Proteolysis-Targeting Chimeras (PROTACs) and Molecular Glue Degraders (MGDs) canonically target proteins for degradation by recruiting them to a single E3 ligase complex. While heterotrivalent PROTACs that can co-opt multiple E3 ligase complexes have been described, to our knowledge all MGDs reported to date are dependent on a single E3. Here, using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can covalently recruit CUL4DCAF16 and CRL1FBXO22 in a parallel and redundant manner to degrade SMARCA2/4. Deep mutational scanning identifies a single cysteine (Cys173) in DCAF16 essential for degrader activity, and intact protein MS confirms covalent adduct at this site. The cryo-EM structure of the DCAF16:SMARCA2:degrader ternary complex reveals a unique binding mode and a distinct interface of neo-interactions, providing insights into degrader specificity. We demonstrate that E3 ligase dependency can be tuned both chemically and genetically. Minimal alterations to the compound's "degradation tail" switches ligase preference from DCAF16 to FBXO22, while a single L59W mutation on DCAF16 is sufficient to drive DCAF16 engagement for otherwise FBXO22-dependent compounds. These results establish a molecular and structural framework for the design of tuneable dual glue degraders that could mitigate challenges from resistance mechanisms in degrader therapies.

ABSTRACT Efficient utilization of complex biomass-derived sugars and tolerance to inhibitors are key requirements for the viability of lignocellulosic-based biorefineries. In this study, a two-stage evolution of an industrial yeast strain engineered with a xylose isomerase pathway yielded strain AceY.14, which exhibited improved fermentative performance and increased tolerance to acetic acid. Whole-genome sequencing of the evolved strain identified SNPs in ZWF1 , a component of the pentose phosphate pathway (PPP), and in the G1 cyclin gene CLN3 , both of which were functionally validated through CRISPR and reverse engineering. The zwf1 E191D mutation reduced xylitol accumulation, alleviating inhibition of xylose isomerase and enhancing flux through the non-oxidative branch of the PPP, while the frameshift cln3 T556fs mutation unexpectedly improved acetic acid tolerance and xylose consumption in the evolved strain, also affecting cell size and growth. Genome sequencing of AceY.14 also revealed a significant reduction in the xylA gene copy number, likely decreasing the metabolic burden associated with high xylose isomerase expression. A synergistic effect was observed in the isu1 Δ /zwf1 Δ double mutant, further boosting xylose consumption rates. A diploid derivative (AceY-2n) demonstrated high productivity and robustness in fermentations using hydrolysates from various lignocellulosic feedstocks, highlighting the strain’s potential for industrial-scale applications. These findings reveal novel metabolic targets for strain optimization and offer valuable insights for the rational engineering of yeast platforms for sustainable biofuel and bioproduct production.

SUMMARY During development, many cell types transition from canonical to non-canonical cell cycles, such as endomitosis and endoreplication, in which they duplicate their DNA but do not divide, giving rise to polyploidy. Little is known on the regulation of endomitosis, where cells enter M phase, but do not perform cytokinesis. Here, we investigate how cells initiate and execute endomitosis in the C. elegans intestine and find that endomitotic cells fail to assemble a central spindle or initiate cytokinetic furrowing. We find that endomitotic cells transcriptionally repress multiple cytokinesis regulators, especially the central spindle factors ZEN-4 Mklp 1 , CYK-4 RacGap 1 and SPD-1 Prc 1 . Intestinal cells lose the capacity to perform cytokinesis in late embryogenesis, and we find that the conserved DREAM (DP, RB-related, E2F and MuvB) complex is involved in the repression of central spindle genes. Together, our work demonstrates that the transition to endomitosis is a well-defined switch that relies on the transcriptional repression of cytokinesis genes.

Engineering animal models with self-sustained luminescence could enable non-invasive longitudinal monitoring of molecular events in living animals. To create animal models that report physiology with autoluminescence, both luciferin biosynthesis enzymes and the luciferase need to be optimised. Previous work on engineering the autoluminescence pathway from fungi resulted in the development of nnLuz_v3, a version of Neonothopanus nambi luciferase with enhanced thermal stability. Here, we generated an nnLuz_v3 reporter of endogenous Cyp1a1 expression as a measure of aryl hydrocarbon receptor (AHR) activation, assessing the performance of nnLuz_v3 in vivo at physiologically relevant expression levels. As AHR dynamically responds to metabolic, environmental and dietary changes it provides a validated platform to assess novel luminescence approaches. In Cyp1a1-nnLuz mice bioluminescence signal was stable, allowing the generation of well-resolved luminescence images both on standard in vivo imaging equipment and consumer-grade cameras. Using mice and nematode models, we demonstrated limited oral availability of the fungal luciferin, potentially compatible with delivering the substrate via food or the microbiome. Our results are an encouraging first step in the generation of an autoluminescent mammalian model of a molecular event and encourage optimisation of other enzymes of the fungal luciferase pathway.

