Oncogenic KRAS mutations underlie some of the deadliest human cancers. Genetic or pharmacological inactivation of mutant KRAS is not sufficient for long-term control of advanced tumors. Using a conceptual framework of pancreatic ductal adenocarcinoma, we find that CRISPR-mediated ablation of mutant KRAS can terminate tumor progression contingent on the concomitant inactivation of STAT3. STAT3 inactivation is needed to ensure that KRAS-ablated tumor cells lose their malignant identity. Mechanistically, the combined loss of mutant KRAS and STAT3 disrupts a core transcriptional program of cancer cells critical to oncogenic competence. This in turn impairs tumor growth in mice and enhances immune rejection, leading to tumor clearance. We propose that the STAT3 transcriptional program operating in cancer cells enforces their malignant identity, rather than providing classical features of transformation, and shapes cancer persistence following KRAS inactivation. Our findings establish STAT3 as a critical enforcer of oncogenic identity in KRAS-ablated tumors, revealing a key vulnerability that could be exploited for combination therapies. Significance The limited clinical success of KRAS inhibitors points to the need to identify means by which tumor cells maintain stemness and immune evasion. We make an unprecedented finding that the STAT3 transcription factor can sustain tumorigenicity of pancreatic cancer cells following depletion of the KRAS oncogenic driver. The results have important implications for successful therapeutic intervention.

The accumulation of protein aggregates has been causatively linked to the pathogenesis of neurodegenerative diseases. In this study, we have conducted a genome-wide CRISPR-Cas9 screen to identify cellular factors that stimulate the degradation of an aggregation-prone reporter protein. Our findings revealed that genes encoding proteins involved in mitochondrial homeostasis, including the translation factor eIF5A, were highly enriched among suppressors of degradation of an aggregation-prone reporter. Conversely, endoplasmic reticulum (ER)-associated ubiquitin ligases facilitated degradation, indicating opposing roles for these cellular compartments in the clearance of aggregation-prone proteins. Genetic or chemical inhibition of eIF5A led to the dissociation of the aggregation-prone substrate from mitochondria, which was accompanied by enhanced degradation through ER-associated ubiquitination. The presence of an aggregation-prone, amphipathic helix that localized the reporter to mitochondria was crucial for the stimulatory effect of eIF5A inhibition. Additionally, the steady-state levels of α-synuclein, a disease-associated protein containing an amphipathic helix that mislocalizes to mitochondria, were reduced upon eIF5A inhibition. We propose that mitochondria behave as a holdout compartment for aggregation-prone proteins, keeping them out of reach of ubiquitin ligases that target them for proteasomal degradation. Therefore, preventing mitochondrial localization of aggregation-prone proteins may offer a viable therapeutic strategy for reducing their levels in neurodegenerative disorders.

The 19S regulatory particle (RP) associates with the 20S core particle (CP) to form the 26S proteasome, an evolutionarily conserved holoenzyme that plays key roles in both physiological and pathological processes. Proteasome inhibitors that target the catalytic subunits within the 20S have proven to be valuable research tools and therapeutics for various cancers. Herein we report the discovery of rapaprotin, a 26S proteasome assembly inhibitor from our natural product-inspired hybrid macrocycle rapafucin library. Rapaprotin induces apoptosis in both myeloma and leukemia cell lines. Genome-wide CRISPR-Cas9 screen identified a cytosolic enzyme, prolyl endopeptidase (PREP) that is required for the pro-apoptotic activity of rapaprotin. Further mechanistic studies revealed that rapaprotin acts as a molecular transformer, changing from an inactive cyclic form into an active linear form, rapaprotin-L, upon PREP cleavage, to block 26S proteasome activity. Time-resolved cryogenic electron microscopy (cryo-EM) revealed that rapaprotin-L induces dissociation of the 19S RP from the 26S holoenzyme, which was verified in cells. Furthermore, rapaprotin exhibits a marked synergistic effect with FDA-approved proteasome inhibitors and resensitizes drug-resistant multiple myeloma cells from patients to bortezomib. Taken together, these results suggest that rapaprotin is a new chemical tool to probe the dynamics of the 26S proteasome assembly and a promising anticancer drug lead.

