The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. PH domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly discovered that PHLPP2 has no catalytic activity against the regulatory phosphorylation sites of Akt, nor the generic substrate para -nitrophenylphosphate. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that current cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1000 Mya, but that lost activity at the base of the metazoan lineage. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a new focus on non-catalytic roles of PHLPP1 and PHLPP2. Significance Statement PHLPP1 and PHLPP2 have previously been reported as protein phosphatases that specifically inactivate Akt, a pro-growth and survival kinase hyperactivated in many human cancers. Unexpectedly, we found that purified PHLPP2 has no detectable enzymatic activity in vitro, an observation which can be rationalized by its unusual active site, which has diverged significantly from that of canonical metal-dependent phosphatases. Furthermore, we show that cancer genomics do not support a role for either PHLPP1 or PHLPP2 in cancer. Our findings argue for the exploration of alternative hypotheses regarding the role of PHLPP in Akt signaling and cancer, with a focus on its non-catalytic functions.

Background Pericytes are crucial for the development, stabilization, and functional regulation of microvasculature, especially in the retina. In diabetic retinopathy (DR), early loss of pericytes is a key event that drives microvascular dysfunction. Despite their critical role, the mechanisms underlying the functional heterogeneity of pericytes in DR remain poorly understood, impeding the development of effective therapeutic strategies. Methods We employed single-cell RNA sequencing to construct a comprehensive single- cell atlas of non-diabetic and diabetic retinas. Using bioinformatic clustering and subcluster analysis, we identified a specific pericyte subcluster associated with diabetic microvascular complications. Differential gene expression analysis and immunofluorescence validation highlighted PTTG1 as a potential key regulator of pericyte dysfunction. To investigate its functional role, we emplyed CRISPR/Cas9 and adenoviral vectors to modulate PTTG1 expression in vitro and in vivo. Combined transcriptomic and metabolomic approaches were used to explore the mechanistic pathways through which PTTG1 influences pericyte biology and vascular function. Results We identified a novel pericyte subcluster characterized by elevated expression of PTTG1, which was strongly correlated with diabetic microvascular dysfunction. Silencing PTTG1 using CRISPR/Cas9 and siRNA in vitro mitigated pericyte dysfunction under high- glucose conditions. Targeted knockdown of PTTG1 using viral vectors improved retinal vascular integrity and reduced neovascularization in diabetic mice. Transcriptomic and untargeted metabolomic analyses revealed that PTTG1 knockdown reprogrammed pericyte energy metabolism by modulating glycolysis pathway genes, reducing oxidative stress, and restoring pericyte function, ultimately alleviating microvascular dysfunction in DR. Conclusions PTTG1 plays a critical role in regulating pericyte dysfunction and maintaining vascular homeostasis in diabetic retinopathy. By modulating key metabolic pathways and pericyte phenotypes, PTTG1 represents a promising therapeutic target for treating diabetic microvascular complications. These insights offer a novel molecular framework for developing targeted therapies aimed at restoring retinal vascular health in diabetic patients.

The nucleobase queuine (q) and its nucleoside queuosine (Q) are micronutrients derived from bacteria that are acquired from the gut microbiome and/or diet in humans. Following cellular uptake, Q is incorporated at the wobble base (position 34) of tRNAs with a GUN anticodon, which is important for efficient translation. Early studies suggested that cytosolic uptake of queuine is mediated by a selective transporter that is regulated by mitogenic signals, but the identity of this transporter has remained elusive. Here, through a cross-species bioinformatic search and genetic validation, we have identified the solute carrier family member SLC35F2 as a unique transporter for both queuine and queuosine in Schizosaccharomyces pombe and Trypanosoma brucei . Furthermore, gene disruption in HeLa cells revealed that SLC35F2 is the sole transporter for queuosine in HeLa cells (K m 174 nM) and a high-affinity transporter for the queuine nucleobase (K m 67 nM), with the presence of another low-affinity transporter (K m 259 nM) in these cells. Competition uptake studies show that SLC35F2 is not a general transporter for other canonical ribonucleobases or ribonucleosides, but selectively imports q and Q. The identification of SLC35F2, an oncogene, as the transporter of both q and Q advances our understanding of how intracellular levels of queuine and queuosine are regulated and how their deficiency contributes to a variety of pathophysiological conditions, including neurological disorders and cancer. Significance Statement The discovery of SLC35F2 as the eukaryotic transporter of queuine and queuosine is key to understanding how these micronutrients are salvaged from the human gut and distributed to different body tissues. Queuosine modification of tRNAs enhances the accuracy and efficiency of codon-anticodon pairing and regulates a range of biological and pathophysiological states, including oxidative stress responses, cancer, learning, memory, and gut homeostasis.