Progranulin, the precursor protein to seven and a half distinct granulin motifs (GRNs), has been implicated in a broad range of diseases. Progranulin depletion is one of the most frequent causes for hereditary Frontotemporal Dementia (FTD). On the other hand, elevated progranulin levels have been associated with increased malignancy of many tumours, manifesting in increased cell proliferation, migration, metastasis formation, and reduced sensitivity to chemotherapeutics. While some functions can be unambiguously attributed to either full-length progranulin or one or multiple of the different GRNs, much about the interplay between progranulin and GRNs remains unknown. Here, we aimed to test the effect of progranulin overexpression on cell-based tumorigenicity assays, assessing proliferation, migration, and colony formation, using the hepatocellular carcinoma cell line HepG2 and the glioblastoma cell line U87. We transduced these cells with lentiviral vectors to overexpress full-length progranulin, two different C-terminally truncated progranulin proteins, lacking either the last two or the last four GRNs, or a triple FLAG-tagged maltose binding protein as a control. We observed increased colony formation in HepG2 overexpressing the full-length progranulin but not the C-terminally truncated constructs. The U87 cell lines were neither affected by an increase in progranulin levels nor by the depletion of progranulin.

ABSTRACT KRAS mutations are among the most prevalent oncogenic drivers in non-small cell lung cancer (NSCLC), yet the mechanisms of therapeutic resistance to KRAS inhibitors in these cancers remains poorly understood. Here, we deploy high-throughput CRISPR base editing screens to systematically map resistance mutations to three mechanistically distinct KRAS-targeted therapies, including KRAS-G12C(OFF) inhibitor (adagrasib), RAS(ON) G12C-selective tri-complex inhibitor (RMC-4998), and RAS(ON) multi-selective tri-complex inhibitor (RMC-7977). Using both a saturation Kras tiling approach and cancer-associated mutation library, we identify common and compound-selective second-site resistance mutations in Kras , as well as gain-of-function and loss-of-function variants across cancer-associated genes that rewire signaling networks in a context-dependent manner. Notably, we identify a recurrent missense mutation in capicua ( Cic ), that promotes resistance to RMC-7977 in vitro and in vivo. Moreover, we show that targeting NFκB signaling in CIC-mutant cells can resensitize them to RAS pathway inhibition and overcome resistance.

Summary Asymmetric localization of mRNAs is a prevalent mechanism for spatial control of protein function and typically involves active transport by cytoskeletal motors. The mechanisms of recognition of localizing mRNAs by motor complexes are poorly understood. Egalitarian is an adaptor protein that binds localization signals in specific RNAs in Drosophila and recruits them to the dynein-dynactin complex for microtubule-based transport. We determined the crystal structure of Egalitarian in complex with the localization signal of the K10 mRNA. Three structural units of Egalitarian, a 3’-5’exonuclease domain, a linker and a C-terminal domain form shape-specific, base-directed and backbone interactions with the RNA. Based on the structure we identified conserved residues recognizing RNA in vitro . Genome-edited flies with mutations in these residues have deficits in Egalitarian function that are congruent with changes in in vitro RNA binding affinities. Our work demonstrates how a minimal RNA localization signal is recognized by an RNA localization factor.