Background and aim Developmental disorders caused by activating mutations in the RAS-MAPK pathway account for nearly 20% of hypertrophic cardiomyopathy (HCM) cases in paediatric patients. Compared to sarcomeric HCM, RAS-HCM presents a higher risk of obstruction and hospitalisation. The myosin inhibitor mavacamten has been approved in the European Union for treating adults with obstructive HCM; however, clinical trials have excluded syndromic HCM. Consequently, this study aimed to characterise the functional and energetic disturbances induced by the RASopathy mutation BRAF p.Thr599Arg in cardiomyocytes and to evaluate the effects of mavacamten treatment. Methods Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a CRISPR-induced BRAF T599R mutation and their isogenic control were employed to model RAS-HCM in vitro. The cell size, contractility, and transcriptomic profile were assessed to determine the phenotype of the cardiomyocytes. Energetics were evaluated using the Mito Stress assay, live ATP levels, and NAD(P)H and FAD+ autofluorescence. Results BRAF-mutant cardiomyocytes demonstrated hypertrophy and hypercontractility. Furthermore, energetic profiling revealed increased mitochondrial NAD(P)H and FAD+ pools and an enhanced energetic state in the Mito Stress assay with increased maximal respiratory capacity. However, they also exhibited a significant ATP drop during rapid pacing compared to the control, suggesting that mitochondrial capacity remains insufficient to meet the ATP demand. Mavacamten treatment normalised excessive ATP consumption during acute pacing, suggesting reduced mitochondrial overactivity. Conclusions BRAF-mutant cardiomyocytes recapitulate the characteristics of HCM in vitro. Mavacamten mitigates dysfunctions and restores energetic balance under stress conditions, indicating it holds potential as a therapeutic option for RASopathy-associated HCM.

Somatic alterations, like mutations and copy number changes, driver oncogenesis and cancer progression. Their inhibition has been exploited in the clinic, with several targeted therapies approved for patients with specific mutations or amplifications. However, the response rate of these treatments remains low. The causes are several, ranging from clonal heterogeneity to off target binding. For this reason, CRISPR assays have been developed to study the exact effect of a gene’s deletion. Still, the results from them are puzzling with the same alterations responding different to knockout even in the same cellular context. For this reason, we have developed SAEG, a novel deep learning architecture for somatic alterations in cancer. Our architecture is able to model mutations and copy number alterations and protein-protein interactions to predict if a cell will be susceptible to a gene knockout. SAEG outperforms other models and we show that it learns patterns that can be traced back to the biochemical and biological properties of genes and amino acids. Code Availability https://github.com/Luisiglm/SAEG Contact luis.iglesiarmatinez@ucd.ie

Human genome sequencing efforts in healthy and diseased individuals continue to identify a broad spectrum of genetic variants associated with predisposition, progression, and therapeutic outcomes for diseases like cancer 1–6 . Insights derived from these studies have significant potential to guide clinical diagnoses and treatment decisions; however, the relative importance and functional impact of most genetic variants remain poorly understood. Precision genome editing technologies like base and prime editing can be used to systematically engineer and interrogate diverse types of endogenous genetic variants in their native context 7–9 . We and others have recently developed and applied scalable sensor-based screening approaches to engineer and measure the phenotypes produced by thousands of endogenous mutations in vitro 10–12 . However, the impact of most genetic variants in the physiological in vivo setting, including contextual differences depending on the tissue or microenvironment, remains unexplored. Here, we integrate new cross-species base editing sensor libraries with syngeneic cancer mouse models to develop a multiplexed in vivo platform for systematic functional analysis of endogenous genetic variants in primary and disseminated malignancies. We used this platform to screen 13,840 guide RNAs designed to engineer 7,783 human cancer-associated mutations mapping to 489 endogenous protein-coding genes, allowing us to construct a rich compendium of putative functional interactions between genes, mutations, and physiological contexts. Our findings suggest that the physiological in vivo environment and cellular organotropism are important contextual determinants of specific gene-variant phenotypes. We also show that many mutations and their in vivo effects fail to be detected with standard CRISPR-Cas9 nuclease approaches and often produce discordant phenotypes, potentially due to site-specific amino acid selection- or separation-of-function mechanisms. This versatile platform could be deployed to investigate how genetic variation impacts diverse in vivo phenotypes associated with cancer and other genetic diseases, as well as identify new potential therapeutic avenues to treat human disease.

