The rapid advancement of genetic editing technologies, such as CRISPR-Cas9, has introduced unprecedented opportunities and challenges within professional sports. This study aims to systematically evaluate the legal and ethical implications associated with the application of gene editing among elite athletes. Employing a mixed-methods design, we conducted a comprehensive survey of 312 stakeholders-including athletes, coaches, legal experts, and ethicists-across five continents. Advanced statistical analyses, including Structural Equation Modeling (SEM) and Multivariate Logistic Regression, were utilized to identify significant predictors of legal risk perception and ethical concern. Results reveal a pronounced divergence in stakeholder attitudes: while 68% of legal professionals emphasize regulatory gaps, 74% of athletes express uncertainty regarding long-term health consequences. The SEM model demonstrated that perceived fairness (β=0.41, p<0.001) and regulatory clarity (β=0.36, p<0.001) are the strongest predictors of overall acceptance. These findings underscore the urgent need for robust international frameworks to address the multifaceted risks of gene editing in sports and highlight the importance of transparent policy-making. Our research provides actionable insights for regulators, sports organizations, and bioethics committees to anticipate and manage the evolving landscape of genetic technologies in athletics.

Lung cancer histological subtypes include lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC). While typically distinct, combined LUAD/SCLC histology tumors occur, and LUAD can transform into SCLC as a resistance mechanism to targeted therapies, especially in EGFR -Mutant LUADs with RB1 / TP53 -inactivation. Although PRC2 complex expression increases during this transformation, its functional role has remained unclear. Using CRISPR-based autochthonous immunocompetent GEMMs, we demonstrate that inactivation of EED, the core PRC2 scaffolding subunit, impairs SCLC tumorigenesis and drives histological transformation from ASCL1-positive SCLC to LUAD through a transient NEUROD1-positive intermediate state. Mechanistically, EED loss de-represses bivalent genes co-marked by H3K27me3 and H3K4me3, including LUAD oncogenic RAS, PI3K, and MAPK pathway genes, to promote transformation to LUAD. Consistently, these same signaling genes are bivalently repressed in human SCLC patient-derived xenograft (PDX) tumors, suggesting a conserved PRC2-dependent mechanism to repress LUAD lineage oncogenic signaling to maintain the SCLC neuroendocrine identity. In a complementary EGFR -mutant LUAD GEMM with RB1/TP53 inactivation, EED was required for LUAD-to-SCLC transformation and distant metastasis upon EGFR withdrawal. These findings identify the PRC2 complex as a key epigenetic enforcer of SCLC neuroendocrine identity and nominate EED inhibition as a potential strategy to block SCLC transformation in high-risk LUAD.

Bacteria encode an enormous diversity of defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a predicted nuclease and an accessory NTPase protein, suggesting additional classes of nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define shared features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid targeting for each system demonstrate protein–protein complex formation is a universal feature of nuclease-NTPase systems and explain patterns of phage targeting and susceptibility. We show that some nuclease-NTPase systems use highly degenerate recognition site preferences to enable exceptionally broad nucleic acid degradation. Our results uncover shared principles of anti-phage defense system function and provide a foundation to explain the widespread role of nuclease-NTPase systems in bacterial immunity.

Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate mRNA would be an advantageous approach to correct gene expression, but has not been evaluated in an in vivo disease model. Here, we investigated if a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identifiy and engineere human proteins capable of increasing mRNA translation using the CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold - key phenotypic indicators of Dravet syndrome. This work validates a new strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential translational activation has to address neurological haploinsufficiency.

ABSTRACT Mutations in the innate immune receptor NOD2 are the greatest single genetic risk factors for Crohn’s disease, yet the mechanisms by which NOD2 regulates intestinal homeostasis remain unclear. Here, we used a CRISPR-generated zebrafish model to determine the impacts of NOD2 deficiency on intestinal health. In a series of cellular, molecular, and transcriptomic studies, we uncovered substantial effects of NOD2 deficiency on epithelial and immune compartments, including deregulated expression of developmental pathways critical for establishment and maintenance of the gut epithelium, and an unexpected increase in the expression of multiple estrogen-response genes. In a series of functional assays, we uncovered a mechanistic link between estrogenic signals and NOD2-deficiency phenotypes, whereby exposure to estrogen alone replicated NOD2-deficiency phenotypes, and exposure to an estrogen receptor antagonist reverted the epithelial defects observed in NOD2 mutants. Our findings identify a critical NOD2-estrogen regulatory axis in the establishment of intestinal homeostasis and suggest that hormonal signaling may contribute to the sex-specific pathogenesis of Crohn’s disease.