Background/Objectives Hepatoblastoma (HB) is the most common form of pediatric liver cancer, with the vast majority of these tumors evidence of mutation and/or deregulation of the oncogenic transcription factors β-catenin (B), YAP (Y) and NRF2 (N). HB research has been hampered by a paucity of established cell lines, particularly those bearing these molecular drivers. All combinations of B, Y and N (i.e. BY, BN, YN and BYN) are tumorigenic when over-expressed in murine livers but it has not been possible to establish cell lines from primary tumors. Recently, we found that concurrent Crispr-mediate targeting of the Cdkn2a tumor suppressor locus allows for such immor-talized cell lines to be generated with high fidelity. Methods We generated 5 immortalized cell lines from primary Cdkn2a -targeted BN and YN HBs and characterized their properties. Notably, 4 of the 5 retain their ability to grow as subcutaneous or pulmonary tumors in the immune-competent mice from which they originated. Most notably, when maintained under hypoxia conditions for as little as 2 days, BN cells reversibly up-regulated the expression of numerous endothelial cell (EC)-specific genes and ac-quired EC-like properties that benefited tumor growth. Conclusions The above approach is currently the only means by which HB cell lines with pre-selected, clinically relevant oncogenic drivers can be generated and the only ones that can be studied in immune-competent mice. Its generic nature should allow HB cell lines with other oncogenic drivers to be derived. A collection of such cell lines will be useful for studying tumor cell-EC trans-differentiation, interactions with the immune environment and drug sensitivities. Simple Summary Most hepatoblastomas (HB) are associated with aberrant expression of β-catenin (B), YAP (Y) and/or NRF2 (N) transcription factors and can be modeled in mice by over-expressing pairwise of triple combination of these. Virtually no human or murine HB cell lines exist that bear these mutations. We describe here an efficient way to generate cell lines from primary BN and YN tumors. Moreover, one of the BN lines shows a remarkable ability to trans-differentiate into endothelial cells under hypoxic conditions that may facilitate angiogenesis. These cell lines along with previousl-derived BN and BYN lines showed similar sensitivities to drugs commonly used to treat HB. Because the approach for cell line derivation we describe is quite general, it should allow for the generation of additional lines driven by less common factors. A collection of such permanent and well-characterized cell lines will facilitate studies that are difficult or impractical to perform in vivo .

Human SAGA is a 20-subunit complex that stimulates transcription and is essential for development. The most prominent addition to SAGA in metazoans compared to yeast is a 150kDa splicing-factor module (SPL). SPL is also a part of the U2snRNP but its role in SAGA is elusive, partially due to absence of high-resolution structural information regarding its incorporation into the complex. In yeast, subunit TAF5 and TAF6 of SAGA are shared with the general transcription factor TFIID. In metazoans, gene duplication created proteins that occur only in SAGA (TAF5L and TAF6L) or in TFIID (TAF5 and TAF6). What functions of SAGA benefit from this protein specialization is unclear. Here we report the structure of endogenous human SAGA purified via an affinity-ligand from cells that were not disturbed by any genomic engineering tools such as CRISPR-Cas9. Our work reveals the high-resolution structure of SPL and of the TAF6L HEAT repeat domain that provides the SPL with a docking surface. We elucidate how SPL and the HEAT repeats are incorporated into SAGA. We find multiple major differences between TAF6L/TAF5L and the canonical paralogues that are directly implicated in structural re-arrangements required to accommodate SPL. Furthermore, SPL binding to SAGA is very different and occupying much less interaction surface than to U2snRNP. However, the two cases still share similar sequences in a helix that is deeply inserted into the SPL. The seemingly weaker interaction of SPL with SAGA raises the possibility that SAGA serves to relay this module to the splicing machinery. Our structure also suggests mutations that could uncouple SPL from SAGA to further interrogate the role of this module.

In human cells, a subset of tRNA-encoding genes contain introns. These are removed by a non-canonical splicing pathway in which the tRNA splicing endonuclease complex catalyzes intron excision and the resulting exons are subsequently ligated by the tRNA-ligase complex (tRNA-LC). Although recent studies have provided insights into the process of intron removal, the molecular mechanisms underpinning tRNA ligation by tRNA-LC remain elusive. The tRNA-LC is a hetero-pentameric protein assembly consisting of Ashwin, CGI-99, FAM98B, the DEAD-box helicase DDX1 and the catalytic subunit RTCB/HSPC117. Using cryo-EM, we have determined an atomic-resolution reconstruction of human tRNA-LC. We find that CGI-99, DDX1 and FAM98B form an alpha-helical bundle that contacts RTCB via an interface located on the opposite side from the location of the ligase active site and tethers DDX1 to the tRNA-LC via its C-terminal helix. FAM98B and CGI-99 extensively interact in an intricately co-folded heterodimer that clamps Ashwin in a pincer-like structure. Interaction analysis using structure-based mutants of tRNA-LC subunits supports the overall architecture of the complex. Finally, we show that the paralogous proteins FAM98A and FAM98C underpin the assembly of compositionally distinct RTCB-containing complexes that lack Ashwin and may have distinct cellular functions. Together, our results provide new insights into the assembly and mechanism of the tRNA ligase complex, shedding light on its functions in tRNA biogenesis and beyond.