Large scale application of single-cell and spatial omics in models and patient samples has led to the discovery of many novel gene sets, particularly those from an immunotherapeutic context. However, the biological meaning of those gene sets has been interpreted anecdotally through over-representation analysis against canonical annotation databases of limited complexity, granularity, and accuracy. Rich functional descriptions of individual genes in an immunological context exist in the literature but are not semantically summarized to perform gene set analysis. To overcome this limitation, we constructed immune cell knowledge graphs (ICKGs) by integrating over 24,000 published abstracts from recent literature using large language models (LLMs). ICKGs effectively integrate knowledge across individual, peer-reviewed studies, enabling accurate, verifiable graph-based reasoning. We validated the quality of ICKGs using functional omics data obtained independently from cytokine stimulation, CRISPR gene knock-out, and protein-protein interaction experiments. Using ICKGs, we achieved rich, holistic, and accurate annotation of immunological gene sets, including those that were unannotated by existing approaches and those that are in use for clinical applications. We created an interactive website ( https://kchen-lab.github.io/immune-knowledgegraph.github.io/ ) to perform ICKG-based gene set annotations and visualize the supporting rationale.

The Plasmodium falciparum sodium efflux pump Pf ATP4 is a leading antimalarial target, but suffers from a lack of high-resolution structural information needed to identify functionally important features in conserved regions and guide rational design of next generation inhibitors. Here, we determine a 3.7Å cryoEM structure of Pf ATP4 purified from CRISPR-engineered P. falciparum parasites, revealing a previously unknown, apicomplexan-specific binding partner, Pf ABP, which forms a conserved, likely modulatory interaction with Pf ATP4. The discovery of Pf ABP presents a new avenue for designing novel Pf ATP4 inhibitors.

ABSTRACT Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Polζ) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT-DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.

Background: Gene editing techniques offer new opportunities to improve important traits in aquaculture. The allergenicity of fish flesh is a major problem in aquaculture. Parvalbumin (Parv) is the most prevalent fish allergen. For instance, in salmonids, a single parvalbumin beta-1 protein (parvb1) has been identified as an allergen in specific patients. Therefore, generating trout carrying two parvb1 alleles deleted from the allergenic peptide-encoding region could prevent allergies in these sensitive individuals. Methods Here, we describe the application of the Crispr/cas9 system in an attempt to delete parvb1 exon 2 encoding the allergenic peptide and, alternatively, to replace exon 2 of parvb1 with exon2 of parvalbumin beta-2 protein (parvb2,) which does not encode the allergenic peptide. Exon skipping and swapping were pursued through microhomology-mediated end-joining (MMEJ) knock-In using specifically designed double-stranded donor DNA. Results Genotyping of approximately 200 F0 fingerlings originating from eggs injected with donor DNA designed for exon 2 skipping led to the identification of only one animal carrying an allele lacking exon 2. Genotyping of approximately 150 fingerlings originating from eggs injected with donor DNA for exon 2 swapping did not result in any trout carrying the expected modified allele. Conclusions These preliminary results indicate the potential difficulties associated with the MMEJ KI experiments performed in farmed fish. Finally, new genomic techniques in aquaculture are further discussed in the context of lively debates taking place in the European parliament regarding a possible revision of the current law that determines the legal status of farm animals modified by genome editing. Gene editing, microhomology-mediated end-joining knock-in, parvalbumin, allergenicity, trout, and genetically modified organisms (GMOs).

Loop-extrusion machinery, comprising the cohesin complex and CCCTC-binding factor CTCF, organizes the interphase chromosomes into topologically associating domains (TADs) and loops, but acute depletion of components of this machinery results in variable transcriptional changes in different cell types, highlighting the complex relationship between chromatin organization and gene regulation. Here, we systematically investigated the role of 3D genome architecture in gene regulation in mouse embryonic stem cells under various perturbation conditions. We found that acute depletion of cohesin or CTCF disrupts the formation of TADs, but affects gene regulation in a gene-specific and context-dependent manner. Furthermore, the loop extrusion machinery was dispensable for transcription from most genes in steady state, consistent with prior results, but became critical for a large number of genes during transition of cellular states. Through a genome-wide CRISPR screen, we uncovered multiple factors that can modulate the role of loop extrusion machinery in gene regulation in a gene-specific manner. Among them were the MORF acetyltransferase complex members (Kat6b, Ing5, Brpf1), which could antagonize the transcriptional insulation mediated by CTCF and cohesin complex at developmental genes. Interestingly, inhibition of Kat6b partially rescues the insulator defects in cells lacking the cohesin loader Nipbl, mutations of which are responsible for the developmental disorder Cornelia de Lange syndrome. Taken together, our findings uncovered interplays between the loop extrusion machinery and histone modifying complex that underscore the context-dependent and gene-specific role of the 3D genome.

