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Mutations in KRAS, particularly at codon 12, are frequent in adenocarcinomas of the colon, lungs and pancreas, driving carcinogenesis by altering cell signalling and reprogramming metabolism. However, the specific mechanisms by which different KRAS G12 alleles initiate distinctive patterns of metabolic reprogramming are unclear. Using isogenic panels of colorectal cell lines harbouring the G12A, G12C, G12D and G12V heterozygous mutations and employing transcriptomics, metabolomics, and extensive biochemical validation, we characterise distinctive features of each allele. We demonstrate that cells harbouring the common G12D and G12V oncogenic mutations significantly alter glutamine metabolism and nitrogen recycling through FOXO1-mediated regulation compared to parental lines. Moreover, with a combination of small molecule inhibitors targeting glutamine and glutamate metabolism, we also identify a common vulnerability that eliminates mutant cells selectively. These results highlight a previously unreported mutant-specific effect of KRAS alleles on metabolism and signalling that could be potentially harnessed for cancer therapy.

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5-null mouse model, which in germinal vesicle (GV) stage oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5-null oocytes, including at some imprinting control regions. This provides evidence for a mechanism of epigenetic impairment in oocytes which could contribute to downstream misregulation of imprinted genes.

Paradoxical activation of wild type RAF by chemical RAF inhibitors (RAFi) is a well-understood 'on-target' biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that requires the translation initiation factor eIF2α. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2α-dependent Integrated Stress Response (ISR). Rather, RAFi exposure activated GCN2, an alternate eIF2α kinase, leading to eIF2α-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2α antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic 'bell-shaped' concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by a GCN2 kinase dead mutation. A M802A GCN2 gatekeeper mutant was activated at lower RAFi concentrations, demonstrating that RAFi bind directly to the GCN2 kinase domain; this is supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation is a previously unappreciated off-target effect that may modulate tumour cell responses.

CD8 T cells target infected or malignant cells via the production of pro-inflammatory cytokines and direct target cell killing. Members of the ZFP36-family of RNA-binding proteins, ZFP36 and ZFP36L1, regulate these functions in T cells via the regulation of mRNA stability and protein translation. We investigate the regulation of ZFP36 and ZFP36L1 expression using in vitro differentiated OT1 TCR transgenic memory-like T cells. We characterise the differential kinetics and sensitivity of ZFP36 and ZFP36L1 to antigen affinity and PMA versus ionomycin stimulation. By selectively inhibiting TCR-induced signalling pathways, we find that p38 MAPK, MEK1/2, and PKC contribute to inducing both ZFP36 and ZFP36L1 expression. By contrast, inhibition of calcineurin using cyclosporin A potently inhibits ZFP36L1 expression while increasing and prolonging ZFP36 expression. The Zfp36 promoter contains many binding sites for the transcription factors ELK-1/4 and few binding sites for NFAT, while the Zfp36l1 promoter contains many NFAT binding sites and few ELK1/4 binding sites. Our findings suggest that the regulation of divergent transcription factors enables calcineurin to act as a signalling node that mediates the differential regulation of ZFP36 and ZFP36L1 during T cell activation.

The germinal center (GC) reaction drives the production of high-affinity antibodies by iterative cycles of B cell somatic hypermutation, selection, and proliferation. How GC B cells undergo rapid cell division while maintaining genome stability is poorly understood. Here, we show that the RNA binding proteins ZFP36L1 and ZFP36L2 act downstream of antigen sensing and protect GC B cells from replication stress by controlling a cell cycle-related posttranscriptional regulon. They safeguard the successful completion of mitosis by balancing CDK1 and p21-mediated regulation of cell-cycle progression. In their absence, GC B cells are prone to arrest in the G-M phase and die by apoptosis, resulting in curtailed GC responses. DNA replication forks stalled at active replication initiation zones, causing replication stress and increased activity of the ATR-CHK1 DNA damage response. Thus, RNA binding proteins guide posttranscriptional gene regulation and maintain a functional G-M checkpoint in GC B cells.