Background The emergence and spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis ( Mtb ) urge the development of novel drugs and efficient therapeutic programs. A recent study aiming to uncover differential beta-lactam susceptibility phenotypes in clinical strains of Mtb found that the M237V substitution in cwlM ( Rv3915 ) was associated with increased susceptibility to amoxicillin. Considering that Mycobacterium smegmatis ( Msm ) is a widely used surrogate model for Mtb , we constructed a cwlM knockdown mutant in Msm using CRISPR interference (CRISPRi) to elucidate the role of CwlM in beta-lactam susceptibility and intracellular survival. Results Quantitative RT-PCR assays confirmed the successful repression of cwlM , while the phenotyping assays confirmed the essentiality of CwlM-related processes for mycobacterial viability. Collectively, the antibiotic susceptibility assays suggested that CwlM SMEG may promote beta-lactam resistance, particularly to meropenem and cefotaxime. Moreover, CwlM SMEG was found to support M. smegmatis intracellular survival within THP-1-derived macrophages. To address conflicting reports regarding its predicted peptidoglycan (PG) hydrolase activity, we purified recombinant CwlM TB . The Micrococcus luteus -derived PG-based zymogram indicated that CwlM TB lacks PG-hydrolytic activity, suggesting it might act as a regulator of PG biosynthesis instead. Conclusions Our findings indicate that CwlM contributes to beta-lactam resistance and intracellular survival, regardless of lacking detectable PG-hydrolytic activity. Overall, CwlM was found to be essential and highly vulnerable, highlighting its potential as a therapeutic target that warrants further investigation.

Abstract Despite its remarkable clinical success in human cancer treatment, immune checkpoint blockade is effective only in a minority of patients. One major obstacle is tumor-driven impairment of T cell priming and early activation, however, the underlying mechanism remains elusive. Here we identify TMEM176B-positive exosomes specifically secreted by cancer cells in plasma of cancer patients rather than healthy donors, high levels of which correlate with worse prognosis and unfavorable outcomes of anti-PD1 therapy. A small-scale CRISPR-Cas9 screen discovers Tmem176b on tumor-derived exosomes as a negative regulator of T cell early activation. Genetic ablation of Tmem176b in mouse cancer cells substantially suppresses tumour growth in a CD8 + T cell-dependent manner. Mechanistically, tumour-derived exosomal Tmem176b attenuates proximal T cell receptor signaling in CD8 + T cells by recruiting tyrosine phosphatase Shp1 to immunological synapse. Blocking TMEM176B, either using neutralizing antibody or using competitive peptide to disrupt Tmem176b-Shp1 interaction, remarkably restrains tumour progression in models of mouse and human cancers, and synergizes with anti-PD1/PD-L1 therapy. Our findings not only uncover tumor-derived Tmem176b as a promising target for cancer immunotherapy, but also provide a potential non-invasive diagnostic tool to detect early cancer and predict clinical response to anti-PD1 therapy.

ABSTRACT DNA polymerase theta (Polθ) plays a crucial role in the repair of DNA double-strand breaks (DSBs) by microhomology-mediated end joining (MMEJ). We previously demonstrated that Polθ inhibition (Polθi) is an effective and well-tolerated approach to sensitise tumours to radiotherapy (RT). Here, we profiled 54 cancer cell lines and found that Polθi induces significant radiosensitisation in most models, though with marked variability not explained by indicators of Polθ activity. To pinpoint molecular determinants of radiosensitisation by Polθi, we performed a CRISPR knockout screen which revealed loss of the TP53BP1/Shieldin pathway component SHLD2 ( FAM35A ) as a vulnerability to Polθi combined with RT. We demonstrated that SHLD2 loss not only increases sensitivity to RT alone, but also enhances the radiosensitising effect of Polθi, both in vitro and in vivo . Importantly, we found that SHLD2 is deleted in a subset of human prostate cancers, often co-occurring with PTEN loss, an adverse prognostic factor. Furthermore, we show that SHLD2 -deficient cancer cells are more reliant on Polθ to prevent DSB accumulation and chromosomal instability. In summary, we discovered SHLD2 loss as a novel collateral vulnerability that can be exploited through combined treatment with Polθi and RT.

