The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific facilities. Pre-prints by Institute authors can be viewed on the Institute's bioRxiv channel. We believe that free and open access to the outputs of publicly‐funded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.
Intrauterine devices (IUDs) are used in mares to suppress oestrous behaviour, but the underlying mechanism is yet to be elucidated. The presence of an embryo or an IUD prevents cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin (PG) release and luteolysis. However, inflammation may also be involved. Endometrial inflammatory markers in uterine lavage fluid were measured on Day 10 (EXP 1, = 25) and Day 15 (EXP 2, = 27) after ovulation in inseminated mares, non-pregnant or pregnant, and in mares in which a small plastic sphere had been inserted into the uterus 4 (EXP 1) or 3 days (EXP 2) after ovulation. Uterine lavage fluid samples were analysed for nitric oxide (NO), prostaglandin E (PGE) (only EXP 1), prostaglandin F (PGF), inhibin A and cytokines, and blood samples for progesterone and oestradiol. On Day 10, the concentration of PGF was lower ( < 0.05) in the IUD group than in pregnant mares. The concentration of the modulatory cytokine IL-10 was significantly higher in the IUD group in comparison to non-pregnant mares, and inhibin A was significantly higher in IUD mares than in the pregnant counterparts on Day 15. The results suggest that the presence of IUD causes endometrial inflammation which is at a resolution stage on Day 15.
Chromosomal translocations are important drivers of hematological malignancies whereby proto-oncogenes are activated by juxtaposition with super-enhancers, often called enhancer hijacking. We analysed the epigenomic consequences of rearrangements between the super-enhancers of the immunoglobulin heavy locus () and proto-oncogene that are common in B cell malignancies. By integrating BLUEPRINT epigenomic data with DNA breakpoint detection, we characterised the normal chromatin landscape of the human locus and its dynamics after pathological genomic rearrangement. We detected an H3K4me3 broad domain (BD) within the locus of healthy B cells that was absent in samples with translocations. The appearance of H3K4me3-BD over in the latter was associated with overexpression and extensive chromatin accessibility of its gene body. We observed similar cancer-specific H3K4me3-BDs associated with super-enhancer hijacking of other common oncogenes in B cell (, and /) and in T-cell malignancies (, and ). Our analysis suggests that H3K4me3-BDs can be created by super-enhancers and supports the new concept of epigenomic translocation, where the relocation of H3K4me3-BDs from cell identity genes to oncogenes accompanies the translocation of super-enhancers.
Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.
Activation of the pro-degenerative protein SARM1 after diverse physical and disease-relevant injuries causes programmed axon degeneration. Original studies indicate that substantially decreased SARM1 levels are required for neuroprotection. However, we demonstrate, in Sarm1 haploinsufficient mice, that lowering SARM1 levels by 50% delays programmed axon degeneration in vivo after sciatic nerve transection and partially prevents neurite outgrowth defects in mice lacking the pro-survival factor NMNAT2. In vitro, the rate of degeneration in response to traumatic, neurotoxic, and genetic triggers of SARM1 activation is also slowed. Finally, we demonstrate that Sarm1 antisense oligonucleotides decrease SARM1 levels by more than 50% in vitro, which delays or prevents programmed axon degeneration. Combining Sarm1 haploinsufficiency with antisense oligonucleotides further decreases SARM1 levels and prolongs protection after neurotoxic injury. These data demonstrate that axon protection occurs in a Sarm1 gene dose-responsive manner and that SARM1-lowering agents have therapeutic potential, making Sarm1-targeting antisense oligonucleotides a promising therapeutic strategy.
Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.
Cell-surface proteins provide excellent biomarkers to identify specific cell types and resolve heterogeneous cell populations. The analysis of cell-surface proteins by flow cytometry produces robust and quantitative information with single-cell resolution, and allows live target cells to be purified and characterized or re-cultured. Studies using antibody screens, proteomics, and candidate analysis have identified a comprehensive set of proteins that are expressed on the surface of naïve and primed human pluripotent stem cells. These findings have led to the development of suitable protein markers and antibodies to accurately distinguish between these two cell types. Here, a detailed protocol is provided that uses multi-color flow cytometry to analyze cell-surface protein expression in naïve and primed human pluripotent stem cells. This method enables the unambiguous identification of pluripotent cell types and the opportunity to sort target cells including during cell state transitions. The protocol can be combined to additionally investigate the expression of reporter genes and other informative features, such as DNA content.
Chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-sequencing) facilitates the genome-wide mapping of DNA sequences that are enriched for specific chromatin-binding proteins or histone post-translational modifications. More recently developed chromatin profiling methods called Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and Cleavage Under Targets and Tagmentation (CUT&Tag) have adapted the ChIP-sequencing approach to produce similar data from a smaller amount of starting material, and while overcoming many of the conventional drawbacks of ChIP-sequencing. Here, we present detailed protocols for ChIP-seq, CUT&RUN, and CUT&Tag to profile genome-wide protein-DNA interactions in naïve human pluripotent stem cells.
Naïve and primed pluripotent stem cells resemble epiblast cells of the pre-implantation and post-implantation embryo, respectively. This chapter describes a simple experimental system for the efficient and consistent transition of human pluripotent stem cells (hPSCs) from the naïve to the primed state, which is a process called capacitation. Naïve hPSCs after capacitation can be differentiated further to somatic lineages, thus reproducing the order of developmental events in the embryo. Protocols for the induction of neuroectoderm, definitive endoderm, and paraxial mesoderm from hPSCs after capacitation and also from conventionally derived primed hPSCs are included in the chapter. Importantly, hPSC capacitation closely recapitulates transcriptional, metabolic, signaling, and cell polarity changes in the epiblast of primate embryos, and therefore offers a unique in vitro model of human peri-implantation development.
Naïve pluripotent stem cells are the in vitro counterparts of pre-implantation embryonic epiblast. During the last few years, several protocols for establishing and maintaining human pluripotent stem cells (hPSCs) with naïve features have been reported, and many of these protocols result in cell populations with different molecular characteristics. As such, choosing the most appropriate method for naïve hPSC maintenance can pose a significant challenge. This chapter presents an optimized system called PXGL for culturing naïve hPSCs. Naïve hPSCs robustly self-renew while retaining a normal karyotype in PXGL, and the protocol is reproducible across different cell lines and independent laboratories.
Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the nicotinamide adenine dinucleotide (NAD)-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death and . We present the crystal structure the SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet know is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.
Inborn errors of immunity (IEI) are a heterogenous group of disorders driven by genetic defects that functionally impact the development and/or function of the innate and/or adaptive immune system. The majority of these disorders are thought to have polygenic background. However, the use of next-generation sequencing in patients with IEI has led to an increasing identification of monogenic causes, unravelling the exact pathophysiology of the disease and allowing the development of more targeted treatments. Monogenic IEI are not only seen in a pediatric population but also in adulthood, either due to the lack of awareness preventing childhood diagnosis or due to a delayed onset where (epi)genetic or environmental factors can play a role. In this review, we discuss the mechanisms accounting for adult-onset presentations and provide an overview of monogenic causes associated with adult-onset IEI.
Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.
Mitochondrial failure has long been associated with programmed axon death (Wallerian degeneration, WD), a widespread and potentially preventable mechanism of axon degeneration. While early findings in axotomised axons indicated that mitochondria are involved during the execution steps of this pathway, recent studies suggest that in addition, mitochondrial dysfunction can initiate programmed axon death without physical injury. As mitochondrial dysfunction is associated with disorders involving early axon loss, including Parkinson's disease, peripheral neuropathies, and multiple sclerosis, the findings that programmed axon death is activated by mitochondrial impairment could indicate the involvement of druggable mechanisms whose disruption may protect axons in such diseases. Here, we review the latest developments linking mitochondrial dysfunction to programmed axon death and discuss their implications for injury and disease.
Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. expression is elevated in human PDA, and deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.
To reflect human development, it is critical to create a substrate that can support long-term cell survival, differentiation, and maturation. Hydrogels are promising materials for 3D cultures. However, a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction. Herein, granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell (hiPSC)-derived neural networks. A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules. Cells and hydrogel granules were combined using a weaker secondary gelation step, forming self-supporting cell laden scaffolds. At three and seven days, granular scaffolds supported higher cell viability compared to bulk hydrogels, whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions (65.52 ± 11.59 μm) after seven days compared to bulk hydrogels (22.90 ± 4.70 μm). Long-term (three-month) cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold. This approach is significant as it provides a simple, rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.