The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/ Cas 9 gRNAs and donor templates via a deno- a ssociated v irus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.

Abstract Background ZWILCH has been reported to prevent cells from prematurely exiting mitosis. However, the underlying mechanisms or involvement of ZWILCH in the tumor immune microenvironment in various cancers remain largely unknown. Methods Generalized dysregulation of ZWILCH was observed through the whole transcriptome analysis in this study. The spatial transcriptome analysis was utilized to identify expressed regions of ZWILCH. Next, cells that mainly expressed ZWILCH in the tumor microenvironment were determined using the single-cell transcriptome analysis. Also, the “cellchat” R package was applied to estimate the effect of ZWILCH on malignant cell communication. Combining multiple analytic approaches including GSEA, GSVA, KEGG enrichment analysis, and Aucell, with TCPA functional protein data, Genome-wide CRISPR screening, potential functions of ZWILCH and the pathways in which ZWILCH participated were thoroughly exploited. Univariate Cox regression analysis calculated the association between ZWILCH and cancer patients’ adverse outcomes. Results ZWILCH is universally highly expressed in tumors. The spatial transcriptome analysis showed that ZWILCH overexpression comes from the tumoral region or mixed tumoral region. At the single-cell level, ZWILCH is chiefly expressed by malignant cells and proliferative T cells. The expression of ZWILCH mRNA is positively correlated with cell proliferation, repair of DNA damage, and cell cycle score. Plenty of metabolic pathways are inhibited in patients with high expression of ZWILCH. Moreover, after ZWILCH knockout, a large number of cancer cell lines are stagnated, inhibited, or died. Additionally, the malignant cells with positive expression of ZWILCH have a stronger ability for cell communication. In short, ZWILCH is meant to be a risk factor for clinical outcomes of multiple tumors. Conclusions ZWILCH is a promising therapeutic target that influences patient prognosis by participating in cell proliferation, enhancing cell communication, and reshaping the tumor microenvironment across different cancers.

Elevated cholesterol poses cardiovascular risks. The glucocorticoid receptor (GR) harbors a still undefined role in cholesterol regulation. Here, we report that a coding single nucleotide polymorphism (SNP) in the gene encoding the GR, rs6190 , associated with increased cholesterol in women according to UK Biobank and All Of Us datasets. In SNP-genocopying mice, we found that the SNP enhanced hepatic GR activity to transactivate Pcsk9 and Bhlhe40 , negative regulators of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) receptors respectively. In mice, the SNP was sufficient to elevate circulating cholesterol across all lipoprotein fractions and the risk and severity of atherosclerotic lesions on the pro-atherogenic hAPOE*2/*2 background. The SNP effect on atherosclerosis was blocked by in vivo liver knockdown of Pcsk9 and Bhlhe40 . Also, corticosterone and tes-tosterone were protective against the mutant GR program in cholesterol and atherosclerosis in male mice, while the SNP effect was additive to estrogen loss in females. Remarkably, we found that the mutant GR program was conserved in human hepatocyte-like cells using CRISPR-engineered, SNP-genocopying human induced pluripotent stem cells (hiPSCs). Taken together, our study leverages a non-rare human variant to uncover a novel GR-dependent mechanism contributing to atherogenic risk, particularly in women. Graphical Abstract