Upstream open reading frames (uORFs) are a widespread class of translated regions (translons) occurring in 5′ leaders of mRNAs and serving critical roles in post-transcriptional regulation. However, their specific biological activities in human cells remains to be fully elucidated. Here, we conducted a genome-wide CRISPR-Cas9 loss-of-function screen of 978 uORFs identified with ribosome profiling, across human cell lines of distinct origin (HAP1, A549 and HEK293T). A total of 155 uORFs were identified as being essential for cell proliferation. These uORFs showed a high cell-type specificity, with only a few being universally essential. Subsequent analysis has revealed that the primary reason underlying the uORF essentiality is not encoded micropeptides, but rather cis -regulatory mechanisms. Moreover, uORFs located within short 5′ UTRs were disproportionately sensitive to frameshift-inducing indels, which frequently lead to the uORF extension and overlap with the coding region (CDS), resulting in translational repression. Finally, by intersecting regions of essential uORF with ClinVar and dbSNP datasets, we identified naturally occurring variants with the potential to disrupt their function and contribute to disease phenotypes. These findings highlight a pervasive and underappreciated layer of translational control in human cells and establish uORFs as critical cis -regulatory elements with potential relevance to human health.

Rapid identification of viral infections and specific variants in patient samples requires a simple and multiplexed RNA detection method that does not rely on DNA sequencing. Although recent direct detection assays based on CRISPR-Cas13a offer rapid RNA detection by avoiding reverse transcription and DNA amplification required of goldstandard PCR assays, these assays are not easily multiplexed to detect multiple viruses or variants without dividing the sample into separate reactions. Here we show that Cas13a acting on single target RNAs exhibits variable nuclease activity that depends on the interaction between the target RNA and crRNA. To exploit this feature for multiplexed detection, we devised a crRNA modification strategy that enables programmable tuning of Cas13a nuclease enzymatic rates. Using a droplet-based Cas13a assay, we demonstrate that kinetic signatures can be harnessed to differentiate among respiratory viruses and SARS-CoV-2 variants in contrived and clinical samples. This kinetic barcoding strategy can be extended to additional RNA targets through simple modification of crRNAs.

Apomixis, a process of clonal reproduction through seed, has the potential to significantly change agriculture by enabling a clonal seed propagation system for hybrid crops. Here, we demonstrate that hybrid seed from synthetically induced apomictic sorghum hybrids can be generated and maintained across multiple seed generations. This was achieved through the combination of avoidance of meiosis and induced parthenogenesis. Avoidance of meiosis was generated by the CRISPR/Cas9 knockout of the sorghum meiosis genes Spo11, Rec8, and OsdL1 and OsdL3. Parthenogenesis was induced in the resultant diploid egg cell using a maize egg cell promoter to express the Cenchrus ASGR-BBML2 gene coding sequence. Two strategies incorporating these components were used to induce synthetic apomixis in two different sorghum hybrids. Each hybrid used Tx623 as a female parent and either Tx430 or the African landrace Macia as a male parent. Seed yields in the induced apomictic hybrids were consistent and stable for multiple generations following self-pollination but reduced relative to the sexual hybrids. Sorghum contains two copies of the Osd1 gene that function in meiotic non-reduction. CRISPR/Cas9 knockout of both OsdL1 and OsdL3 loci was sufficient to produce clonal hybrid progeny in conjunction with the other apomixis induction components, but this led to a significant reduction in seed set. By contrast, a single in-frame edit of either OsdL1 or OsdL3 significantly improved seed set of clonal hybrid progeny. Fine-tuning OsdL activity appears to be essential to optimizing fertility. As the efficiency of seed set in the induced synthetic sorghum apomicts was lower than that of the sexual hybrid control, additional improvements are required to unlock the agronomic potential of synthetically induced apomictic sorghum in the field.