Synthetic phosphatidylinositol phosphate (PtdIns ) derivatives play a pivotal role in broadening our understanding of PtdIns metabolism. However, the development of such tools is reliant on efficient enantioselective and regioselective synthetic strategies. Here we report the development of a divergent synthetic route applicable to the synthesis of deuterated PtdIns4 and PtdIns5 derivatives. The synthetic strategy developed involves a key enzymatic desymmetrisation step using Lipozyme TL-IM®. In addition, we optimised the large-scale synthesis of deuterated -inositol, allowing for the preparation of a series of saturated and unsaturated deuterated PtdIns4 and PtdIns5 derivatives. Experiments in MCF7 cells demonstrated that these deuterated probes enable quantification of the corresponding endogenous phospholipids in a cellular setting. Overall, these deuterated probes will be powerful tools to help improve our understanding of the role played by PtdIns in physiology and disease.
Obesity leads to ovarian dysfunction and the establishment of local leptin resistance. The aim of our study was to characterize the levels of NOD-like receptor protein 3 (NLRP3) inflammasome activation in ovaries and liver of mice during obesity progression. Furthermore, we tested the putative role of leptin on NLRP3 regulation in those organs. C57BL/6J female mice were treated with equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) for estrous cycle synchronization and ovary collection. In diet-induced obesity (DIO) protocol, mice were fed chow diet (CD) or high-fat diet (HFD) for 4 or 16 weeks, whereas in the hyperleptinemic model (LEPT), mice were injected with leptin for 16 days (16 L) or saline (16 C). Finally, the genetic obese leptin-deficient (+/? and -/-) mice were fed CD for 4 week. Either ovaries and liver were collected, as well as cumulus cells (CCs) after superovulation from DIO and LEPT. The estrus cycle synchronization protocol showed increased protein levels of NLRP3 and interleukin (IL)-18 in diestrus, with this stage used for further sample collections. In DIO, protein expression of NLRP3 inflammasome components was increased in 4 week HFD, but decreased in 16 week HFD. Moreover, NLRP3 and IL-1β were upregulated in 16 L and downregulated in Transcriptome analysis of CC showed common genes between LEPT and 4 week HFD modulating NLRP3 inflammasome. Liver analysis showed NLRP3 protein upregulation after 16 week HFD in DIO, but also its downregulation in . We showed the link between leptin signaling and NLRP3 inflammasome activation in the ovary throughout obesity progression in mice, elucidating the molecular mechanisms underpinning ovarian failure in maternal obesity.
SARM1, a protein with critical NADase activity, is a central executioner in a conserved programme of axon degeneration. We report seven rare missense or in-frame microdeletion human variant alleles in patients with amyotrophic lateral sclerosis (ALS) or other motor nerve disorders that alter the SARM1 auto-inhibitory ARM domain and constitutively hyperactivate SARM1 NADase activity. The constitutive NADase activity of these seven variants is similar to that of SARM1 lacking the entire ARM domain and greatly exceeds the activity of wild-type SARM1, even in the presence of nicotinamide mononucleotide (NMN), its physiological activator. This rise in constitutive activity alone is enough to promote neuronal degeneration in response to otherwise non-harmful, mild stress. Importantly, these strong gain-of-function alleles are completely patient-specific in the cohorts studied and show a highly significant association with disease at the single gene level. These findings of disease-associated coding variants that alter SARM1 function build on previously reported genome-wide significant association with ALS for a neighbouring, more common intragenic single nucleotide polymorphism (SNP) to support a contributory role of SARM1 in these disorders. A broad phenotypic heterogeneity and variable age-of-onset of disease among patients with these alleles also raises intriguing questions about the pathogenic mechanism of hyperactive SARM1 variants.
The extreme diversity of the human immune system, forged and maintained throughout evolutionary history, provides a potent defense against opportunistic pathogens. At the same time, this immune variation is the substrate upon which a plethora of immune-associated diseases develop. Genetic analysis suggests that thousands of individually weak loci together drive up to half of the observed immune variation. Intense selection maintains this genetic diversity, even selecting for the introgressed Neanderthal or Denisovan alleles that have reintroduced variation lost during the out-of-Africa migration. Variations in age, sex, diet, environmental exposure, and microbiome each potentially explain the residual variation, with proof-of-concept studies demonstrating both plausible mechanisms and correlative associations. The confounding interaction of many of these variables currently makes it difficult to assign definitive contributions. Here, we review the current state of play in the field, identify the key unknowns in the causality of immune variation, and identify the multidisciplinary pathways toward an improved understanding.
