The marking of DNA, histones, and RNA is central to gene expression regulation in development and disease. Recent evidence links N6-methyladenosine (m6A), installed on RNA by the METTL3-METTL14 methyltransferase complex, to histone modifications, but the link between m6A and DNA methylation remains scarcely explored. This study shows that METTL3-METTL14 recruits the DNA methyltransferase DNMT1 to chromatin for gene-body methylation. We identify a set of genes whose expression is fine-tuned by both gene-body 5mC, which promotes transcription, and m6A, which destabilizes transcripts. We demonstrate that METTL3-METTL14-dependent 5mC and m6A are both essential for the differentiation of embryonic stem cells into embryoid bodies and that the upregulation of key differentiation genes during early differentiation depends on the dynamic balance between increased 5mC and decreased m6A. Our findings add a surprising dimension to our understanding of how epigenetics and epitranscriptomics combine to regulate gene expression and impact development and likely other biological processes.

Upon infection, human immunodeficiency virus type 1 (HIV-1) releases its cone-shaped capsid into the cytoplasm of infected T cells and macrophages. The capsid enters the nuclear pore complex (NPC), driven by interactions with numerous phenylalanine-glycine (FG)-repeat nucleoporins (FG-Nups). Whether NPCs structurally adapt to capsid passage and whether capsids are modified during passage remains unknown, however. Here, we combined super-resolution and correlative microscopy with cryoelectron tomography and molecular simulations to study the nuclear entry of HIV-1 capsids in primary human macrophages. Our data indicate that cytosolically bound cyclophilin A is stripped off capsids entering the NPC, and the capsid hexagonal lattice remains largely intact inside and beyond the central channel. Strikingly, the NPC scaffold rings frequently crack during capsid passage, consistent with computer simulations indicating the need for NPC widening. The unique cone shape of the HIV-1 capsid facilitates its entry into NPCs and helps to crack their rings.

Recently, oncolytic virus (OV) therapy has shown great promise in treating malignancies. However, intravenous safety and inherent lack of immunity are two significant limitations in clinical practice. Herein, we successfully developed a recombinant Newcastle disease virus with porcine α1,3GT gene (NDV-GT) triggering hyperacute rejection. We demonstrated its feasibility in preclinical studies. The intravenous NDV-GT showed superior ability to eradicate tumor cells in our innovative CRISPR-mediated primary hepatocellular carcinoma monkeys. Importantly, the interventional clinical trial treating 20 patients with relapsed/refractory metastatic cancer (Chinese Clinical Trial Registry of WHO, ChiCTR2000031980) showed a high rate (90.00%) of disease control and durable responses, without serious adverse events and clinically functional neutralizing antibodies, further suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of NDV-GT for immunovirotherapy. Collectively, our results demonstrate the high safety and efficacy of intravenous NDV-GT, thus providing an innovative technology for OV therapy in oncological therapeutics and beyond.

Structural maintenance of chromosomes (SMC) complexes organize the genome via DNA loop extrusion. Although some SMCs were reported to do so symmetrically, reeling DNA from both sides into the extruded DNA loop simultaneously, others perform loop extrusion asymmetrically toward one direction only. The mechanism underlying this variability remains unclear. Here, we examine the directionality of DNA loop extrusion by SMCs using in vitro single-molecule experiments. We find that cohesin and SMC5/6 do not reel in DNA from both sides, as reported before, but instead extrude DNA asymmetrically, although the direction can switch over time. Asymmetric DNA loop extrusion thus is the shared mechanism across all eukaryotic SMC complexes. For cohesin, direction switches strongly correlate with the turnover of the subunit NIPBL, during which DNA strand switching may occur. Apart from expanding by extrusion, loops frequently diffuse and shrink. The findings reveal that SMCs, surprisingly, can switch directions.

During early mammalian development, the endoderm germ layer forms the foundation of the respiratory and digestive systems through complex patterning. This intricate process, guided by a series of cell fate decisions, remains only partially understood. Our study introduces innovative genetic tracing codes for 14 distinct endodermal regions using novel mouse strains. By integrating high-throughput and high-precision single-cell RNA sequencing with sophisticated imaging, we detailed the spatiotemporal and genetic lineage differentiation of the endoderm at single-cell resolution. We discovered an unexpected multipotentiality within early endodermal regions, allowing differentiation into various organ primordia. This research illuminates the complex and underestimated phenomenon where endodermal organs develop from multiple origins, prompting a reevaluation of traditional differentiation models. Our findings advance understanding in developmental biology and have significant implications for regenerative medicine and the development of advanced organoid models, providing insights into the intricate mechanisms that guide organogenesis.