The autoimmune disease systemic lupus erythematosus (SLE) is associated with genetic variants in the X-linked gene CXORF21 , which encodes the protein TASL. TASL acts as an adaptor in the IRF5 pathway and is necessary for the phosphorylation of IRF5 in response to TLR7 or TLR9 stimulation. Here, we investigate the role of TASL in the humoral immune response, and in the development of lupus in the B6.MRL lpr murine model of SLE. We find that while TASL is dispensable for their development, it is required for the full activation of B cells via endolysosomal TLR stimulation, and consequent interferon signalling and inflammatory cytokine expression. Additionally, TASL is crucial for the emergence of age-associated B cells (ABCs), a B cell population derived from the extrafollicular response that increases with age and is expanded in autoimmune disease, and the production of IgG2c antibodies. We also find that deletion of TASL prevents the onset of autoimmunity in the genetically-determined B6.MRL lpr model of lupus.

Type 1 diabetes can be cured by β–cell replacement in principle, yet recurrent autoimmunity and transplantation barriers rapidly destroy implanted cells. Genome–wide CRISPR screening by Cai et al . highlighted RNLS and HIVEP2 as candidate genes, but their value outside an autoimmune setting is unknown. Here, it was evaluated whether single-gene knockout of RNLS or HIVEP2 could similarly protect β-cell grafts against allo- and xenogeneic rejection. Murine β–TC–6 and human EndoC–βH1 cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2, and editing efficiencies were confirmed via T7 endonuclease I assay and TIDE analysis. Functional characterization indicated that RNLS deletion modestly impaired glucose-stimulated insulin secretion in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. For in vivo assessment, genetically edited β-cell spheroids were subcutaneously transplanted into CD-1 mice to model allo- (murine β-cells) and xenogeneic (human β-cells) rejection scenarios. Bioluminescence imaging revealed no protective effects of RNLS or HIVEP2 deletion, with grafts from both knockout groups displaying identical rejection kinetics compared to controls. These findings indicate that single-gene deletions of RNLS or HIVEP2 are insufficient for conferring meaningful protection against allo- or xenorejection, highlighting the necessity for combinatorial genome editing strategies or complementary biomaterial-based immunomodulation to achieve effective and sustained β-cell graft survival.

Anoikis is an apoptotic cell death program triggered upon detachment from surrounding extracellular structures. However, the ability to evade cell death by anoikis in the presence of apoptosis-inducing stimuli is necessary for the formation of malignant tumors and progression to metastasis. Our findings indicate that the BRN2 (POU3F2) transcription factor is associated with anoikis resistance in melanoma cells. However, the BRN2 signaling cascade driving anoikis resistance remains unknown. Herein, we employed genome-wide CRISPR screens to validate BRN2 as a driver of anoikis resistance. Small molecule inhibition of BRN2 in melanoma cell lines with acquired anoikis resistance resensitized to death by anoikis in ultra-low attachment conditions. Our quantitative mass spectrometry analysis revealed that BRN2 functionally impacts oxidative phosphorylation and mitochondrial activity, whereby probes designed to inhibit BRN2 induced apoptosis and mitochondrial fragmentation through the MAPK and NF-κB signaling pathways and reduction in PPARɣ expression. Our study suggests that inhibition of BRN2 might allow the targeting of metastatic cells in circulation, and sensitizes cells to BRAF-targeted therapy, improving the prognosis for melanoma patients. Abstract Figure Graphical Abstract. Role of BRN2 in driving anoikis resistance in melanoma. Upon detachment from the extra-cellular matrix (ECM) melanoma cells must evade cell death by anoikis to seed distant metastases. This study expanded the understanding of the role of the BRN2 transcription factor as a driver of resistance to anoikis in melanoma. The use of small molecule inhibitors targeting BRN2 revealed that the transcription factor drives anoikis resistance via the MAPK and NF-κB signaling pathways, resulting in PPARγ dysregulation and subsequently driving mitochondrial dysfunction. Green boxes = previously published drivers of anoikis resistance in melanoma. Blue box = changes to mitochondrial function following inhibition of BRN2 as determined by proteomics analysis.

CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically-encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied (‘homed’). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (i.e. copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drive’s efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo -targeting ‘split’ homing-drive in the West Nile virus mosquito Culex quinquefasciatus , we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimisation of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.