High-quality clinical research is dependent on adequate design, methodology, and data collection. The utilization of electronic data capture (EDC) systems is recommended to optimize research data through proper management. This paper's objective is to present the procedures of REDCap (Research Electronic Data Capture), which supports research development, and to promote the utilization of this software among the scientific community.
Epigenetic inheritance of gene expression states enables a single genome to maintain distinct cellular identities. How histone modifications contribute to this process remains unclear. Using global chromatin perturbations and local, time-controlled modulation of transcription, we establish the existence of epigenetic memory of transcriptional activation for genes that can be silenced by the Polycomb group. This property emerges during cell differentiation and allows genes to be stably switched after a transient transcriptional stimulus. This transcriptional memory state at Polycomb targets operates in cis; however, rather than relying solely on read-and-write propagation of histone modifications, the memory is also linked to the strength of activating inputs opposing Polycomb proteins, and therefore varies with the cellular context. Our data and computational simulations suggest a model whereby transcriptional memory arises from double-negative feedback between Polycomb-mediated silencing and active transcription. Transcriptional memory at Polycomb targets thus depends not only on histone modifications but also on the gene-regulatory network and underlying identity of a cell.
Neuroimmunology in the broadest sense is the study of interactions between the nervous and the immune systems. These interactions play important roles in health from supporting neural development, homeostasis and plasticity to modifying behaviour. Neuroimmunology is increasingly recognised as a field with the potential to deliver a significant positive impact on human health and treatment for neurological and psychiatric disorders. Yet, translation to the clinic is hindered by fundamental knowledge gaps on the underlying mechanisms of action or the optimal timing of an intervention, and a lack of appropriate tools to visualise and modulate both systems. Here we propose ten key disease-agnostic research questions that, if addressed, could lead to significant progress within neuroimmunology in the short to medium term. We also discuss four cross-cutting themes to be considered when addressing each question: i) bi-directionality of neuroimmune interactions; ii) the biological context in which the questions are addressed (e.g. health vs disease vs across the lifespan); iii) tools and technologies required to fully answer the questions; and iv) translation into the clinic. We acknowledge that these ten questions cannot represent the full breadth of gaps in our understanding; rather they focus on areas which, if addressed, may have the most broad and immediate impacts. By defining these neuroimmunology priorities, we hope to unite existing and future research teams, who can make meaningful progress through a collaborative and cross-disciplinary effort.
The germinal centre (GC) is required for the generation of high affinity antibodies and immunological memory. Here we show that the RNA binding protein HuR has an essential function in GC B cells to sustain the GC response. In its absence, the GC reaction and production of high-affinity antibody is severely impaired. Mechanistically, HuR affects the transcriptome qualitatively and quantitatively. The expression and splicing patterns of hundreds of genes are altered in the absence of HuR. Among these genes, HuR is required for the expression of Myc and a Myc-dependent transcriptional program that controls GC B cell proliferation and Ig somatic hypermutation. Additionally, HuR regulates the splicing and abundance of mRNAs required for entry into and transition through the S phase of the cell cycle, and it modulates a gene signature associated with DNA deamination protecting GC B cells from DNA damage and cell death.
Vaccines are a highly effective intervention for conferring protection against infections and reducing the associated morbidity and mortality in vaccinated individuals. However, ageing is often associated with a functional decline in the immune system that results in poor antibody production in older individuals after vaccination. A key contributing factor of this age-related decline in vaccine efficacy is the reduced size and function of the germinal centre (GC) response. GCs are specialised microstructures where B cells undergo affinity maturation and diversification of their antibody genes, before differentiating into long-lived antibody-secreting plasma cells and memory B cells. The GC response requires the coordinated interaction of many different cell types, including B cells, T follicular helper (Tfh) cells, T follicular regulatory (Tfr) cells and stromal cell subsets like follicular dendritic cells (FDCs). This review discusses how ageing affects different components of the GC reaction that contribute to its limited output and ultimately impaired antibody responses in older individuals after vaccination. An understanding of the mechanisms underpinning the age-related decline in the GC response is crucial in informing strategies to improve vaccine efficacy and extend the healthy lifespan among older people.