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.

Li J, Bergmann L, Rafael de Almeda A, Webb KM, Gogol MM, Voigt P, Liu Y, Liang H, Smolle MM Epigenetics

The Isw1b chromatin-remodeling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeler. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promotes the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodeling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs.

+view abstract Nucleic acids research , PMID: 35188579

Ronsmans S, Sørig Hougaard K, Nawrot TS, Plusquin M, Huaux F, Jesús Cruz M, Moldovan H, Verpaele S, Jayapala M, Tunney M, Humblet-Baron S, Dirven H, Cecilie Nygaard U, Lindeman B, Duale N, Liston A, Meulengracht Flachs E, Kastaniegaard K, Ketzel M, Goetz J, Vanoirbeek J, Ghosh M, Hoet PHM Immunology

Immune-mediated, noncommunicable diseases-such as autoimmune and inflammatory diseases-are chronic disorders, in which the interaction between environmental exposures and the immune system plays an important role. The prevalence and societal costs of these diseases are rising in the European Union. The EXIMIOUS consortium-gathering experts in immunology, toxicology, occupational health, clinical medicine, exposure science, epidemiology, bioinformatics, and sensor development-will study eleven European study populations, covering the entire lifespan, including prenatal life. Innovative ways of characterizing and quantifying the exposome will be combined with high-dimensional immunophenotyping and -profiling platforms to map the immune effects (immunome) induced by the exposome. We will use two main approaches that "meet in the middle"-one starting from the exposome, the other starting from health effects. Novel bioinformatics tools, based on systems immunology and machine learning, will be used to integrate and analyze these large datasets to identify immune fingerprints that reflect a person's lifetime exposome or that are early predictors of disease. This will allow researchers, policymakers, and clinicians to grasp the impact of the exposome on the immune system at the level of individuals and populations.

+view abstract Environmental Epidemiology , PMID: 35169671

Hanna CW, Huang J, Belton C, Reinhardt S, Dahl A, Andrews S, Stewart AF, Kranz A, Kelsey G Epigenetics , Bioinformatics

Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.

+view abstract Nucleic acids research , PMID: 35137160

Yurchenko AA, Pop OT, Ighilahriz M, Padioleau I, Rajabi F, Sharpe HJ, Poulalhon N, Dreno B, Khammari A, Delord M, Alberdi A, Soufir N, Battistella M, Mourah S, Bouquet F, Savina A, Besse A, Mendez-Lopez M, Grange F, Monestier S, Mortier L, Meyer N, Dutriaux C, Robert C, Saïag P, Herms F, Lambert J, de Sauvage F, Dumaz N, Flatz L, Basset-Seguin N, Nikolaev SI Signalling

Vismodegib is approved for the treatment of locally advanced basal cell carcinoma (laBCC), but some cases demonstrate intrinsic resistance (IR) to the drug. We sought to assess the frequency of IR to vismodegib in laBCC and its underlying genomic mechanisms.

+view abstract Clinical Cancer Research , PMID: 35078858

Suriyalaksh M, Raimondi C, Mains A, Segonds-Pichon A, Mukhtar S, Murdoch S, Aldunate R, Krueger F, Guimerà R, Andrews S, Sales-Pardo M, Casanueva O Epigenetics , Bioinformatics

We design a "wisdom-of-the-crowds" GRN inference pipeline and couple it to complex network analysis to understand the organizational principles governing gene regulation in long-lived /Notch . The GRN has three layers (input, core, and output) and is topologically equivalent to bow-tie/hourglass structures prevalent among metabolic networks. To assess the functional importance of structural layers, we screened 80% of regulators and discovered 50 new aging genes, 86% with human orthologues. Genes essential for longevity-including ones involved in insulin-like signaling (ILS)-are at the core, indicating that GRN's structure is predictive of functionality. We used reporters and a novel functional network covering 5,497 genetic interactions to make mechanistic predictions. We used genetic epistasis to test some of these predictions, uncovering a novel transcriptional regulator, , that works alongside DAF-16/FOXO. We present a framework with predictive power that can accelerate discovery in and potentially humans.