Heterochromatin is a repressive epigenetic state that suppresses transcription and safeguards genomic integrity. However, the full mechanism of how it is regulated remains elusive. Here, we focus on a previously described Pol II variant called rpb2-N44Y, which has a single substitution mutation within the Rpb2 subunit of Pol II that reduces RNAi-dependent heterochromatin. Through CRISPR-mediated site-directed mutagenesis, we find that rpb2-N44Y is a gain-of-function mutation. Furthermore, the heterochromatin defects of the rpb2-N44Y mutant requires a subunit of the Elongator complex called Elongator Protein 1 (Elp1), a protein that canonically promotes in mcm5s2U34 tRNA modifications. Intriguingly, we find that loss of Elp1, but not of other Elongator subunits such as Elp3, can robustly suppress heterochromatin defects in the rpb2-N44Y mutant. Elp1 acts independently of the mcm5s2 U34 tRNA modification to suppress RNAi-dependent heterochromatin at the pericentromere and the levels of small interfering RNAs (siRNAs) at affected heterochromatin. Overall, our study reveals two distinct Rpb2-centric pathways, via RNAi or Elp1, that can positively or negatively regulate heterochromatin, respectively. Furthermore, our findings reveal the first evidence of a chromatin function for Elp1 that is distinct from its canonical role in tRNA modifications. This work expands our understanding of how Elp1 can influence chromatin biology.

ABSTRACT Transcription in eukaryotes is regulated by chromatin-based mechanisms that control nucleosome occupancy, chromatin modifications, and transcription factor binding. We have previously shown that the transcription factor ADNP forms the ChAHP complex with the chromatin remodeler CHD4 and HP1 proteins, acting as a site-specific regulator of transcription and antagonist of CTCF binding. However, the molecular basis of these functions remained unclear. Here, we demonstrate that the CHD4 subunit is essential to antagonize CTCF and silence transcription of transposons, while HP1 proteins are dispensable. Although the remodeling activity of CHD4 is not required for ChAHP chromatin association, it is critical for both transposon repression and CTCF antagonism. Our findings support a model wherein ADNP recruits chromatin remodeling activity in a sequence-specific manner, enabling transcriptional control and local modulation of chromatin architecture.

Long-read sequencing enables the incorporation of isoform-level expression into single-cell transcriptomic studies, offering detail beyond those accessible with short-read methods. Although insightful, these approaches have typically been costly and yielded limited data for each individual cell. Recent advances in library preparation approaches and sequencing throughput have brought long-read single-cell studies closer to the mainstream. Here, we present a comparative analysis of commercial approaches for single-cell long-read sequencing. We have performed parallel analyses of the same cDNA material, generated using the 10X genomics platform, on Illumina short-read, and PacBio and Oxford Nanopore long-read platforms. We also demonstrate the impact of CRISPR-based depletion of libraries, to remove highly expressed transcripts, prior to long-read sequencing in these experiments. By analysing single-source cDNA libraries in parallel, we enable a direct comparison of each platform, evaluating standard metrics alongside concordance in clustering and cell type identification. While each approach generates usable gene and isoform expression data, we identify limitations common across platforms, primarily linked to cDNA synthesis inefficiencies and read filtering strategies. Our work demonstrates the increasing utility of single-cell long-read sequencing for isoform-resolved analyses, such as direct immunoglobulin chain reconstruction without additional amplification, and the detection of alternative splicing patterns across immune cell subtypes in CD45, a key gene for immune cell activation and differentiation. Our benchmarking of current platform options provides a foundation for researchers looking to adopt single-cell long-read sequencing into their transcriptomic studies, providing a framework for its integration into diverse biological questions.

The recent discovery of TIGR-Tas (Tandem Interspaced Guide RNA–Targeting Systems) marks a major advance in the field of genome editing, introducing a new class of compact, programmable DNA-targeting systems that function independently of traditional CRISPR-Cas pathways. TIGR-Tas effectors use a novel dual-spacer guide RNA (tigRNA) to recognize both strands of target DNA without requiring a protospacer adjacent motif (PAM). These Tas proteins introduce double-stranded DNA cuts with characteristic 8-nucleotide 3′ overhangs and are significantly smaller than Cas9, offering delivery advantages for in vivo editing. Structural analyses reveal homology to box C/D snoRNP proteins, suggesting a previously unrecognized evolutionary lineage of RNA-guided nucleases. This review positions TIGR-Tas at the forefront of a new wave of RNA-programmable genome-editing technologies. In parallel,I provide comparative insight into the diverse and increasingly modular CRISPR-Cas systems, including Cas9, Cas12, Cas13, and emerging effectors like Cas3, Cas10, CasΦ, and Cas14. While the CRISPR-Cas universe has revolutionized molecular biology, TIGR-Tas systems open a complementary and potentially more versatile path for programmable genome manipulation. I discuss mechanistic distinctions, evolutionary implications, and potential applications in human cells, synthetic biology, and therapeutic genome engineering.