Abstract Tn7 mobile genetic elements are known for their sophisticated target-site selection mechanisms and, in some cases, programmability. Recognition of target sites is mediated by designated transposon-encoded proteins and modulated by host factor proteins. In the case of the CRISPR-associated Tn7 elements from the type V-K, the ribosomal protein uS15 is an integral component of recruitment complex that promotes R-loop completion. Previous biochemical work also revealed that the ribosomal protein uL29 and the acyl carrier protein (ACP) influence Tn7 transposition frequency in vitro . However, how uL29 and ACP regulate the formation of the Tn7 targeting complex remains unclear. The prototypical Tn7 element encodes a heteromeric transposase (TnsAB), a AAA+ adaptor (TnsC), and two target-site selection proteins (TnsD and TnsE). TnsD targets a highly conserved site at the end of the glmS gene ( attTn7 ). However, poor protein stability has precluded the molecular characterization of how TnsD recognizes its target site. Here, we show that ACP and uL29 interact with the C-terminal region of TnsD through reciprocal electrostatic interactions, in turn, mitigating its tendency to aggregate. Additionally, we identify the uL29 and ACP residues that mediate the interaction with TnsD and stimulate DNA binding. These results unveil unique features of the TnsD-mediated target-site selection complex.

CRISPR/Cas9-based genome editing in the model bryophyte Physcomitrium patens (commonly known as Physcomitrella) is widely used for gene knockout via small insertions or deletions (indels). However, this approach may leave residual gene activity and typically requires sequencing-based validation. In this study, we established an efficient strategy for generating large, targeted deletions across multiple genes using dual-gRNA targeting. We first compared the efficiency of polycistronic tRNA-gRNA arrays to conventional gRNA constructs expressed under individual promoters, using the checkpoint protein gene MAD2 as a target. We found that a polycistronic construct doubled the frequency of large gene deletions compared to a conventional design. We then demonstrated that simultaneous deletion of two or four genes, targeting the katanin and TPX2 gene families, respectively, can be achieved in a single transformation event. The polycistronic system also increased deletion frequencies in the multiplex context, with up to 42% efficiency for individual genes and successful recovery of quadruple mutants. As a drawback, we confirmed that deletion efficiency varied substantially among individual gRNA pairs, indicating that gRNA design remains a critical factor in multiplex editing. This study establishes a versatile and scalable framework for generating multi-gene deletion mutants in P. patens , facilitating functional genomics and biotechnological applications requiring precise gene removal.

In contrast to animals, plants have a high regenerative capacity, and they can form new organs and even complete individuals from a few cells present in adult tissues, either in response to injury or to the alteration of their environment. In this study, we describe the isolation and characterization of the more adventitious roots1-1 ( mars1-1 ) mutant, which exhibits enhanced regenerative potential upon wounding in tomato hypocotyl explants. Additionally, the mars1-1 fruits exhibited a rough surface due to the ectopic proliferation of subepidermal cells, which formed callus-like structures on the cuticle. The MARS1/ROUGH gene encodes a conserved lysine-specific histone demethylase, SlLSD1, which regulates a variety of processes in metazoans, including cell proliferation, stem cell pluripotency, and embryogenesis. Two CRISPR/Cas9 null alleles, mars1-2 and mars1-3 , were generated and their pleiotropic phenotype was characterized. We found elevated levels of H3K4me1 in mars1/rough seedlings, which suggests that SlLSD1 is required for the demethylation of this histone mark. To ascertain the impact of altered epigenetic marks in the mars1/rough mutants on gene expression regulation, we conducted a transcriptome analysis using a variety of RNA-Seq studies on tomato hypocotyls. By employing specific bioinformatic workflows and leveraging on the resolution of directional RNA-Seq data, we have identified over several dozen distinct genomic regions that exhibit de novo expression in the mars1/rough mutants. One such region includes a novel B-type cyclin gene, which is upregulated in the mars1/rough mutants and may account for the observed phenotypes. Our findings indicate that SlLSD1 plays a role in the establishment and maintenance of silencing in specific genomic regions that are essential for tissue-specific reprogramming.

Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5’ or 3’ ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV- Dmd chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.