+view abstract iScience , PMID: 35036864

Prescott JA, Balmanno K, Mitchell JP, Okkenhaug H, Cook SJ Signalling , Imaging

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

+view abstract The Biochemical journal , PMID: 35029639

Velten B, Braunger JM, Argelaguet R, Arnol D, Wirbel J, Bredikhin D, Zeller G, Stegle O Epigenetics

Factor analysis is a widely used method for dimensionality reduction in genome biology, with applications from personalized health to single-cell biology. Existing factor analysis models assume independence of the observed samples, an assumption that fails in spatio-temporal profiling studies. Here we present MEFISTO, a flexible and versatile toolbox for modeling high-dimensional data when spatial or temporal dependencies between the samples are known. MEFISTO maintains the established benefits of factor analysis for multimodal data, but enables the performance of spatio-temporally informed dimensionality reduction, interpolation, and separation of smooth from non-smooth patterns of variation. Moreover, MEFISTO can integrate multiple related datasets by simultaneously identifying and aligning the underlying patterns of variation in a data-driven manner. To illustrate MEFISTO, we apply the model to different datasets with spatial or temporal resolution, including an evolutionary atlas of organ development, a longitudinal microbiome study, a single-cell multi-omics atlas of mouse gastrulation and spatially resolved transcriptomics.

+view abstract Nature methods , PMID: 35027765

Whale AJ, King M, Hull RM, Krueger F, Houseley J Epigenetics

Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 can cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32 and Pif1. CUP1 amplification is also critically dependent on late-firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after activation, leaving a tract of H3K56-acetylated chromatin that promotes secondary recombination events during elongation after replication fork re-start events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

+view abstract Nucleic acids research , PMID: 35018465

Misheva M, Kotzamanis K, Davies LC, Tyrrell VJ, Rodrigues PRS, Benavides GA, Hinz C, Murphy RC, Kennedy P, Taylor PR, Rosas M, Jones SA, McLaren JE, Deshpande S, Andrews R, Schebb NH, Czubala MA, Gurney M, Aldrovandi M, Meckelmann SW, Ghazal P, Darley-Usmar V, White DA, O'Donnell VB Signalling

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

+view abstract Nature communications , PMID: 35013270

Crainiciuc G, Palomino-Segura M, Molina-Moreno M, Sicilia J, Aragones DG, Li JLY, Madurga R, Adrover JM, Aroca-Crevillén A, Martin-Salamanca S, Del Valle AS, Castillo SD, Welch HCE, Soehnlein O, Graupera M, Sánchez-Cabo F, Zarbock A, Smithgall TE, Di Pilato M, Mempel TR, Tharaux PL, González SF, Ayuso-Sacido A, Ng LG, Calvo GF, González-Díaz I, Díaz-de-María F, Hidalgo A

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution.

+view abstract Nature , PMID: 34987220

Lozano T, Conde E, Martín-Otal C, Navarro F, Lasarte-Cia A, Nasrallah R, Alignani D, Gorraiz M, Sarobe P, Romero JP, Vilas A, Roychoudhuri R, Hervás-Stubbs S, Casares N, Lasarte JJ Immunology

Adoptive cell transfer therapy using CD8 T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8 T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8 T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8 T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8 T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-γ, IFN-α, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8 T cells with respect to FOXP3-wt CD8 T cells. Our results suggest that transient expression of FOXP3 by CD8 T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy.

+view abstract Cancer letters , PMID: 34973390

Sandovici I, Georgopoulou A, Pérez-García V, Hufnagel A, López-Tello J, Lam BYH, Schiefer SN, Gaudreau C, Santos F, Hoelle K, Yeo GSH, Burling K, Reiterer M, Fowden AL, Burton GJ, Branco CM, Sferruzzi-Perri AN, Constância M Epigenetics

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here, we demonstrate, through fetal, endothelial, hematopoietic, and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The angiocrine effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signaling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis.