Abstract Pigment cells in fish species play crucial roles in forming colour patterns of each species and other physiological characteristics including photoprotection. Research on photoprotection by pigment cells in animals has primarily concentrated on black pigment cells, known as melanophores. However, the roles of other pigment cells and their synergistic effects on UV protection remain poorly understood. In this study, we use the Arabian killifish embryos as a model for studying the mechanisms of UV protection by different pigment cells. This species features highly fluorescent pigment cells called fluoroleucophores and black pigment cells known as melanophores. The fluorescent pigments and black melanin pigments are generated by genes gch (GTP cyclohydrolase) and tyr (tyrosinase) respectively. We generated gch(-/-) and gch/tyr(-/-) double mutant lines using CRISPR/Cas9 genome editing and examined the UV sensitivity of these mutant embryos. Both morphology and gene expression data revealed that the gch/tyr(-/-) double mutant line exhibited the highest UV sensitivity, and the gch(-/-) line also demonstrated a greater stress response compared to wild type (WT). From the study, we have identified the synergistic role of black and fluorescent pigment cells in providing effective UV protection from the early stages of embryonic development.

Summary Enhancers play a critical role in regulating transcription. Nearly 90% of human genetic variants identified in genome-wide association studies (GWAS) are located in distal regions, underscoring the importance of enhancers in human development, diseases, and traits. It is widely suggested that mammalian enhancers frequently skip active genes, and thus, linear proximity is a poor predictor of their targets. A key unresolved question is how often mammalian enhancers skip proximal active genes to specifically target distal genes. Genome-wide enhancer-promoter mapping shows that enhancers frequently bypass active genes, while ultra-deep locus-specific analyses reveal extensive multi-way interactions between enhancers and promoters, forming nested microcompartments. The functional significance of these seemingly contrasting phenomena remains unclear. Here, we compared hundreds of enhancer-target gene pairs identified using enhancer-promoter chromatin contact maps, enhancer-promoter RNA interaction data, and genome-scale CRISPR interference (CRISPRi) perturbations. Our findings reveal limited overlap between active gene-skipping enhancer-gene pairs identified through physical interaction mapping and CRISPRi. Additionally, promoters involved in multi-way enhancer interactions are not co-regulated by shared coactivators. Notably, gene-skipping and non-skipping enhancers identified via CRISPRi differ fundamentally in chromatin features, gene activation strength, false discovery rates, target gene distance, coactivator requirements, and cell-type specificity of target genes. These results suggest that gene-skipping enhancer-promoter interactions observed in chromatin and RNA-based analyses do not reliably predict functional enhancer-gene relationships. We propose that linear enhancer-promoter proximity and coactivator dependency offer a simple, scalable, and cost-effective method for genome-wide prediction of enhancer and GWAS targets, with accuracy comparable to state-of-the-art experimental techniques. While enhancers can skip active genes, such deliberate skipping appears to be the exception rather than the rule.

Summary TET1, TET2 and TET3 are DNA demethylases with critical roles in development and differentiation. To assess the contributions of TET proteins to cell function during early development, single and compound knockouts of Tet genes in mouse pluripotent embryonic stem cells (ESCs) were generated. Here we show that TET proteins are not required to transit between naïve, formative and primed pluripotency. Moreover, ESCs with double-knockouts of Tet1 and Tet2 or triple-knockouts of Tet1, Tet2 and Tet3 are phenotypically indistinguishable. These TET-deficient ESCs exhibit differentiate defects; they fail to activate somatic gene expression and retain expression of pluripotency transcription factors. Therefore, TET1 and TET2, but not TET3 act redundantly to facilitate somatic differentiation. Importantly however, TET-deficient ESCs can differentiate into primordial germ cell-like cells (PGCLCs), and do so at high efficiency in the presence or absence of PGC-promoting cytokines. Moreover, acquisition of a PGCLC transcriptional programme occurs more rapidly in TET-deficient cells. These results establish that TET proteins act at the juncture between somatic and germline fates: without TET proteins, epiblast cell differentiation defaults to the germline.