The transcription factor STAT3 plays broad roles in epithelial biology, yet its function in human esophageal development remains undefined. Using 2D and 3D human induced pluripotent stem cell (hiPSC)-derived platforms, we investigated how STAT3 regulates esophageal epithelial differentiation. We find that STAT3 is dispensable for definitive endoderm and anterior foregut endoderm specification but becomes essential during the transition to esophageal progenitor cells (EPCs). Inhibition of STAT3, via CRISPR-mediated knockout or siRNA, impairs the expression of key EPC and differentiation markers, including TP63 , and disrupts 3D organoid formation. These defects are accompanied by reduced epithelial proliferation. Notably, STAT3 is highly expressed in human fetal esophageal tissues and hiPSC-derived organoids, while its deletion in the developing mouse esophagus does not affect epithelial architecture, highlighting species-specific differences. Together, these findings identify STAT3 as a critical determinant of basal cell identity and epithelial morphogenesis, revealing a developmental checkpoint in early human esophageal lineage commitment.

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved -- specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

Upstream open reading frames (uORFs) are short translated regions that occur in the 5□ untranslated regions (5□ UTRs) of mRNA transcripts where they primarily serve to repress expression translation of the downstream primary open reading frame (pORF). Their widespread presence across mammalian transcriptomes suggests an important role in shaping the proteome, although the mechanistic basis of their regulatory effects remain incompletely understood. Here we present an integrated experimental and computational investigation into the features that govern uORF-mediated translation control. Using high-resolution proteomics data from 29 healthy human tissues and machine learning-based simulations, we have systematically dissected how features including uORF length, amino acid composition, start codon position, stop codon position, and Kozak context influence repressive activity, and performed experimental validation using reporter gene constructs. We also investigated how multiple uORFs within a single 5□ UTR can interact in synergistic or antagonistic ways, with the potential to produce counterintuitive effects on pORF translation. From these studies, we present a model of uORF function, suggesting a hierarchy of uORF feature importance, and proposing that a combination of uORF translation initiation probability, ribosome recycling rate, intercistronic ternary complex recharging requirements, and ribosome stalling mechanisms underlie uORF repressive activity. Together, these studies provide a comprehensive view of the molecular logic underlying uORF activity, offering new insights into their endogenous and highlighting their potential as targets for drug development.

Zonula occludens-1 (ZO-1), encoded by the TJP1 gene, is a crucial scaffolding protein within tight junctions that maintains epithelial and endothelial barrier integrity. In addition to its structural role, ZO-1 participates in signal transduction pathways that influence various cellular processes such as proliferation, differentiation, and apoptosis. Increasing evidence suggests that tight junction proteins, including ZO-1, play important regulatory roles in tumor progression, particularly by modulating metastasis, cell polarity, and vascular remodeling. Ovarian cancer, the most lethal gynecologic malignancy, is characterized by rapid growth, peritoneal dissemination, and a strong reliance on tumor angiogenesis. However, the specific role of ZO-1 in regulating angiogenesis within ovarian cancer remains poorly defined. In this study, we used CRISPR-Cas9-mediated gene editing to generate TJP1 knockout (KO) ovarian cancer cell lines and investigated the impact of ZO-1 loss on the expression of angiogenesis-related genes. Transcriptomic and qRT-PCR analyses revealed upregulation of KLF5 and IL-8, both of which are well-established pro-angiogenic factors. Furthermore, functional assessment using a Matrigel™ tube formation assay demonstrated that conditioned media from ZO-1-deficient cells significantly enhanced endothelial tube formation. These findings indicate that ZO-1 loss promotes a pro-angiogenic tumor microenvironment, likely through modulation of key signaling molecules such as KLF5 and IL-8. Therefore, ZO-1 may serve as a potential suppressor of angiogenesis and a therapeutic target in ovarian cancer.

