes in the endoparasitic Acanthocephala, the most reduced group we studied. Together, the loss of [~]400 protein-coding genes and [~]10 metazoan core gene losses went along with one microRNA family loss. Furthermore, the loss of [~]4 microRNA families or [~]34 metazoan core genes associated with one lost morphological feature. These are the first quantitative insights into the regulatory impact of microRNAs on organismic complexity as a predictable consequence in regressive evolution of parasites.

evolutionary biology↗

bioRxiv2024

Predicting substrates for orphan Solute Carrier Proteins using multi-omics datasets

Solute carriers (SLC) are integral membrane proteins responsible for transporting a wide variety of metabolites, signaling molecules and drugs across cellular membranes. Despite key roles in metabolism, signaling and pharmacology, around one third of SLC proteins are orphans whose substrates are unknown. Experimental determination of SLC substrates is technically challenging given the wide range of possible physiological candidates. Here, we develop a predictive algorithm to identify correlations between SLC expression levels and intracellular metabolite concentrations by leveraging existing cancer multi-omics datasets. Our predictions recovered known SLC-substrate pairs with high sensitivity and specificity compared to simulated random pairs. CRISPR loss-of-function screen data and metabolic pathway adjacency data further improved the performance of our algorithm. In parallel, we combined drug sensitivity data with SLC expression profiles to predict new SLC-drug interactions. Together, we provide a novel bioinformatic pipeline to predict new substrate predictions for SLCs, offering new opportunities to de-orphanise SLCs with important implications for understanding their roles in health and disease.

molecular biology↗

bioRxiv2023

Epigenetic context predicts gene expression variation and reproductive traits across genetically identical individuals

In recent decades, genome-wide association studies (GWAS) have been the major approach to understand the biological basis of individual differences in traits and diseases. However, GWAS approaches have limited predictive power to explain individual differences, particularly for complex traits and diseases in which environmental factors play a substantial role in their etiology. Indeed, individual differences persist even in genetically identical individuals, although fully separating genetic and environmental causation is difficult in most organisms. To understand the basis of individual differences in the absence of genetic differences, we measured two quantitative reproductive traits in 180 genetically identical young adult Caenorhabditis elegans roundworms in a shared environment and performed single-individual transcriptomics on each worm. We identified hundreds of genes for which expression variation was strongly associated with reproductive traits, some of which depended on individuals historical environments and some of which was random. Multiple small sets of genes together were highly predictive of reproductive traits, explaining on average over half and over a quarter of variation in the two traits. We manipulated mRNA levels of predictive genes to identify a set of causal genes, demonstrating the utility of this approach for both prediction and understanding underlying biology. Finally, we found that the chromatin environment of predictive genes was enriched for H3K27 trimethylation, suggesting that gene expression variation may be driven in part by chromatin structure. Together, this work shows that individual, non-genetic differences in gene expression are both highly predictive and causal in shaping reproductive traits.

genomics↗

bioRxiv2023

Interchromosomal contacts between regulatory regions trigger stable transgenerational epigenetic inheritance in Drosophila

Non-genetic information can be inherited across generations by the process of Transgenerational Epigenetic Inheritance (TEI). TEI can be established by various triggering events, including transient genetic perturbations. In Drosophila, hemizygosity of the Fab-7 regulatory element triggers inheritance of the histone mark H3K27me3 at a homologous locus on another chromosome, resulting in heritable epigenetic differences in eye colour. By mutating transcription factor binding sites within the Fab-7 element, we demonstrate the importance of two proteins in the establishment and maintenance of TEI: GAGA-factor and Pleiohomeotic. We show that these proteins function by recruiting the Polycomb Repressive Complex 2 and by mediating interchromosomal chromatin contacts between Fab-7 and its homologous locus. Finally, using an in vivo synthetic biology system to induce them, we show that chromatin contacts alone can establish TEI, providing a mechanism by which hemizygosity of one locus can establish epigenetic memory at another in trans through long-distance chromatin contacts.

genetics↗

bioRxiv2023

Chronic cisplatin exposure does not affect epimutations in C. elegans but induces fluctuations in tRNA-derived small non-coding RNAs.

The individual lifestyle and environment of an organism can influence its phenotype and potentially the phenotype of its offspring. The different genetic and non-genetic components of the inheritance system and their mutual interactions are key mechanisms to generate inherited phenotypic changes. Epigenetic changes can be transmitted between generations independently from changes in DNA sequence. In C. elegans, epigenetic differences, i.e., epimutations, mediated by small non-coding RNAs, particularly 22G-RNAs, as well as chromatin have been identified and their average persistence is 3 to 5 generations. In addition, previous research showed that some epimutations had a longer duration and concerned genes that were enriched for multiple components of xenobiotic response pathways. These results raise the possibility that environmental stresses might change the rate at which epimutations occur, with potential significance for adaptation. In this work, we explore this question by propagating C. elegans lines either in control conditions or in moderate or high doses of cisplatin, which introduces genotoxic stress by damaging DNA. Our results show that cisplatin has a limited effect on global small non-coding RNAs epimutations and epimutations in gene expression levels. However, cisplatin exposure leads to increased fluctuations in the levels of small non-coding RNAs derived from tRNA cleavage. We show that changes in tRNA-derived small RNAs may be associated with gene expression changes. Our work shows that epimutations are not substantially altered by cisplatin exposure but identifies transient changes in tRNA-derived small RNAs as a potential source of transcriptional variation induced by genotoxic stress.

