P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation.

Innate lymphoid cells (ILCs) are guardians of mucosal immunity, yet the transcriptional networks that support their function remain poorly understood. We used inducible combinatorial deletion of key transcription factors (TFs) required for ILC development (RORγt, RORα and T-bet) to determine their necessity in maintaining ILC3 identity and function. Both RORγt and RORα were required to preserve optimum effector functions; however, RORα was sufficient to support robust interleukin-22 production among the lymphoid tissue inducer (LTi)-like ILC3 subset, but not natural cytotoxicity receptor (NCR) ILC3s. Lymphoid tissue inducer-like ILC3s persisted with only selective loss of phenotype and effector functions even after the loss of both TFs. In contrast, continued RORγt expression was essential to restrain transcriptional networks associated with type 1 immunity within NCR ILC3s, which coexpress T-bet. Full differentiation to an ILC1-like population required the additional loss of RORα. Together, these data demonstrate how TF networks integrate within mature ILCs after development to sustain effector functions, imprint phenotype and restrict alternative differentiation programs.

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8-12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal-ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

The histone modification H3K27me3 plays a central role in Polycomb-mediated epigenetic silencing. H3K27me3 recruits and allosterically activates Polycomb Repressive Complex 2 (PRC2), which adds this modification to nearby histones, providing a read/write mechanism for inheritance through DNA replication. However, for some PRC2 targets, a purely histone-based system for epigenetic inheritance may be insufficient. We address this issue at the Polycomb target in , as a narrow nucleation region of only ~three nucleosomes within mediates epigenetic state switching and subsequent memory over many cell cycles. To explain the memory's unexpected persistence, we introduce a mathematical model incorporating extra protein memory storage elements with positive feedback that persist at the locus through DNA replication, in addition to histone modifications. Our hybrid model explains many features of epigenetic switching/memory at and encapsulates generic mechanisms that may be widely applicable.

The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFβ/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFβ signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFβ signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFβ signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFβ pathway function in human pluripotency spanning a developmental window from naive to primed states.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Genomic imprinting is an epigenetic marking process that results in the monoallelic expression of a subset of genes. Many of these 'imprinted' genes in mice and humans are involved in embryonic and extraembryonic growth and development, and some have life-long impacts on metabolism. During mammalian development, the genome undergoes waves of (re)programming of DNA methylation and other epigenetic marks. Disturbances in these events can cause imprinting disorders and compromise development. Multi-locus imprinting disturbance (MLID) is a condition by which imprinting defects touch more than one locus. Although most cases with MLID present with clinical features characteristic of one imprinting disorder. Imprinting defects also occur in 'molar' pregnancies-which are characterized by highly compromised embryonic development-and in other forms of reproductive compromise presenting clinically as infertility or early pregnancy loss. Pathogenic variants in some of the genes encoding proteins of the subcortical maternal complex (SCMC), a multi-protein complex in the mammalian oocyte, are responsible for a rare subgroup of moles, biparental complete hydatidiform mole (BiCHM), and other adverse reproductive outcomes which have been associated with altered imprinting status of the oocyte, embryo and/or placenta. The finding that defects in a cytoplasmic protein complex could have severe impacts on genomic methylation at critical times in gamete or early embryo development has wider implications beyond these relatively rare disorders. It signifies a potential for adverse maternal physiology, nutrition, or assisted reproduction to cause epigenetic defects at imprinted or other genes. Here, we review key milestones in DNA methylation patterning in the female germline and the embryo focusing on humans. We provide an overview of recent findings regarding DNA methylation deficits causing BiCHM, MLID, and early embryonic arrest. We also summarize identified SCMC mutations with regard to early embryonic arrest, BiCHM, and MLID.

Perinatal mortality is a major issue in laboratory mouse breeding. We compared a counting method using daily checks (DAILY_CHECK) with a method combining daily checks with detailed video analyses to detect cannibalisms (VIDEO_TRACK) for estimating the number of C57BL/6 pups that were born, that died and that were weaned in 193 litters from trios with (TRIO-OVERLAP) or without (TRIO-NO_OVERLAP) the presence of another litter. Linear mixed models were used at litter level. To understand whether cannibalism was associated with active killing (infanticide), we analysed VIDEO_TRACK recordings of 109 litters from TRIO-OVERLAP, TRIO-NO_OVERLAP or SOLO (single dams). We used Kaplan-Meier method and logistic regression at pup level. For DAILY_CHECK, the mean litter size was 35% smaller than for VIDEO_TRACK ( < 0.0001) and the number of dead pups was twice lower ( < 0.0001). The risk of pup loss was higher for TRIO-OVERLAP than TRIO-NO_OVERLAP ( < 0.0001). A high number of pup losses occurred between birth and the first cage check. Analyses of VIDEO_TRACK data indicated that pups were clearly dead at the start of most of the cannibalism events and infanticide was rare. As most pups die and disappear before the first cage check, many breeding facilities are likely to be unaware of their real rates of mouse pup mortality.