Maintenance of T cell population size, which is important for immune homeostasis, is controlled by interleukin-7 (IL-7) and low-affinity TCR/MHC interactions that provide limited survival cues. Using arrayed CRISPR screening of miR-17∼92 targets, Bio-ID proximity labeling and proteomics we identified Tmem127 as an essential regulator of the T cell surface proteome. We validated interaction with the common gamma chain (IL-2Rγ) in a multi-protein complex. Tmem127 reduces IL-7 receptor surface expression to restrict homeostatic proliferation, thereby controlling naïve and central memory T cell population sizes. Tmem127 germline knockout (KO) mice display splenomegaly, accelerated experimental autoimmune encephalomyelitis and Tmem127-deficient bone marrow displays a competitive advantage over wildtype cells. Thus, we identified Tmem127 as an important regulator of the common gamma chain and immune homeostasis.

ABSTRACT Developmental decisions rely on cells making accurate transcriptional responses to signals they receive, as with Notch pathway activity. Local condensates or transcription factor hubs are a proposed mechanism for facilitating gene activation by nuclear complexes. To investigate their importance in endogenous Notch signalling, we deployed multi-colour live-imaging to measure Notch transcription-complex enrichment at a target gene locus in combination with the transcription dynamics. The co-activator Mastermind (Mam) was present in signalling-dependent nuclear foci, during Notch active developmental stages. Tracking these highly-dynamic Mam hubs together with transcription in the same nucleus, revealed that their condensation precedes and correlates with the profile of transcription and becomes stabilized if transcription is inhibited. Manipulations to signalling levels had concordant effects on hub intensities and transcription profiles, altering their probability and amplitude. Together the results argue that signalling induces the formation of transcription hubs whose properties are instrumental in the quantitative gene expression response to Notch activation.

Dengue virus (DENV) continues to pose a major global health burden, yet therapeutic options remain limited due to the virus's capacity for immune evasion, serotype variability, and persistence. While exosomes have been implicated as vehicles for viral dissemination and immune evasion, the cellular mechanisms underlying their generation during DENV infection remain poorly defined. Here, we identify the endoplasmic reticulum (ER)-associated host protein Reticulon 3 (RTN3), particularly its short isoform RTN3S, as a critical regulator of replication-competent viral cargo loading during infectious exosome biogenesis in DENV infection. Using hepatic and monocytic cell models, we revealed that RTN3S expression is induced upon infection and that RTN3S directly associates with DENV replication complexes, facilitating the packaging of replication-competent viral RNA and host proteins into infectious exosomes. Loss of RTN3 function via CRISPR-Cas9 markedly attenuated exosome production and reduced the transfer of infectious viral components to recipient naïve cells. Mutational analyses of RTN3S further revealed that both its N-terminal amphipathic and C-terminal domains are essential for exosomal loading of viral material. Single-cell RNA-sequencing of peripheral blood mononuclear cells (PBMCs) from DENV-infected individuals confirmed RTN3 upregulation in monocytes, particularly in those displaying intermediate/classical phenotypes, and revealed a transcriptional signature linking RTN3 to ER stress, vesicle trafficking, and impaired antiviral responses. These findings uncover a previously unrecognized RTN3-centered mechanism by which DENV hijacks the host exosomal machinery to propagate infection and potentially escape immune surveillance. Thus, our findings demonstrate a novel function for RTN3 in orchestrating the biogenesis of infectious exosomes, providing mechanistic insight and identifying a new therapeutic axis for combating flavivirus infections through host-directed approaches.