molecular biology↗

bioRxiv2023

Identification of proteins that bind extracellular microRNAs secreted by the parasitic nematode Trichinella spiralis

Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently we characterised the parasitic nematode Trichinella spiralis as a model to study miRNA secretion. T. spiralis larvae secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds non-selectively to miRNA. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely.

biochemistry↗

bioRxiv2023

Histone methylation has a direct metabolic role in human cells

The N-terminal tails of eukaryotic histones are frequently post-translationally modified. The role of these modifications in transcriptional regulation is well-documented. However, the extent to which the enzymatic process of histone post-translational modification itself contributes to metabolic regulation is less clear. Here we investigated the metabolic role of histone methylation using metabolomics, proteomics and RNA-seq data from cancer cell lines, primary tumour samples and healthy tissue samples. In cancer the transcription of histone methyltransferases was inversely correlated to the activity of NNMT, an enzyme previously characterised as a methyl sink that disposes of excess methyl groups carried by the universal methyl donor S-adenosyl methionine (SAM or AdoMet). In healthy tissues histone methylation was inversely correlated to the levels of an alternative methyl sink, PEMT. These associations affected the levels of multiple histone marks on chromatin genome-wide but had no detectable impact on transcriptional regulation. We show that histone methyltransferases with a variety of different associations to transcription are co-regulated by the Retinoblastoma (Rb) tumour suppressor in human cells. Total HMT expression is increased in Rb-mutant cancers, and this leads to NNMT downregulation. Together, our results suggest a direct metabolic role for histone methylation in SAM homeostasis, independent of transcriptional regulation.

bioinformatics↗

bioRxiv2022

Fluctuations in chromatin state at regulatory loci occur spontaneously under relaxed selection and are associated with epigenetically inherited variation in C. elegans gene expression.

Some epigenetic information can be transmitted between generations without changes in the underlying DNA sequence. Changes in epigenetic regulation, termed epimutations, can occur spontaneously and be propagated in populations in a manner reminiscent of DNA mutations. Epimutations mediated by small non-coding RNAs occur in C. elegans and persist for around 3-5 generations on average. Here, we explored whether chromatin states also undergo spontaneous change and whether this could be a potential alternative mechanism for transgenerational epigenetic inheritance. We compared the chromatin and gene expression profiles at matched time points from three independent lineages of C. elegans propagated at minimal population size. Spontaneous changes in chromatin occurred in around 1% of regulatory regions each generation. Some were heritable epimutations and were significantly enriched for heritable changes in expression of nearby protein-coding genes. Most chromatin-mediated epimutations were short-lived but a subset had longer duration. Genes subject to long-lived epimutations were enriched for multiple components of xenobiotic response pathways. This points to a possible role for epimutations in adaptation to environmental stressors.

molecular biology↗

bioRxiv2022

Loss of the E3 ubiquitin ligases UBR-5 or HECD-1 restores Caenorhabditis elegans development in the absence of SWI/SNF function

SWItch/Sucrose Non-Fermenting (SWI/SNF) complexes are a family of chromatin remodellers that are conserved across eukaryotes. Mutations in subunits of SWI/SNF cause a multitude of different developmental disorders in humans, most of which have no current treatment options. Here we identify an alanine to valine causing mutation in the SWI/SNF subunit snfc-5 (SMARCB1 in humans) that prevents embryonic lethality in C. elegans nematodes harbouring a loss-of-function mutation in the SWI/SNF subunit swsn-1 (SMARCC1/2 in humans). Furthermore, we found that the combination of this specific mutation in snfc-5 and a loss-of-function mutation in either of the E3 ubiquitin ligases ubr-5 (UBR5 in humans) or hecd-1 (HECTD1 in humans) can restore development to adulthood in swsn-1 loss-of-function mutants that otherwise die as embryos. Using these mutant models, we established a set of 335 genes that are dysregulated in SWI/SNF mutants that arrest their development embryonically but exhibit near wild-type levels of expression in the presence of suppressor mutations that prevent embryonic lethality, suggesting that SWI/SNF promotes development by regulating this specific subset of genes. In addition, we show that SWI/SNF protein levels are reduced in swsn-1; snfc-5 double mutants and partly restored to wild-type levels in swsn-1; snfc-5; ubr-5 triple mutants, consistent with a model in which UBR-5 regulates SWI/SNF levels by tagging the complex for proteasomal degradation. Our findings establish a link between two E3 ubiquitin ligases and SWI/SNF function and suggest that UBR5 and HECTD1 might be viable therapeutic targets for the many developmental disorders caused by missense mutations in SWI/SNF subunits.

developmental biology↗