In healthy cells, AXIN1 organizes assembly of a large destruction complex that mediates proteolysis of the transcriptional co-activator β-catenin to prevent inappropriate Wnt/β-catenin pathway activation. In hepatocellular carcinoma (HCC), AXIN1 mutations (11%) associate with a poor-prognosis subtype that is molecularly distinct from β-catenin-mutant HCC (28-40%). How AXIN1 deficiency drives HCC formation has remained highly debated. Here, we address this issue by introducing HCC-associated AXIN1 and CTNNB1 mutations in human liver cancer cells and liver-derived organoids. We show that different mutant AXIN1 classes activate varying degrees of Wnt signaling, although at lower overall levels than CTNNB1 mutations. Strikingly, premature stop codons in 5’ coding regions do not classify as knock-out mutations but drive alternative translation of an N-terminally truncated AXIN1 variant with partially retained suppressor activity. All AXIN1 variants endow liver progenitor organoids with the capacity to grow in the absence of R-spondin and Wnt, indicative of aberrant Wnt/β-catenin pathway activation. Additionally, induced Wnt/β-catenin pathway activation inversely correlates with YAP/TAZ-mediated signaling, thus leaving higher residual YAP/TAZ activity in AXIN1 -mutant versus CTNNB1 -mutant cells. We conclude that AXIN1 mutations drive physiologically relevant Wnt/β-catenin signaling in HCC, while providing a permissive environment for YAP/YAZ signaling, thereby distinguishing AXIN1 mutations from those in CTNNB1 .

Enhancers are discrete DNA elements that regulate the expression of eukaryotic genes. They are important not only for their regulatory function, but also as loci that are frequently associated with disease traits. Despite their significance, our conceptual understanding of how enhancers work remains limited. CRISPR-interference methods have recently provided the means to systematically screen for enhancers in cell culture, from which a formula for predicting whether an enhancer regulates a gene, the Activity-by-Contact (ABC) Score, has emerged and has been widely adopted. While useful as a binary classifier, it is less effective at predicting the quantitative effect of an enhancer on gene expression. It is also unclear how the algebraic form of the ABC Score arises from the underlying molecular mechanisms and what assumptions are needed for it to hold. Here, we use the graph-theoretic linear framework, previously introduced to analyze gene regulation, to formulate the default model , a mathematical model of how multiple enhancers independently regulate a gene. We show that the algebraic form of the ABC Score arises from this model. However, the default model assumptions also imply that enhancers act additively on steady-state gene expression. This is known to be false for certain genes and we show how modifying the assumptions can accommodate this discrepancy. Overall, our approach lays a rigorous, biophysical foundation for future studies of enhancer-gene regulation.

Genetic modifications using CRISPR-Cas9 have revolutionized cancer research and other pre-clinical studies. Exceptionally, these efficient tools are inadequate in a few disease models and cell lines due to the aberrant differentiation states and the accumulation of excessive somatic mutations that compromise the robustness of viral gene delivery and stable transduction. A couple of B lymphoma cell lines fall into this category where lentiviral transfection becomes inefficient and exhibits variable efficiency. Additionally, lentiviral delivery requires high biosafety levels. To address this challenge, we have developed a two-step strategy that supports CRISPR-Cas9 through lentivirus and murine ecotropic γ-retrovirus. By engineering B lymphoma cell lines to express Cas9 and mCat-1, a specific receptor for ecotropic retroviruses, we enable efficient and safe gene editing through ecotropic γ-retrovirus. We demonstrate the efficacy of this method by generating IgM-deficient B lymphoma cell lines. This innovative approach simplifies protocols, enhances accessibility, and paves the way for standardized gene manipulation of B cell lymphoma models for molecular cell biology research.