Selenocysteine (Sec), the 21st amino acid, is co-translationally inserted at UGA codons via a specialized machinery requiring SECIS elements, Sec-tRNA^Sec, eEFSec, and SECIS-binding protein 2 (SBP2). While SBP2 is essential for Sec incorporation in vitro and in vivo, the function of its paralog, SECISBP2L, remains incompletely defined. In this study, we investigated the distinct roles of SBP2 and SECISBP2L in the human hepatocellular carcinoma cell line HepG2, which expresses a broad selenoproteome. Using CRISPR-Cas9 genome editing, we generated SBP2 and SECISBP2L edited cell lines. Consistent with previous findings, SBP2 targeting impaired selenoprotein mRNA and protein expression, whereas SECISBP2L targeting did not. However, transcriptomic profiling by RNA-seq revealed that SECISBP2L targeting induced differential expression of over 800 genes, with significant enrichment in pathways related to extracellular matrix organization and cell adhesion. In contrast, SBP2 targeting produced a distinct transcriptomic signature enriched for metabolic and ion transport processes. Notably, only limited overlap in differentially expressed genes was observed between the two knockout models. Mass spectrometry and immunoblot data indicated that CRISPR-targeted SECISBP2L cells produce a truncated protein via internal translation initiation, suggesting that observed gene expression changes may be attributable to loss of a portion of the SECISBP2L N-terminus. These findings support a model in which SECISBP2L plays a noncanonical role in regulating gene expression independent of selenoprotein synthesis. Given prior associations between SECISBP2L downregulation or mutation and cancer progression, our data raise the possibility that SECISBP2L modulates cell adhesion and extracellular matrix gene networks relevant to metastatic potential. This work establishes a foundation for further mechanistic studies into SECISBP2L’s role in gene regulation and disease.

Abstract Synthetic lethality (SL) underlies the success of PARP1 inhibitors (PARPi) in treating homologous recombination (HR) deficient cancers, but extending this paradigm to other DNA damage response (DDR) deficiencies has proven challenging. We performed an in vivo CRISPR screen to identify DDR gene mutations that both enhance tumorigenesis and confer sensitivity to PARPi. Our screen identified FANCA deficiency as a driver of PARPi SL that was validated across diverse human cancer models. FANCA deficiency does not impair HR but disrupts Okazaki fragment maturation (OFM), causing lagging strand gaps and RPA exhaustion upon PARPi treatment. These effects require FANCA interaction with FEN1, independently of its canonical role in interstrand crosslink repair. We find FANCA-mediated FEN1 recruitment is required for OFM at oncogene-associated R loops during PARPi treatment. These findings establish a novel and non-canonical function for FANCA in FEN1-mediated OFM that can be leveraged for PARPi synthetic lethality in FANCA-mutant cancers.

During zygotic genome activation (ZGA) in Drosophila, broad domains of Polycomb-modified chromatin are rapidly established across the genome. Here, we investigate the spatial and temporal dynamics by which Polycomb group (PcG) histone modifications, H3K27me3 and H2Aub, emerge during early embryogenesis. Using ChIP-seq and live imaging of CRISPR-engineered GFP-tagged PcG components, we show that PRC2-dependent H3K27me3 accumulates adjacent to a subset of E(z)-bound prospective Polycomb Response Elements (PREs) beginning in nuclear cycle 14 (NC14), with patterns indicative of nucleation followed by spreading. Surprisingly, PRE-binding factors Pho, Combgap, and GAGA-factor are excluded from interphase nuclei prior to NC10 despite nuclear localization of E(z) throughout early interphases. Loss-of-function studies further demonstrate that GAGA-factor is largely dispensable for PcG domain establishment, whereas the pioneer factor Zelda is required for proper deposition of H3K27me3 and H2Aub at a subset of Polycomb domains. The role of Zelda at Polycomb domains is context-dependent; a large subset of targets requires Zelda not for PcG factor recruitment, but instead to license a loaded PRE to deposit H3K27me3 and H2Aub. Our findings support a model where licensing of PcG domains is an initial step in the regulatory processes governing Polycomb-regulated developmental genes.