The guanine-nucleotide exchange factor (GEF) P-Rex1 mediates G protein-coupled receptor (GPCR) signaling by activating the small GTPase Rac. We show here that P-Rex1 also controls GPCR trafficking. P-Rex1 inhibits the agonist-stimulated internalization of the GPCR S1PR1 independently of its Rac-GEF activity, through its PDZ, DEP, and inositol polyphosphate 4-phosphatase domains. P-Rex1 also limits the agonist-induced trafficking of CXCR4, PAR4, and GLP1R but does not control steady-state GPCR levels, nor the agonist-induced internalization of the receptor tyrosine kinases PDGFR and EGFR. P-Rex1 blocks the phosphorylation required for GPCR internalization. P-Rex1 binds G protein-coupled receptor kinase 2 (Grk2), both in vitro and in cells, but does not appear to regulate Grk2 activity. We propose that P-Rex1 limits the agonist-induced internalization of GPCRs through its interaction with Grk2 to maintain high levels of active GPCRs at the plasma membrane. Therefore, P-Rex1 plays a dual role in promoting GPCR responses by controlling GPCR trafficking through an adapter function as well as by mediating GPCR signaling through its Rac-GEF activity.

P-Rex1 is a guanine-nucleotide factor for the small GTPase Rac (Rac-GEF) that is known to mediate neutrophil migration and ROS production in response to the activation of GPCRs. These roles of P-Rex1 are assumed to require its activation of Rac.

DNA methylation was the earliest epigenetic mark discovered-it is essential for mammalian development and forms a molecular memory that can transcend generations, as in the phenomenon of genomic imprinting. Set against this long-term potential, methylation is dynamic across the life cycle, with genome-wide changes at germ-cell specification, gametogenesis, and preimplantation development accompanying major shifts in cell potency. With a tool kit of precision genetic reagents, the mouse has been a mainstay in developing mechanistic understanding of how methylation is targeted to the genome and in exploring its susceptibility to environmental factors, such as parental diet. The availability of genome sequence from many more species combined with the ability to profile methylation and other epigenetic marks in very small numbers of cells now provides rich epigenomic information from other mammals. This information has begun to reveal both similarities as well as surprising differences in the way in which methylation is patterned across the genome among mammals. Such knowledge will be critical in assessing the outcomes of interventions during assisted reproduction in human clinical practice and livestock production.

Changes in transcript abundance and isoforms, mediated by epigenetic and post-transcriptional mechanisms, are a hallmark of the development, activation, and effector functions of immune cells. How epigenetic and post-transcriptional processes are orchestrated to regulate transcription and pre-mRNA processing, and their interplay with metabolism, is emerging as important for immunity. DNA and histone modifications recruit RNA-binding proteins (RBPs) to mediate co-transcriptional RNA processing at specific chromatin loci. Simultaneously, RBPs influence the deposition of epigenetic modifications by regulating the expression of chromatin-modifying enzymes and enzymes that control the amounts of metabolites. These are used as substrates by chromatin-modifying enzymes and can influence RBP activity; thus, modulation of metabolic pathways represents a mechanism to regulate the epigenetic landscape and pre-mRNA processing. A body of work identifies emerging regulatory principles that address the interplay between epigenetics and RBPs in the nucleus, and of cytoplasmic post-transcriptional mechanisms that regulate metabolism and epigenetics. In this review, we focus on the interconnections between RBP-mediated processes, chromatin modifications, and metabolic pathways, highlighting the role that such circuits have in T- and B-lymphocytes, and in autoimmunity.

We investigated the roles of Rac guanine-nucleotide exchange factor (Rac-GEF) P-Rex1 in glucose homeostasis using Prex1 and catalytically inactive Prex1 mice. P-Rex1 maintains fasting blood glucose levels and insulin sensitivity through its Rac-GEF activity but limits glucose clearance independently of its catalytic activity, throughout aging. Prex1 mice on a high-fat diet are protected from diabetes. The increased glucose clearance in Prex1 mice may stem in part from constitutively enhanced hepatic glucose uptake. P-Rex1 controls Glut2 surface levels and mitochondrial morphology, membrane potential, and ATP production in hepatocytes, independently of its catalytic activity. The inverse agonist GRA2 showed that P-Rex1 suppresses glucose uptake and mitochondrial ATP production in hepatocytes through the orphan GPCR Gpr21. Cell fractionation showed that P-Rex1 controls Gpr21 trafficking, independently of its catalytic activity. We propose that P-Rex1 limits hepatocyte glucose uptake by retaining Gpr21 at the plasma membrane. These findings delineate new strategies for controlling glucose homeostasis.