+view abstract Developmental cell , PMID: 34963058

Runfola M, Perni M, Yang X, Marchese M, Bacci A, Mero S, Santorelli FM, Polini B, Chiellini G, Giuliani D, Vilella A, Bodria M, Daini E, Vandini E, Rudge S, Gul S, Wakelam MOJ, Vendruscolo M, Rapposelli S Signalling

The identification of effective pharmacological tools for Alzheimer's disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that can restore cellular homeostasis. To investigate this strategy, we designed and synthetized SG2, a compound related to the thyroid hormone thyroxine, that shares a pleiotropic activity with its endogenous parent compound, including autophagic flux promotion, neuroprotection, and metabolic reprogramming. We demonstrate herein that SG2 acts in a pleiotropic manner to induce recovery in a model of AD based on the overexpression of Aβ42 and improves learning abilities in the 5XFAD mouse model of AD. Further, in vitro ADME-Tox profiling and toxicological studies in zebrafish confirmed the low toxicity of this compound, which represents a chemical starting point for AD drug development.

+view abstract Pharmaceuticals , PMID: 34959730

Rivera Del Alamo MM, Reilas T, Lukasik K, Galvão AM, Yeste M, Katila T Epigenetics

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.

+view abstract Animals , PMID: 34944269

Mikulasova A, Kent D, Trevisan-Herraz M, Karataraki N, Fung KTM, Ashby C, Cieslak A, Yaccoby S, van Rhee F, Zangari M, Thanendrarajan S, Schinke C, Morgan GJ, Asnafi V, Spicuglia S, Brackley CA, Corcoran AE, Hambleton S, Walker BA, Rico D, Russell LJ Immunology

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.

+view abstract Genome research , PMID: 34933939

Kubinyecz O, Santos F, Drage D, Reik W, Eckersley-Maslin MA Epigenetics

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.

+view abstract Development , PMID: 34931676

Gould SA, Gilley J, Ling K, Jafar-Nejad P, Rigo F, Coleman M

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.

+view abstract Cell reports , PMID: 34910914

Huang TC, Wang YF, Vazquez-Ferrer E, Theofel I, Requena CE, Hanna CW, Kelsey G, Hajkova P Epigenetics

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.

+view abstract Nature , PMID: 34880491

Rugg-Gunn PJ Epigenetics

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.

+view abstract Methods in molecular biology , PMID: 34870841

Bendall A, Semprich CI Epigenetics

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.

+view abstract Methods in molecular biology , PMID: 34870837

Rostovskaya M Epigenetics

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.

+view abstract Methods in molecular biology , PMID: 34870834

Rostovskaya M Epigenetics

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.

+view abstract Methods in molecular biology , PMID: 34870831

Rugg-Gunn PJ Epigenetics

Human pluripotent stem cells exist in naïve and primed states that recapitulate the distinct molecular and cellular properties of pre- and post-implantation epiblast cells, respectively. Naïve pluripotent stem cells can be captured directly from blastocysts but, more commonly, the cells are reprogrammed from primed cells in a process called "resetting". Several methods to achieve resetting have been described. Chemical resetting of primed cells to a naïve pluripotent state is one such method and has come to the forefront as a simple, efficient, and transgene-free method to induce naïve pluripotency. The process involves the transient application of a histone deacetylase inhibitor to initiate resetting, followed by the emergence of nascent naïve pluripotent stem cells in supportive conditions, and finally the stabilization and expansion of naïve pluripotent stem cell cultures. Here, a detailed protocol is provided for chemical resetting starting from plating primed cells until a stable culture of naïve pluripotent stem cells is established.

+view abstract Methods in molecular biology (Clifton, N.J.) , PMID: 34870828

Loreto A, Angeletti C, Gu W, Osborne A, Nieuwenhuis B, Gilley J, Arthur-Farraj P, Merlini E, Amici A, Luo Z, Hartley-Tassell L, Ve T, Desrochers LM, Wang Q, Kobe B, Orsomando G, Coleman MP

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.

+view abstract eLife , PMID: 34870595