ABSTRACT Invasive candidiasis affects 1.6 million people annually, implicating high mortality and morbidity in immunocompromised and hospitalized patients. Echinocandins, inhibitors of β-1,3-glucan synthesis, are used as a first-line treatment; however, their efficacy is increasingly compromised by resistance and tolerance. To understand how echinocandins remodel Candida cell wall structures, thereby reducing drug effectiveness, this study compares the effects of echinocandin exposure on the cell walls of the prevalent pathogen Candida albicans and the recently emerged multidrug-resistant superbug Candida auris . High-resolution solid-state NMR analysis revealed a conserved cell wall structure in both species, with a rigid inner layer composed of closely associated chitin microfibrils and β-1,3-glucans, supported by a flexible network of β-1,6-glucans and additional β-1,3-glucans. Despite the presence of N -mannan fibrils in the outer layer, mannan components are mobile and rely on α-1,2-linked mannoside sidechains to maintain contact with chitin and β-1,3-glucans. Caspofungin treatment rigidifies certain mannan sidechains and β-1, 6-glucans to reinforce the cell wall in response to the depletion of most β-1,3-glucans. While caspofungin treatment reduced water permeability in both species, only C. albicans responded by inducing cell wall thickening and changes in chitin and β-1,3-glucan dynamics. Furthermore, the deletion of KRE6 genes encoding β-1,6-glucan synthase reduced the echinocandin susceptibility of C. auris, and the impaired β-1,6-glucan biosynthesis were offset by compensatory upregulation of this wall component due to caspofungin treatment. The profound alterations induced by caspofungin in Candida cell wall architecture suggest that cell wall structural contribute substantially to drug resistance and tolerance.

Abstract Adaptive thermogenesis in brown adipose tissue is regulated by PPARGC1A (PGC-1α), which could be affected by common polymorphism (rs8192678, Gly482Ser) that associates with metabolic disorders. We used CRISPR-Cas9 in immortalized human brown adipocyte cell line to edit the rs8192678 locus to evaluate the allele-dependent effects on cold response of brown adipocytes. In both 482Ser- and 482Gly-edited brown adipocytes, UCP1 expression increased after 6-hour cold exposure, but the increase was less pronounced in 482Gly cells, which also had lower mitochondrial activity. Furthermore, 482Gly PGC-1α had an almost two-fold longer half-life at 37°C, and four-fold longer half-life at 32°C, compared with the 482Ser variant. Despite the longer half-life, the 482Gly PGC-1α had lower activity, and was more prone to be retained in the cytoplasm after cold exposure. Our experimental data support gene x environment interactions between Gly482Ser and cold exposure in human brown adipocytes. These data support a novel molecular basis for the epidemiological observations that link the Gly482Ser polymorphism with obesity and related metabolic disorders.

With the advent of a variety of vaccines against viral infections, there are multiple viruses that can be prevented via vaccination. However, breakthrough infection or uncovered strains can still cause vaccine-preventable viral infections (VPVI). Therefore, timely diagnosis, treatment and surveillance of these viruses is critical to patient care and public health. Point-of-care (POC) viral diagnostics tools have brought significant improvements in the detection and management of VPVI. These cutting-edge technologies enable prompt, accurate results, enhancing patient care by facilitating timely treatment decisions. This review delves into the advancements in POC testing, including antigen/antibody detection and molecular assays, while focusing on their impact on diagnosis, treatment and surveillance of VPVI such as mpox, viral hepatitis, influenza, flaviviruses (Dengue, Zika and Yellow fever virus), and COVID-19. The role of POC tests in monitoring viral infection is crucial for tracking disease progression and managing outbreaks. Furthermore, the application of POC diagnostics has shown to be vital for public health strategies. In this review, we also highlight emerging POC technologies like CRISPR-based diagnostics and smartphone-integrated POC devices, which have proven particularly beneficial in resource-limited settings. We underscore the importance of continued research to optimize these diagnostic tools for wider global use for mpox, viral hepatitis, influenza, flaviviruses, and COVID-19, while also addressing current challenges related to their sensitivity, specificity, availability, efficiency, and others.