Chinese hamster ovary (CHO) cells are the leading mammalian system for recombinant therapeutic protein production. However, optimizing transgene expression remains challenging due to the limited understanding of the regulatory mechanisms controlling gene expression in CHO cells. Towards overcoming this barrier, here we provide a systematic characterization of cis-regulatory elements in CHO cells. Using genome-wide STARR-seq, a high-throughput method for quantifying enhancer strength, we identified regions with enhancer activity in the CHO cell genome. By integrating these data with ATAC-seq and histone modification profiles, we were able to characterize the chromatin state of these regions. Our analysis revealed thousands of newly identified enhancer sequences. The most active sequences could drive transgene expression at levels similar to or higher than strong viral enhancers. Notably, half of the regions found to have enhancer activity were within inaccessible chromatin in their native context. We observed that accessible enhancers were primarily near to transcriptional start sites and associated with ubiquitously-expressed genes, whereas inaccessible enhancers were predominantly intergenic and associated with tissue-specific genes. Additionally, through a deep-learning-based approach ETS and YY1 transcription factor (TF) binding motifs were identified as key determinants of enhancer identity and strength. Disrupting YY1 binding motifs led to reduced enhancer activity, thereby highlighting the importance of YY1 as a transcriptional activator in CHO cells. Our study demonstrates the first comprehensive map of functionally-validated enhancers in CHO cells and generates new insights into gene regulation and the role of TFs in determining enhancer strength. This study helps to lay the foundation for strategic engineering of CHO cell transcriptional networks to achieve enhanced biopharmaceutical production.

Tertiary lymphoid structures (TLSs) arise in non-lymphoid tissues in response to persistent antigen stimulation and chronic inflammation. Spanning organs from lung and liver to meninges, skin, and beyond, TLSs range from loose aggregates of immune cells to fully mature structures containing functional germinal centers (GC). In this review, we provide a comprehensive overview of TLS formation, architecture, and function across diverse tissues, highlighting both shared features and tissue-specific adaptations. We then explore the clinical relevance of TLS in infections, autoimmunity, cancer, and allergy, emphasizing their dual roles in mediating protective immunity and driving pathology. Finally, we discuss emerging technologies that are transforming our ability to dissect TLSs at high resolution (including spatial multi-omics, advanced imaging, and digital pathology), enabling mechanism-guided strategies to modulate TLSs therapeutically. Framing TLSs through the lens of maturation and tissue context provides a foundation for interpreting their clinical associations and for enhancing or dismantling these niches according to need.

In the twenty years since extrafollicular B cell responses were originally described, much has been learned about B cell biology. With this progress, the term "extrafollicular" has expanded beyond its initial use to describe a variety of B cell processes, resulting in ambiguity over the term. Extrafollicular responses are often not identified by location, convoluting the criteria being used to define the pathway. Here, we discuss the current understanding of B cell responses as relevant to the current uses of the term "extrafollicular." In this context, we propose a framework to classify evolving concepts in B cell biology. The use of this framework moving forward is expected to help harmonize and clarify the discussion in the field.

The diversity of antibodies underpins robust immune responses. During the formation of the antibody repertoire in early bone marrow B-cells, random antibody heavy-chain proteins are generated from recombined VH, DH, and JH gene segments. Many are non-functional and are negatively selected. To understand this process in normal mice, we have undertaken an in-depth analysis of heavy-chain selection at this pre-B cell transition. We find independent selection acting on three regions of the complementarity-determining region 3 (CDR3) antigen-binding site, with particularly heavy counter-selection against certain productive VH/JH combinations. This led us to hypothesise that VH-replacement, where the VH gene segment in an existing VDJ combination is replaced, targets productive VDJ rearrangements that code for non-functional heavy chains. We detect VH-replacement recombination products that closely follow the pattern of selection of functional and non-functional VDJ rearrangements. This reveals a physiological role for VH-replacement in the developmental release of B-cells that are stalled by non-functional heavy-chains. This leads to re-modelling of the restricted early VDJ repertoire toward the use of other VH gene segments throughout the IgH locus.