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

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Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified. Furthermore, using our 'HPV16-specific Region Capture Hi-C' technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a 'cancer-causing gene' is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.

The exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease. ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

Inhibition of ferroptosis via selenium supplementation promotes the survival of follicular helper T cells, boosting the germinal center and antibody response following vaccination in mice and people.

Streptococcal pneumonia is a worldwide health problem that kills ∼2 million people each year, particularly young children, the elderly, and immunosuppressed individuals. Alveolar macrophages and neutrophils provide the early innate immune response to clear pneumococcus from infected lungs. However, the level of neutrophil involvement is context dependent, both in humans and in mouse models of the disease, influenced by factors such as bacterial load, age, and coinfections. Here, we show that the G protein-coupled receptor (GPCR) adaptor protein norbin (neurochondrin, NCDN), which was hitherto known as a regulator of neuronal function, is a suppressor of neutrophil-mediated innate immunity. Myeloid norbin deficiency improved the immunity of mice to pneumococcal infection by increasing the involvement of neutrophils in clearing the bacteria, without affecting neutrophil recruitment or causing autoinflammation. It also improved immunity during Escherichia coli-induced septic peritonitis. It increased the responsiveness of neutrophils to a range of stimuli, promoting their ability to kill bacteria in a reactive oxygen species-dependent manner, enhancing degranulation, phagocytosis, and the production of reactive oxygen species and neutrophil extracellular traps, raising the cell surface levels of selected GPCRs, and increasing GPCR-dependent Rac and Erk signaling. The Rac guanine-nucleotide exchange factor Prex1, a known effector of norbin, was dispensable for most of these effects, which suggested that norbin controls additional downstream targets. We identified the Rac guanine-nucleotide exchange factor Vav as one of these effectors. In summary, our study presents the GPCR adaptor protein norbin as an immune suppressor that limits the ability of neutrophils to clear bacterial infections.

Capture Hi-C is widely used to obtain high-resolution profiles of chromosomal interactions involving, at least on one end, regions of interest such as gene promoters. Signal detection in Capture Hi-C data is challenging and cannot be adequately accomplished with tools developed for other chromosome conformation capture methods, including standard Hi-C. Capture Hi-C Analysis of Genomic Organization (CHiCAGO) is a computational pipeline developed specifically for Capture Hi-C analysis. It implements a statistical model accounting for biological and technical background components, as well as bespoke normalization and multiple testing procedures for this data type. Here we provide a step-by-step guide to the CHiCAGO workflow that is aimed at users with basic experience of the command line and R. We also describe more advanced strategies for tuning the key parameters for custom experiments and provide guidance on data preprocessing and downstream analysis using companion tools. In a typical experiment, CHiCAGO takes ~2-3 h to run, although pre- and postprocessing steps may take much longer.

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The spinal cord receives input from peripheral sensory neurons and controls motor output by regulating muscle innervating motor neurons. These functions are carried out by neural circuits comprising molecularly distinct neuronal subtypes generated in a characteristic spatiotemporal arrangement from progenitors in the embryonic neural tube. To gain insight into the diversity and complexity of cells in the developing human neural tube, we used single-cell mRNA sequencing to profile cervical and thoracic regions in four human embryos of Carnegie stages (CS) CS12, CS14, CS17 and CS19 from gestational weeks 4-7. Analysis of progenitor and neuronal populations from the neural tube and dorsal root ganglia identified dozens of distinct cell types and facilitated the reconstruction of the differentiation pathways of specific neuronal subtypes. Comparison with mouse revealed overall similarity of mammalian neural tube development while highlighting some human-specific features. These data provide a catalogue of gene expression and cell type identity in the human neural tube that will support future studies of sensory and motor control systems. The data can be explored at https://shiny.crick.ac.uk/scviewer/neuraltube/.

Selective elimination of damaged mitochondria via macroautophagy (mitophagy) is a conserved cellular process that plays an important role in organismal health. In recent years mitophagy has been studied in parallel to the more general, non-selective autophagy pathway induced in response to amino acid starvation with important similarities and differences noted between the two. The elaborate sequence of membrane rearrangements that give rise to autophagosomes in the non-selective pathway have their counterpart in mitophagy, but with the addition of other factors, such as a ubiquitin mark and mitophagy receptors, which mediate cargo recognition. In some types of mitophagy such as the one induced by ivermectin, the forming autophagosomal structure contains six different elements: the targeted mitochondrial fragment, a section of endoplasmic reticulum that provides a cradle, a ubiquitin layer, the mitophagy receptors and the early and late autophagosomal proteins/membranes. Super-resolution microscopy is ideally suited to investigate the spatial relationships between these elements that converge together but retain some distinctive localization, and we provide here a general protocol that can be used for mammalian cells.