The possibility of CRISPR/Cas gene editing system regulation is an important point for creation of effective molecular biological instrument. Allosteric regulation at the level of guide RNAs is one of possible approach to regulate gene editing system. In this work, we designed chimeric guide RNAs (sgRNA, crRNA, and tracrRNA) containing an aptamer to theophylline in their structure in order to create gene editing systems whose activity can be regulated (increased or decreased) allosterically. Two approaches were used to design the guide RNAs. In the first variant, aptamer sequence was a part of the main elements of guide RNAs. In the second variant, the aptamer sequence was added to the 3'- or 5'-end of the guide RNAs. Changes in Cas9 nuclease activity in the presence of constructed chimeric guide RNAs and the effect of theophylline on the efficiency of model DNA cleavage were studied. Several promising candidates for the role of components of the theophylline-activated and deactivated CRISPR/Cas9 system have been discovered.

Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a significant challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the system’s versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents a significant advancement in the feasibility and scalability of gene-editing delivery technologies.

Podocyte dysfunction is central to various glomerular diseases, necessitating reliable biomarkers for early detection and diagnosis. This study investigates the regulatory mechanisms of membrane-associated guanylate kinase inverted 2 (MAGI2) and its potential as a biomarker for podocytopathies. The expression of the gene coding for the scaffolding protein MAGI2 was examined across four species and demonstrated to be conserved within the podocyte filtration slit. In vitro and in vivo studies using isolated glomeruli and mammalian animal models of glomerular disease, including DOCA-salt hypertension, nephrotoxic serum nephritis, and puromycin aminonucleoside nephropathy, demonstrated significant downregulation of MAGI2 in injured podocytes. This downregulation was also conserved in a zebrafish model of focal and segmental glomerulosclerosis (FSGS), and the podocyte-specific MAGI2 ortholog Magi2a was reduced post podocyte injury. CRISPR/Cas9-generated zebrafish mutants for magi2a exhibited marked glomerular filtration barrier defects and downregulation of nephrin, underscoring MAGI2’s critical role in podocyte function. Human biopsy analyses revealed differential MAGI2 expression: it was increased in minimal change disease (MCD) patients but significantly decreased in primary, but not secondary FSGS cases. As MAGI2 localization did not change in disease states it is an alternative marker for super-resolution microscopy-based morphometry of the filtration slit, correlating with nephrin-based measurements. These findings highlight the potential of MAGI2 as a sensitive biomarker for podocyte injury and its diagnostic utility in differentiating between primary FSGS and MCD.