ABSTRACT Embryonic development is precisely shaped by maternal and zygotic factors. These maternal factors exert their influence through maternal effects, a phenomenon where an offspring’s phenotype is determined, at least in part, by the mother’s environment and genotype. While environmental maternal effects can cause phenotypes that present both early and later in life, genetic maternal effects generally induce phenotypes in the earliest embryonic stages. Here, we reveal a genetic maternal effect that influences the development of cells that arise after early embryogenesis, highlighting that specific cell types can be susceptible to late-onset genetic maternal effects. Using zebrafish to study microglia, the resident immune cells of the brain, we identified a mutation in sry-related HMG box gene-17 (sox17) that exhibits a maternal effect phenotype that presents as a reduction of microglia in the brain and precursors in the yolk sac. We demonstrate that sox17 is expressed in microglia and their yolk sac precursors and is maternally-loaded. We show that sox17 restoration via embryonic injection reverses the maternal effect on microglia and yolk sac cells in sox17 mutants. To identify additional genes interacting with sox17 , we nominated genes from scRNA sequencing analysis of mouse embryonic microglia to perform a genetic screen using CRISPR mutagenesis and a custom-built robot that captures confocal images of the zebrafish brain in high-throughput. This screen identified f11r.1, gas6, and mpp1 as modifiers of microglia abundance in the embryonic brain, which we demonstrated are also expressed in zebrafish microglia. Transcriptional and mutant analyses with these new modifiers suggest that sox17 positively regulates mpp1 transcription. These results demonstrate that microglia are susceptible to genetic maternal effects, in addition to their known sensitivity to environmental maternal effects. Our findings reveal a late-onset phenotype associated with the maternal genotype, expanding the recognized impact of genetic maternal effects beyond initial embryo viability and into long-term vigor.

Pathogenic KCNQ2 variants are associated with developmental and epileptic encephalopathy (KCNQ2-DEE), a devastating disorder characterized by neonatal-onset seizures and impaired neurodevelopment with no effective treatments. KCNQ2 encodes the voltage-gated potassium channel K V 7.2, which regulates action potential threshold and repolarization. However, the relationship between K V 7.2 dysfunction and abnormal neuronal activity remains unclear. Here, we use human induced pluripotent stem (iPSC)-derived neurons from 5 KCNQ2-DEE patients with pathogenic variants and CRISPR/Cas9-corrected isogenic controls to investigate pathophysiological mechanisms. We identify a common dyshomeostatic enhancement of Ca 2+ -activated small conductance potassium (SK) channels, which drives larger post-burst afterhyperpolarizations in KCNQ2-DEE neurons. Using microelectrode arrays (MEAs), we recorded over 18 million extracellular spikes from >8,000 neurons during 5 weeks in culture and then applied supervised and unsupervised machine learning algorithms to dissect time-dependent functional neuronal phenotypes that defined both patient-specific and shared firing features among KCNQ2-DEE patients. Our analysis identified irregular spike timing and enhanced bursting as functional biomarkers of KCNQ2-DEE and demonstrated the significant influence of genetic background on phenotypic diversity. Importantly, using unbiased machine learning models, we showed that chronic treatment with the K V 7 activator retigabine rescues the disease-associated functional phenotypes with variable efficacy. Our findings highlight SK channel upregulation as a critical pathophysiological mechanism underlying KCNQ2-DEE and provide a robust MEA-based machine learning platform useful for deciphering phenotypic diversity amongst patients, discovering functional disease biomarkers, and evaluating precision medicine interventions in personalized iPSC neuronal models.

TGF-β-mediated signaling controls mast cell (MC) development and exerts anti-inflammatory functions, while antigen/allergen (Ag)-triggered FcεRI activation commands pro-inflammatory reactions. TGF-β induces strong C-terminal and low linker phosphorylation of SMAD2. In contrast, Ag triggers immediate, MEK-dependent SMAD2 linker phosphorylation only. Both stimuli can positively or negatively influence each other’s effects on MC activation in a gene-dependent manner. However, the molecular and cellular mechanisms of SMAD2 in MCs still need to be elucidated. To decipher the role(s) of SMAD2 in MCs, SMAD2 was ablated in PMC-306 MCs using CRISPR/Cas9, and the effects were studied after TGF-β and/or Ag stimulation. The absence of SMAD2 led to increased proliferation and survival, as well as decreased transcription of target genes like Smad7 and Jun in steady state and after TGF-β treatment. Interestingly, SMAD2 was found to regulate the strength and kinetics of TGF-β-mediated SMAD1/5 activation, resulting in augmented expression of genes like Id2 and Id3 in SMAD2-deficient MCs. Unexpectedly, SMAD2 was observed to license Ag-triggered production of pro-inflammatory cytokines, such as IL-6 and TNF, by monitoring expression of secondary repressive signaling elements. Re-introducing SMAD2 restored these events with varying sensitivity depending on the receptor system triggered. Our findings reveal SMAD2 as an initial hub in TGF-β-SMAD1/5 and Ag-FcεRI signaling, offering new possibilities for therapeutic intervention in both TGF-β-controlled and Ag-triggered MC functions using potential SMAD2 activators or inhibitors.