E-cadherin downregulation is an epithelial-mesenchymal transition hallmark canonically attributed to transcriptional repression. Here we delineate a metabolite-driven endocytic route of E-cadherin downregulation in inflammation-associated colorectal cancer (CRC). Specifically, IP kinase-2 (IP6K2), a 5-diphosphoinositol pentakisphosphate (5-IP) synthase upregulated in patients with CRC, is activated via a ROS-Src phosphorylation axis elicited by dextran sulfate sodium (DSS), generating 5-IP around adherens junction (AJ) to promote E-cadherin endocytosis and the transcriptional activities of β-catenin. Mechanistically, 5-IP inhibits inositol 5-phosphatases such as OCRL to promote PI(4,5)P-mediated endocytic adaptor recruitment. Depleting 5-IP or overexpressing a 5-IP binding-deficient OCRL mutant confers resistance to DSS-elicited AJ disruption. Intestinal epithelium-specific IP6K2 deletion attenuates DSS-induced colitis/CRC, whereas an IP6K2 isoform-selective inhibitor protects wild-type but not IP6K2 mice against DSS insult. Thus, 5-IP is an oncometabolite whose stimulus-dependent synthesis relieves a PI(4,5)P dephosphorylation-based endocytic checkpoint, leading to AJ disassembly and protumorigenic β-catenin activation. Targeting IP6K2 could strengthen intestinal epithelial barrier against inflammation and cancer.

Genetic fusion of protein tags is widely used to study protein functions in vivo. It is well known that tag fusion can cause unwanted changes in protein stability, but whether this is an inherent property of the tagged protein, or can be influenced by the cell and tissue environment, is unclear. Using a series of genome edited mouse models, we show that tag-dependent changes in protein expression can vary across different primary cell and tissue contexts. In one case (Ncaph2), a C-terminal auxin-inducible degron fusion strongly increased protein stability in some tissues but decreased it in others. Destabilisation resulted from tissue-specific 'leakage' of the auxin-inducible degron, which depended on TIR1 expression, and occurred selectively in the small intestine where basal concentrations of auxin/ indole-3-acetic acid can reach levels that are sufficient to trigger protein degradation in cultured cells. Stabilisation occurred in post-mitotic cells via an endogenous degradation signal situated at the NCAPH2 C-terminus, which normally undergoes activation upon cell cycle exit, but is inactivated by C-terminal tag fusion. Our results highlight the underappreciated importance of cell and tissue environment in determining the consequences of tag fusions on protein expression, which may be particularly important in animal models that contain diverse cell types.

Influenza remains a significant threat to human and animal health. Assessing serological protection against influenza has relied upon haemagglutinin inhibition (HAI) assays, which are used to gauge existing immune landscapes, seasonal vaccine decisions and in systems vaccinology studies. HAI assays were first described in the 1940s. Here, we adapt our high-throughput live virus microneutralisation (LV-N) assay for SARS-CoV-2, benchmark against HAI assays, and report serological vaccine responsiveness in a cohort of older (> 65 yo) community dwelling adults.

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Profiling combinations of histone modifications identifies gene regulatory elements in different states and discovers features controlling transcriptional and epigenetic programs. However, efforts to map chromatin states in complex, heterogeneous samples are hindered by the lack of methods that can profile multiple histone modifications together with transcriptomes in individual cells. Here, we describe single-cell multitargets and mRNA sequencing (scMTR-seq), a high-throughput method that enables simultaneous profiling of six histone modifications and transcriptome in single cells. We apply scMTR-seq to uncover dynamic and coordinated changes in chromatin states and transcriptomes during human endoderm differentiation. We also use scMTR-seq to produce lineage-resolved chromatin maps and gene regulatory networks in mouse blastocysts, revealing epigenetic asymmetries at gene regulatory regions between the three embryo lineages and identifying Trps1 as a potential repressor in epiblast cells of trophectoderm-associated enhancer networks and their target genes. Together, scMTR-seq enables investigation of combinatorial chromatin landscapes in a broad range of heterogeneous samples, providing insights into epigenetic regulatory systems.