Missing heritability in hereditary diffuse gastric cancer (HDGC) ranges from 60 to 90%. These HDGC-like families, despite complying with HDGC clinical criteria, lack CDH1 and CTNNA1 actionable germline variants, and are not offered HDGC-targeted life-saving disease prevention measures. Herein, we explored novel HDGC predisposition mechanisms affecting the CDH1 -regulatory network. We called single-nucleotide (SNV) and copy-number variants (CNV) from 19 HDGC-like probands from whole-genome sequencing data and performed gene-ontology analysis. Chromatin enhancer marks and CDH1 promoter interactions were evaluated in normal stomach by ChIP-seq, ATAC-seq and 4C-seq, variant causality was assessed by RT-PCR, immunohistochemistry and microsatellite instability (MSI) analysis in tumours. Functional analysis was performed using CRISPR-Cas9, RT-PCR and flow cytometry in cell lines, and enhancer assays using mouse embryos. Within the CDH1 topologically associating domain (TAD), we found two deletions in Family F4 and F9. F4 carried a heterozygous CDH3 20kb-CNV triggering CDH1 mRNA/protein loss in homozygosity by CRISPR-Cas9 editing, similarly to a CDH1 coding deletion. This 20kb sequence encloses two hypomorphic tissue-specific regulatory elements (REs), each contributing 50% to CDH1 expression regulation. F9 carried a heterozygous 39bp-intergenic CNV downstream of CDH1 , triggering CDH1 mRNA/protein loss by CRISPR-Cas9. F15, presenting gastric but not colorectal cancer, carried an MLH1 heterozygous 2.7Kb germline CNV overlapping a stomach-specific RE found by ChIP-seq. The gastric tumour of mixed histology displayed Microsatellite instability (MSI), reduced MLH1 mRNA and protein, and reduced CDH1 and E-cadherin protein. CRISPR-Cas9 clones mimicking the MLH1 heterozygous CNV, triggered loss of MLH1 and CDH1 /E-cadherin mRNA and protein, similar to a coding deletion. Beyond the CDH1 TAD and tumour risk syndrome genes, multiple deletions of stomach accessible chromatin sequences were found in particularly young-affected individuals from additional 6 families. This oligogenic pattern impaired specifically mucin genes and multiple immune-related pathways. Herein, we pinpointed novel mechanisms behind HDGC predisposition. One involves deletions of CDH1 -REs in the TAD or stomach-specific CDH1 -REs in the MLH1 locus. The second involves multiple deletions of stomach REs affecting mucin and immune-related genes, favouring a gastric immune-deficient phenotype. Altogether, by combining stomach-specific chromatin accessibility and promoter interactions with whole genome sequencing, we solved the missing heritability in 47% of HDGC-like families within our cohort.

Mammalian cells frequently enter mitosis before DNA replication has finished, necessitating the rapid processing of replication forks to facilitate chromosome segregation. The TRAIP ubiquitin ligase induces mitotic replisome disassembly, fork cleavage, and repair via ‘Mitotic DNA Synthesis’ (MiDAS). Until now, it was unclear how TRAIP is regulated in mitotic cells. Here we show that TRAIP phosphorylation mediates a complex with the TTF2 ATPase and DNA Polymerase ε (Polε). Whereas TTF2 ATPase activity removes RNA polymerase II from mitotic chromosomes, replisome disassembly involves an unanticipated mechanism. The TTF2 amino terminus couples TRAIP to Polε, via tandem Zinc fingers that recognise phosphorylated TRAIP, and a motif that binds to POLE2. Thereby, TTF2 and Polε cause TRAIP to ubiquitylate the CDC45-MCM-GINS (CMG) helicase, triggering replisome disassembly and MiDAS. These data identify TTF2 as a multifunctional regulator of chromatin transactions during mitosis.

The regulation of inflammatory gene expression involves complex interactions between transcription factors (TFs), signaling pathways and epigenetic chromatin-mediated mechanisms. This study investigated mechanisms by which by IFN-γ-mediated priming augments TLR-induced expression of NF-κB target genes in primary human monocytes. IFN-γ priming enhanced the expression of signature inflammatory genes such as IL6 , TNF , IL1B , and CXCL10 when monocytes were exposed to various TLR agonists. RNA-seq analysis identified genes synergistically activated by IFN-γ and LPS, which were enriched in inflammatory pathways. Similar synergistic activation was observed with the TLR1/2 agonist PAM3CYS, suggesting a shared regulatory mechanism. ATAC-seq analysis revealed that TLR ligands induce IRF1 TF activity independently of IFN-γ. JAK1/2 inhibitor (iJAK) treatment reduced IRF1 expression and protein levels, especially in IFN-γ-treated monocytes, but not in LPS-stimulated monocytes, suggesting LPS-induced IRF1 may compensate for loss of IFN-γ-induced IRF1. We applied CRISPR-Cas9 to knock out IRF1 in primary human monocytes and found loss of IRF1 abrogates synergistic activation of key inflammatory genes, suggesting a pivotal role for IRF1. This genetic data was corroborated by IRF1 CUT&RUN data showing resistance of IRF1 binding to JAK inhibition under (IFN-γ + LPS) costimulated conditions, and co-occupancy of IRF1 binding sites by NF-κB. This study enhances our understanding of inflammatory gene regulation, highlighting IRF1 as a key player and a potential therapeutic target for inflammatory diseases.

