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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.

Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving -targeting microRNAs. Additional PTEN loss in double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

Regulatory T cells (T) are indispensable for the control of immune homeostasis and have clinical potential as a cell therapy for treating autoimmunity. T can lose expression of the lineage-defining Foxp3 transcription factor and acquire effector T cell (T) characteristics, a process referred to as T plasticity. The extent and reversibility of such plasticity during immune responses remain unknown. Here, using a murine genetic fate-mapping system, we show that T stability is maintained even during exposure to a complex microbial/antigenic environment. Furthermore, we demonstrate that the observed plasticity of T after adoptive transfer into a lymphopenic environment is a property limited to only a subset of the T population, with the nonconverting majority of T being resistant to plasticity upon secondary stability challenge. The unstable T fraction is a complex mixture of phenotypically distinct T, enriched for naïve and neuropilin-1-negative T, and includes peripherally induced T and recent thymic emigrant T These results suggest that a "purging" process can be used to purify stable T that are capable of robust fate retention, with potential implications for improving cell transfer therapy.

Cells must adapt to changes in their environment to maintain cell, tissue and organismal integrity in the face of mechanical, chemical or microbiological stress. Nuclear factor-κB (NF-κB) is one of the most important transcription factors that controls inducible gene expression as cells attempt to restore homeostasis. It plays critical roles in the immune system, from acute inflammation to the development of secondary lymphoid organs, and also has roles in cell survival, proliferation and differentiation. Given its role in such critical processes, NF-κB signalling must be subject to strict spatiotemporal control to ensure measured and context-specific cellular responses. Indeed, deregulation of NF-κB signalling can result in debilitating and even lethal inflammation and also underpins some forms of cancer. In this review, we describe the homeostatic feedback mechanisms that limit and 're-set' inducible activation of NF-κB. We first describe the key components of the signalling pathways leading to activation of NF-κB, including the prominent role of protein phosphorylation and protein ubiquitylation, before briefly introducing the key features of feedback control mechanisms. We then describe the array of negative feedback loops targeting different components of the NF-κB signalling cascade including controls at the receptor level, post-receptor signalosome complexes, direct regulation of the critical 'inhibitor of κB kinases' (IKKs) and inhibitory feedforward regulation of NF-κB-dependent transcriptional responses. We also review post-transcriptional feedback controls affecting RNA stability and translation. Finally, we describe the deregulation of these feedback controls in human disease and consider how feedback may be a challenge to the efficacy of inhibitors.

Endoplasmic reticulum (ER) stress promotes placental dysmorphogenesis and is associated with poor pregnancy outcomes. We show that unfolded protein response signalling pathways located in the ER drive differentiation of mouse trophoblast stem cells into trophoblast subtypes involved in development of the placental labyrinth zone and trophoblast invasion. In a mouse model of chronic ER stress (Eif2s1 ), higher ER stress in homozygous blastocysts is accompanied by reduced trophectoderm cell number, developmental delay, and is associated with an increased incidence of early pregnancy loss. Administration of the chemical chaperone, tauroursodeoxycholic acid, to Eif2s1 heterozygous females during pregnancy alleviated ER stress in the mutant placenta, restored normal trophoblast populations and reduced the frequency of early pregnancy loss. Our results suggest that alleviation of intrauterine ER stress could provide a potential therapeutic target to improve pregnancy outcome in women with pre-gestational metabolic or gynaecologic conditions. ABSTRACT: Women with pre-gestational health conditions (e.g., obesity, diabetes) or gynaecological problems (e.g., endometriosis) are at increased risk of adverse pregnancy outcomes including miscarriage, preeclampsia and fetal growth restriction. Increasing evidence suggests that unfavourable intrauterine conditions leading to poor implantation and/or defective placentation are a possible causative factor. The endoplasmic reticulum (ER) unfolded protein response (UPR ) signalling pathways are a convergence point of various physiological stress stimuli that can be triggered by an unfavourable intrauterine environment. Therefore, we explored the impact of ER stress on mouse trophoblast differentiation in vitro, mouse blastocyst formation and early placenta development in the Eif2s1 mutant mouse model of chronic ER stress. Chemically-manipulated ER stress or activation of UPR pathways in a mouse trophoblast stem cell line promoted lineage-specific differentiation. Co-treatment with specific UPR pathway inhibitors rescued this effect. While the inner cell mass was unaffected, the trophectoderm of homozygous Eif2s1 blastocysts exhibited ER stress associated with a reduced cell number. Furthermore, one-third of Eif2s1 homozygous blastocysts exhibited severe developmental defects. We have previously reported a reduced trophoblast population and premature trophoblast differentiation in Eif2s1 homozygous placentas at mid-gestation. Here, we demonstrate that treatment of Eif2s1 heterozygous pregnant females with the chemical chaperone tauroursodeoxycholic acid alleviated ER stress, restored the trophoblast population, and reduced the frequency of embryonic lethality. Our data suggest that therapeutic targeting of ER stress may improve pregnancy outcome in women with pre-gestational metabolic or gynaecologic conditions. This article is protected by copyright. All rights reserved.

Recombination activating genes (RAGs), consisting of RAG1 and RAG2, are stringently regulated lymphoid-specific genes, which initiate V(D)J recombination in developing lymphocytes. We report the regulation of RAG1 through a microRNA (miRNA), miR-29c, in a B cell stage-specific manner in mice and humans. Various lines of experimentation, including CRISPR-Cas9 genome editing, demonstrate the target specificity and direct interaction of miR-29c to RAG1. Modulation of miR-29c levels leads to change in V(D)J recombination efficiency in pre-B cells. The miR-29c expression is inversely proportional to RAG1 in a B cell developmental stage-specific manner, and miR-29c null mice exhibit a reduction in mature B cells. A negative correlation of miR-29c and RAG1 levels is also observed in leukemia patients, suggesting the potential use of miR-29c as a biomarker and a therapeutic target. Thus, our results reveal the role of miRNA in the regulation of RAG1 and its relevance in cancer.