P-Rex2 is a Rac guanine-nucleotide factor (Rac-GEF) that controls glucose homeostasis. This role is thought to be mediated through its adaptor function inhibiting Pten rather than through its Rac-GEF activity, but this remains to be demonstrated. To examine this question, we have investigated the roles of P-Rex2 in glucose homeostasis using Prex2 and catalytically-inactive Prex2 mice. We show that P-Rex2 is required for insulin sensitivity but limits glucose clearance, suppressing glucose uptake into liver and skeletal muscle independently of its catalytic activity. In hepatocytes, P-Rex2 suppresses Glut2 cell surface levels, mitochondrial membrane potential and mitochondrial ATP production. We identify the orphan GPCR Gpr21 as a P-Rex2 target and propose that P-Rex2 limits hepatic glucose clearance by controlling Gpr21 trafficking. In skeletal muscle cells, P-Rex2 suppresses glucose uptake through a separate adaptor function, independently of Gpr21. Additionally, P-Rex2 suppresses insulin secretion by pancreatic islets and plasma insulin levels. Finally, P-Rex2 plays distinct Rac-GEF activity dependent and independent roles in PIP production in liver and skeletal muscle, respectively. Together, our study identifies complex roles of P-Rex2 in glucose homeostasis, mediated through largely GEF-activity independent mechanisms which include the GPCR Gpr21 in hepatocytes and but are not obviously linked to the regulation of Pten.

T follicular helper (T) cells are a helper T-cell subset that is defined by their localisation to B-cell areas of secondary lymphoid tissues, enabling them to provide their B-cell helper function. Precursors of T cells migrate to the B-cell follicles by upregulating CXCR5 and downregulating CCR7, a process that can be blocked by S1PR1 overexpression. T cells and their precursors also express the early activation antigen CD69, which is a negative regulator of S1PR1. In this study, we tested the hypothesis that CD69 expression by T cells is important for their differentiation and localisation after immunization. Genetic deletion of CD69 on T cells and a proportion of their precursors did not alter their formation, nor their ability to support high-affinity B-cell responses. This demonstrates that although CD69 is expressed highly on T cells, it is not necessary for their formation or their B-cell helper functions in lymph nodes (LNs).

The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized the study of gene expression in individual cells, providing unprecedented insights into cellular heterogeneity and developmental processes. The application of scRNA-seq to oocyte biology has facilitated the identification of species-specific transcriptional signatures and developmental trajectories, enhancing our understanding of oogenesis. This chapter presents a detailed protocol for scRNA-seq analysis of growing bovine oocytes.

During gastrulation, mouse epiblast cells form the three germ layers that establish the body plan and initiate organogenesis. While single-cell atlases have advanced our understanding of lineage diversification, spatial aspects of differentiation remain poorly defined. Here, we applied spatial transcriptomics to mouse embryos at embryonic (E) E7.25 and E7.5 days and integrated these data with existing E8.5 spatial and E6.5-E9.5 single-cell RNA-seq atlases. This resulted in a spatiotemporal atlas of over 150,000 cells with 82 refined cell-type annotations. The resource enables exploration of gene expression dynamics across anterior-posterior and dorsal-ventral axes, uncovering spatial logic guiding mesodermal fate decisions in the primitive streak. We also developed a computational pipeline to project additional single-cell datasets into this framework for comparative analysis. Freely accessible through an interactive web portal, this atlas offers a valuable tool for the developmental and stem cell biology communities to investigate mouse embryogenesis in a spatial and temporal context.

Although the regulation of branching morphogenesis by spatially distributed cues is well established, the underlying intracellular signaling mechanisms are not well understood. The development of the lacrimal gland is driven by fibroblast growth factor (FGF) signaling, which activates phospholipase C gamma (PLCγ). Here, we showed that mutating the PLCγ1 binding site on Fgfr2 leads to ectopic branching and hyperplasia in the lacrimal gland, which was phenocopied by either deleting PLCγ1 or disabling any of its SH2 domains. PLCγ1 inactivation did not change the level of Fgfr2 or affect mitogen-activated protein kinase (MAPK) signaling but instead led to sustained AKT phosphorylation due to increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) production. Consistent with this, the PLCγ1 mutant phenotype can be reproduced by the elevation of phosphatidylinositol 3-kinase (PI3K) signaling in Pten knockout and attenuated by blocking AKT signaling. Our findings demonstrate that FGF-activated PLCγ modulates PI3K signaling by shifting phosphoinositide metabolism, revealing the crucial role of PLCγ in branching morphogenesis and organ size control.