Summary Genome editing using CRISPR/Cas is a key technology for speeding up breeding for climate-resilient, high-yielding crops (Scheben et al ., 2017). However, efficient targeted mutagenesis requires implementing stable transformation methods and establishing a CRISPR/Cas setup suitable for the species of interest (Shan et al ., 2020). The availability of such methods is a significant bottleneck to advancing many promising, albeit under-researched, crops. Testing and establishing vectors for efficient application of CRISPR/Cas in non-model crops could boost research and breeding of new valuable crops (Ye and Fan, 2021). We edited key pathway genes in the betalain biosynthesis pathway of grain amaranth, i.e., A. hypochondriacus L ., to prove how targeted mutagenesis can be implemented in an orphan crop using the CasCADE modular cloning system (Hoffie, 2022). Grain amaranth is a resilient C 4 dicot orphan crop with excellent nutritional composition. These properties make amaranth a well-suited candidate to be bred as a climate-resilient crop (Joshi et al ., 2018). However, no efficient and reproducible protocol for successful application of CRISPR/Cas9 or stable transformation and regeneration, has been demonstrated in A. hypochondriacus (Castellanos-Arévalo et al ., 2020).

ABSTRACT The axonal membrane-associated periodic skeletal (MPS) consisting of evenly spaced F-actin rings crosslinked by spectrin heterotetramers has been observed in many neuronal subtypes across multiple species. The MPS has been implicated in a diversity of functions ranging from a load-bearing tension buffer to a periodic ruler positioning channels and signalling complexes. However, the initiation and early development of the axonal MPS are poorly understood. Using superresolution imaging of embryonic dorsal root ganglion axons, we show that the early development of the βII-spectrin MPS involves recruitment and stabilisation of the βII-spectrin to the axonal cortex followed by progressive establishment of long-range periodic order. Microtubule dynamics are essential for MPS formation in the early stages, while microtubules have a passive stabilizing function in the mature MPS. We show that the early subplasmalemmal recruitment and confinement of βII-spectrin is dependent on cortical actin. Further, active nucleation of F-actin is required in early development but dispensable for the maintenance of the mature MPS. Finally, using a βII-spectrin knockout model, we demonstrate that the actin-binding and lipid interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. Our study provides insights into the mechanisms governing the development and maturation of the axonal MPS, highlighting the critical role of cytoskeletal dynamics.

SUMMARY Wound infection is a major disruptor of wound healing. Keratinocytes, critical in repair and microbial responses, require the L-arginine hydrolysing enzyme arginase1, for effective healing. Wound pathogens such as Pseudomonas aeruginosa may also need L-arginine. We therefore investigated host-microbial interactions in the context of wound healing and L-arginine metabolism. Arginase-inhibited murine wounds challenged with P. aeruginosa, exhibited significantly delayed re-epithelialisation. This finding was recapitulated in vitro using P. aeruginosa- challenged, arginase1 deficient ( shARG1) keratinocytes, associated with reduced epithelial proliferation and viability, and heightened inflammation. Whilst P. aeruginosa challenge promoted host metabolism of L-arginine, this was perturbed in wounded shARG1 keratinocytes. There was, however, heightened downstream polyamine metabolism in shARG1 cells when under P. aeruginosa challenge. Host keratinocyte arginase1 deficiency promoted bacterial growth in vitro , in line with a failure to upregulate the anti-microbial peptides, β-defensins, in shARG1 scratches. This work demonstrates a pivotal role for keratinocyte arginase1 in wound infection. HIGHLIGHTS Host arginase is required for effective healing under P. aeruginosa challenge. P. aeruginosa enhances host keratinocyte L-arginine metabolism upon scratch. P. aeruginosa promotes polyamine metabolism in arginase1 deficient wounds in vitro . Arginase1 is required for keratinocyte anti-microbial defence against P. aeruginosa .