Transcriptome profiling of bladder cancer has revealed distinct basal-like and luminal-like molecular subtypes, which may be correlated with pathological subtypes of different patient outcomes. However, whether these molecular subtypes originate from the corresponding cell types in the normal urothelium and whether different cells of origin influence bladder cancer progression remain unclear. Here, we conducted cell-type-specific lineage tracing in CRISPR/Cas9-induced mouse bladder cancer models of Pten and Trp53 targeting. We show that although basal, intermediate, and superficial umbrella cells can all serve as the cell of origin for bladder cancer, transformed umbrella cells were gradually displaced by tumor cells from inner layers, particularly transformed basal cells, which had highest stemness. Histological and single cell RNA-sequencing data comparing basal- and intermediate-cell-induced bladder tumors revealed that basal-induced tumors displayed higher heterogeneity, and contained unique cell clusters including Krt14+Ki67+ highly proliferative basal cells, squamous cell carcinoma, and transitioning cells towards the Gata3+ luminal subtype. Trajectory analysis confirmed the cell lineage differentiation hierarchy uncovered in lineage tracing. Moreover, human bladder cancer molecular subtype signatures were highly enriched in mouse tumor cell clusters of the corresponding cell of origin, and a gene signature derived from the unique basal-induced clusters is predictive of worse patient outcome. Overall, our results support that the basal and luminal molecular subtypes of bladder cancer have the corresponding cells of origin as their basis, and that urothelial basal cells are intrinsically more competitive than intermediate and umbrella cells in generating aggressive bladder cancer subtypes.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is commonly present in all eukaryotic organisms and tissue tested so far. EccDNA populations exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inherence of eccDNA between cells or a deficiency the methods by which eccDNA is detected. This study revisits the Circle-seq approach for enrichment of eccDNA to address if these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time. Linear DNA is digested by increasing exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to restriction enzymes. A key finding is the critical role of random hexamer primer concentration and genomic DNA input in Rolling Circle Amplification (RCA) for generating high-quality long amplicons from eccDNA (concatemeric tandem copy, CTC), essential for confident de novo eccDNA construction from long-read sequencing data. Lower primer concentrations substantially increased the percentage of CTC-derived eccDNA and improved the overlap of identified eccDNAs in technical replicates. Applying this revisited approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated that the optimized conditions enhanced the overlap of detected eccDNA up to over 50% overlap which substantially improved over previous studies (less than 1%). Moreover, Additionally, the oncogenic signature of eccDNAs can be identified across all replicates. These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology and its potential clinical applications.

Mutations of EIF2AK4 , which encodes the eIF2α kinase GCN2, cause a severe inherited form of pulmonary hypertension called pulmonary veno-occlusive disease (PVOD). Some pathogenic variants of GCN2 are amenable to pharmacological reactivation by low concentrations of ATP-pocket binding inhibitors. Kinase inhibition at modestly elevated concentrations limits the clinical utility of these drugs against PVOD. We therefore performed an in cellulo chemical screen for GCN2 activators and identified three structurally distinct compounds with low micromolar stimulatory activities. Unlike previously described GCN2 activators, one of these molecules activated GCN2 independently of GCN1. Modelling supported by structure activity screens suggested it binds within the ATP-pocket of GCN2, but unlike existing ligands does not protrude inward into the allosteric pocket or outward into the solvent. This overcomes a key requirement of other GCN2 activators.

Abstract Deficiency of the Monocarboxylate Transporter 8 (MCT8) severely impairs thyroid hormone (TH) transport into the brain, disrupting brain development as well as peripheral TH homeostasis. Studies assessing MCT8 expression patterns and tissue-specific pathologies induced by local TH-deficiency are often inconclusive due to unreliable antibody staining and the lack of functional tools to specifically target MCT8-expressing cells. For this purpose, we generated non-inducible Mct8-Cre and tamoxifen-inducible Mct8-CreERT2 mice. Mct8-Cre;Sun1-sfGFP mice demonstrated ubiquitous Sun1-sfGFP expression, due to early recombination driven by Mct8 gene expression at the stage of trophoblast implantation. Tamoxifen injection in 6-week-old Mct8-CreERT2 mice induced reporter expression specifically in Mct8-expressing cells in the brain and peripherally in liver, kidney, and thyroid, without leaky reporter expression in vehicle controls. Using vDISCO tissue clearing and 3D-imaging of GFP-nanobody-boosted mice, we further identified the sublingual salivary gland and the prostate as prominent Mct8-expressing organs. Nuclei from Mct8-expressing cells could selectively be enriched using fluorescence-activated nuclei sorting on Mct8-CreERT2;Sun1-sfGFP mice and characterized as choroid plexus cells and tanycytes. Our new inducible Mct8-CreERT2 line provides researchers with a tool to reliably mark, enrich, and characterize Mct8-expressing cells and to genetically modify genes specifically in these cells to study thyroid hormone transport and function.