Glioblastoma (GBM) is an aggressive brain tumour with limited treatment options and poor prognosis, largely due to its heterogeneity and the involvement of multiple intracellular signalling pathways that contribute to drug resistance. Standard therapies have not significantly improved patient outcomes over the past two decades. While recent advancements in targeted drug combination therapies, such as dabrafenib and trametinib, show promise for certain GBM subgroups, identifying drug combinations effective across the broader GBM population remains a challenge. Integrin-mediated signalling, particularly through Focal Adhesion Kinase (FAK), plays a pivotal role in GBM pathogenesis and invasion, making it a potential therapeutic target [1]. In our study, we utilized a chemogenomic screening approach to identify synergistic drug combinations that target FAK in glioblastoma. We initially employed a CRISPR-engineered GBM model to assess the effects of FAK depletion and discovered that combining FAK inhibitors with MEK inhibitors, particularly trametinib, demonstrated synergistic effects. This potent combination was validated through various 2D & 3D assays, including cell viability/apoptotic assessment, synergistic analysis, cellular imaging, and target engagement assays. The combination also effectively inhibited spheroid growth and invasion across a diverse panel of patient derived GBM stem cells. Molecular mechanisms underlying these effects included suppression of multiple kinase signalling pathways and enhanced apoptosis, elucidated using Reverse Phase Protein Array (RPPA) profiling and western blot validation. In vivo , the combination therapy significantly reduced tumour volume in orthotopic transplantation models. These findings suggest that combining FAK and MEK inhibitors represent a promising therapeutic strategy to overcome the challenges of GBM treatment.

Methylome profiling is an emerging clinical tool for tumor classification and liquid biopsies. Here, we developed FLEXseq, a genome-wide methylation profiler that enriches and sequences the fragments of DNA flanking the CCGG motif. FLEXseq strongly correlates (Pearson’s r = 0.97) with whole genome bisulfite sequencing (WGBS) while enriching 18-fold. To demonstrate the broad applicability of FLEXseq, we verified its usage across cells, body fluids, and formalin-fixed paraffin-embedded (FFPE) tissues. DNA dilutions down to 250 pg decreased CpG coverage, but bias in methylation remained low (Pearson’s r ≥ 0.90) compared to a 10 ng input. FLEXseq offers a cost-efficient, base-pair resolution methylome with potential as a diagnostic tool for tissue and liquid biopsies.

Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of patients with IRD or other inherited diseases. Graphical abstract

Achieving a diversity of neuronal cell types and circuits during brain development requires alternative splicing of developmentally regulated mRNA transcripts. Microexons are a type of alternatively spliced exon that are 3–27 nucleotides in length and are predominantly expressed in neuronal tissues. A key regulator of microexon splicing is the RNA-binding protein Serine/arginine repetitive matrix 4 ( Srrm4 ). Srrm4 is a highly conserved, vertebrate splicing factor that is part of an ancient family of splicing proteins. To better understand the function of Srrm4 during brain development, we examined neural expression of zebrafish srrm4 from days 1–5 of development using fluorescence in situ hybridization. We found that srrm4 has a dynamically changing expression pattern, with expression in diverse cell types and stages during development. We then used CRISPR-based mutagenesis to generate zebrafish srrm4 mutants. Unlike previously described morphant phenotypes, srrm4 mutants did not show overt morphological defects. Moreover, sequencing of wild-type and mutant transcriptomes revealed only minor changes in splicing. srrm4 thus appears to have a limited role in zebrafish neural development.