Interferons (IFN) are cytokines that regulate the expression of hundreds of genes during viral infections to generate a broadly antiviral environment in the stimulated cell. Antiviral breadth is provided by the concurrent expression of many individual IFN-stimulated genes (ISG), each encoding a protein with often exquisite antiviral specificity. Here, we show that mechanistic plasticity at a single genetic locus is a novel mechanism to diversify the antiviral profile of human cells. Through alternative splicing, the OAS2 gene encodes two antiviral molecules with distinct target specificities. The shorter OAS2 p69 isoform blocks the replication of seasonal human coronavirus OC43 (HCoV-OC43), while the longer p71 isoform restricts the replication of picornavirus Cardiovirus A (EMCV). The restriction profile is determined by the variable length OAS2 C-terminal tail. Remarkably, the antiviral mechanisms underlying these distinct antiviral profiles are either RNase L dependent or independent, suggesting that splicing divides ‘classic restriction’ versus ‘virus sensing’ systems across two distinct OAS2 polypeptides. Together, our data reveal that the human OAS2 locus uses alternative splicing and mechanistic plasticity to diversify antiviral profiles.

ABSTRACT To gain insight into biological mechanisms that cause resistance to DNA damage, we performed parallel pooled genetic CRISPR-Cas9 screening for survival in high risk HNSCC subtypes. Surprisingly, and in addition to ATM, DNAPK, and NFKB signaling, JAK1 was identified as a driver of tumor cell radiosensitivity. Knockout of JAK1 in HNSCC increases cell survival by enhancing the DNA damage-induced G2 arrest, and both knockout and JAK1 inhibition with abrocitinib prevent subsequent formation of radiation-induced micronuclei. Loss of JAK1 function does not affect canonical CDK1 signaling but does reduce activation of PLK1 and AURKA, kinases that regulate both G2 and M phase progression. Correspondingly, JAK1 KO was found to cause mitotic defects using both EdU labeling and live cell imaging techniques. Given this insight, we evaluated Kif18a inhibition as an approach to exacerbate mitotic stress and enhance the efficacy of radiation. These studies establish Kif18a inhibition as a novel strategy to counteract therapeutic resistance to DNA damage mediated by G2 cell cycle arrest.

Design: ing drugs that can restore a diseased cell to its healthy state is an emerging approach in systems pharmacology to address medical needs that conventional target-based drug discovery paradigms have failed to meet. Single-cell transcriptomics can comprehensively map the differences between diseased and healthy cellular states, making it a valuable technique for systems pharmacology. However, single-cell omics data is noisy, heterogeneous, scarce, and high-dimensional. As a result, no machine learning methods currently exist to use single-cell omics data to design new drug molecules. We have developed a new deep generative framework named MolGene-E to tackle this challenge. MolGene-E combines two novel models: 1) a cross-modal model that can harmonize and denoise chemical-perturbed bulk and single-cell transcriptomics data, and 2) a contrastive learning-based generative model that can generate new molecules based on the transcriptomics data. MolGene-E consistently outperforms baseline methods in generating high-quality, hit-like molecules from gene expression profiles obtained from single-cell datasets as validated by target knock-out experiments using CRISPR. This superior performance is demonstrated across diverse de novo molecule generation metrics. Extensive evaluations demonstrate that MolGene-E achieves state-of-the-art performance for zero-shot molecular generations. This makes MolGene-E a potentially powerful new tool for drug discovery.

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size (6–54 kDa) and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours ( e.g ., viral and non-viral vectors) or are not applicable in vivo ( e.g ., nucleofection), limiting therapeutic applications. To address these problems, we developed the first protein-based anti-CRISPR delivery platform, LF N -Acr/PA, which delivers Acrs into cells within minutes. LF N -Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that LF N -Acr binds and uses to enter the cell. In the presence of PA, LF N -Acr enters human cells at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LF N -Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LF N -Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

ABSTRACT Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and molecular biochemistry in vivo . Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel expression system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins — from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1 — while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system’s utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal significant, dose-dependent changes in Cas1 mobility following UV-light induced damage, providing evidence for its involvement in DNA repair processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.

Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5′-exoribonuclease RNase J was identified as additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them PNPase. Despite a measured, strong 3’ exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase is a component of the cyanobacterial degradosome that can recruit either RNase E or RNase J, together with additional enriched proteins.

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 GV 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. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID) and hydatidiform moles.

Summary Transitions between subsets of differentiating hematopoietic cells are widely regarded as unidirectional in vivo . Here, we introduce clonal phylogenetic tracer (CP-tracer) that sequentially introduces genetic barcodes, enabling high-resolution analysis of ∼100,000 subclones derived from ∼500 individual hematopoietic stem cells (HSC). This revealed previously uncharacterized HSC functional subsets and identified bidirectional fate transitions between myeloid-biased and lineage-balanced HSC. Contrary to the prevailing view that the more self-renewing My-HSCs unidirectionally transition to balanced-HSCs, phylogenetic tracing revealed durable lineage bidirectionality with the transition favoring My-HSC accumulation over time 1,2 . Further, balanced-HSCs mature through distinct intermediates—My-HSCs and lymphoid-biased-HSCs—with lymphoid competence here shown by CRISPR/Cas9 screening to be dependent on the homeobox gene, Hhex . Hhex enables Ly-HSC differentiation, but its expression declines with age. These findings establish HSC plasticity and Hhex as a determinant of myeloid-lymphoid balance with each changing over time to favor the age-related myeloid bias of the elderly. Highlights Sequenctial introduction of DNA barcodes in vivo was developed to assess time dependent changes in cell fate. Clonal phylogenetic tracer (CP-tracer) enabled high-resolution phylogenetic analysis of ∼100,000 subclones derived from ∼500 individual hematopoietic stem cells (HSC). Bidirectional fate transitions between myeloid-biased haematopoietic stem cells (My-HSCs) and lineage-balanced haematopoietic stem cells (balanced-HSCs) were observed. Hhex was identified as a molecular driver of HSC lymphoid competence.

ABSTRACT The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) regulates cell differentiation, proliferation, and function in various tissues, including the liver, adipose tissue, skin, lung, and hematopoietic system. Studies in rats, mice, humans, and chickens have shown that CEBPA mRNA undergoes alternative translation initiation, producing three C/EBPα protein isoforms. Two of these isoforms act as full-length transcription factors with N-terminal transactivation domains and a C-terminal dimerization and DNA-binding domains. The third isoform is an N-terminally truncated variant, translated from a downstream AUG codon. It competes with full-length isoforms for DNA binding, thereby antagonizing their activity. Expression of the truncated C/EBPα isoform depends on the initial translation of a short upstream open reading frame (uORF) in CEBPA mRNA and subsequent re-initiation at a downstream AUG codon, a process stimulated by mTORC1 signaling. We investigated whether the ortholog of the CEBPA gene in the evolutionarily distant, short-lived African turquoise killifish ( Nothobranchius furzeri ) is regulated by similar mechanisms. Our findings reveal that the uORF- mediated regulation of C/EBPα isoform expression is conserved in killifish. Disruption of the uORF selectively eliminates the truncated isoform, leading to unrestrained activity of the full-length C/EBPα isoforms. This genetic modification significantly extended both the median and maximal lifespan and improved the healthspan of male N. furzeri . These results highlight a conserved mechanism of CEBPA gene regulation across species and its potential role in modulating the lifespan and aging phenotypes.

SUMMARY Biallelic loss-of-function variants in the adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans -Golgi network, including the autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the ‘druggable genome’, in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans -Golgi network, suggesting they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

The chromatin regulator MLL2 (KMT2B) is the primary histone 3 lysine 4 (H3K4) trimethyltransferase acting at bivalent promoters in embryonic stem cells (ESCs) and is required for differentiation toward neuroectoderm. Here, we demonstrate that this requirement occurs during exit from naïve pluripotency, days before neuroectoderm differentiation is impaired. During exit, the effect of MLL2 on transcription is subtle, increasing the expression of a few important neuroectodermal transcription factors. In contrast, MLL2’s effect on chromatin architecture is substantial, stabilising loops associated with bivalent promoters in primed ESCs. MLL2 H3K4 catalytic activity is dispensable for stabilising these loops during ESC exit and for neuroectoderm differentiation. We therefore identify a non-catalytic function for MLL2 in stabilising 3D chromatin architecture, which has implications for lineage specification. Because MLL2 shares features with all four MLLs, we propose that chromatin tethering, rather than H3K4 methylation, represents a primary function for MLLs during lineage commitment decisions.