Xist RNA initiates X inactivation as it spreads in cis across the chromosome. Here, we reveal a biophysical basis for its cis-limited diffusion. Xist RNA and HNRNPK together drive a liquid-liquid phase separation (LLPS) that encapsulates the chromosome. HNRNPK droplets pull on Xist and internalize the RNA. Once internalized, Xist induces a further phase transition and "softens" the HNRNPK droplet. Xist alters the condensate's deformability, adhesiveness, and wetting properties in vitro. Other Xist-interacting proteins are internalized and entrapped within the droplet, resulting in a concentration of Xist and protein partners within the condensate. We attribute LLPS to HNRNPK's RGG and Xist's repeat B (RepB) motifs. Mutating these motifs causes Xist diffusion, disrupts polycomb recruitment, and precludes the required mixing of chromosomal compartments for Xist's migration. Thus, we hypothesize that phase transitions in HNRNPK condensates allow Xist to locally concentrate silencing factors and to spread through internal channels of the HNRNPK-encapsulated chromosome.

In Huntington's disease (HD), striatal projection neurons (SPNs) degenerate during midlife; the core biological question involves how the disease-causing DNA repeat (CAG)n in the huntingtin (HTT) gene leads to neurodegeneration after decades of biological latency. We developed a single-cell method for measuring this repeat's length alongside genome-wide RNA expression. We found that the HTT CAG repeat expands somatically from 40-45 to 100-500+ CAGs in SPNs. Somatic expansion from 40 to 150 CAGs had no apparent cell-autonomous effect, but SPNs with 150-500+ CAGs lost positive and then negative features of neuronal identity, de-repressed senescence/apoptosis genes, and were lost. Our results suggest that somatic repeat expansion beyond 150 CAGs causes SPNs to degenerate quickly and asynchronously. We conclude that in HD, at any one time, most neurons have an innocuous but unstable HTT gene and that HD pathogenesis is a DNA process for almost all of a neuron's life.

This study investigated the cervicovaginal microbiome's (CVM's) impact on Chlamydia trachomatis (CT) infection among Black and Hispanic adolescent and young adult women. A total of 187 women with incident CT were matched to 373 controls, and the CVM was characterized before, during, and after CT infection. The findings highlight that a specific subtype of bacterial vaginosis (BV), identified from 16S rRNA gene reads using the molBV algorithm and community state type (CST) clustering, is a significant risk factor for CT acquisition. A microbial risk score (MRS) further identified a network of bacterial genera associated with increased CT risk. Post treatment, the CVM associated with CT acquisition re-emerged in a different subset of cases leading to reinfection. Additionally, the analysis showed a connection between post-treatment CVM and the development of pelvic inflammatory disease (PID) and miscarriage, further underscoring the CVM's contributing role to incident CT natural history and highlighting its consideration as a therapeutic target.

The gut microbiota is a powerful influencer of systemic immunity, with its impact on distal organs like the lungs garnering increasing attention. In this issue of Cell, Burrows et al. report that a gut protozoan plays a key role in shaping the immunological steady state of the lung.

Osteosarcoma is the most common primary cancer of the bone, with a peak incidence in children and young adults. Using multi-region whole-genome sequencing, we find that chromothripsis is an ongoing mutational process, occurring subclonally in 74% of osteosarcomas. Chromothripsis generates highly unstable derivative chromosomes, the ongoing evolution of which drives the acquisition of oncogenic mutations, clonal diversification, and intra-tumor heterogeneity across diverse sarcomas and carcinomas. In addition, we characterize a new mechanism, termed loss-translocation-amplification (LTA) chromothripsis, which mediates punctuated evolution in about half of pediatric and adult high-grade osteosarcomas. LTA chromothripsis occurs when a single double-strand break triggers concomitant TP53 inactivation and oncogene amplification through breakage-fusion-bridge cycles. It is particularly prevalent in osteosarcoma and is not detected in other cancers driven by TP53 mutation. Finally, we identify the level of genome-wide loss of heterozygosity as a strong prognostic indicator for high-grade osteosarcoma.

In a genome-wide association study (GWAS) meta-analysis of 688,808 individuals with major depression (MD) and 4,364,225 controls from 29 countries across diverse and admixed ancestries, we identify 697 associations at 635 loci, 293 of which are novel. Using fine-mapping and functional tools, we find 308 high-confidence gene associations and enrichment of postsynaptic density and receptor clustering. A neural cell-type enrichment analysis utilizing single-cell data implicates excitatory, inhibitory, and medium spiny neurons and the involvement of amygdala neurons in both mouse and human single-cell analyses. The associations are enriched for antidepressant targets and provide potential repurposing opportunities. Polygenic scores trained using European or multi-ancestry data predicted MD status across all ancestries, explaining up to 5.8% of MD liability variance in Europeans. These findings advance our global understanding of MD and reveal biological targets that may be used to target and develop pharmacotherapies addressing the unmet need for effective treatment.

Understanding protein function would be facilitated by direct, real-time observation of chemical kinetics in the atomic structure. The selectivity filter (SF) of the K+ channel provides an ideal model, catalyzing the dehydration and transport of K+ ions across the cell membrane through a narrow pore. We used a "pump-probe" method called electric-field-stimulated time-resolved X-ray crystallography (EFX) to initiate and observe K+ conduction in the NaK2K channel in both directions on the timescale of the transport process. We observe both known and potentially new features in the high-energy conformations visited along the conduction pathway, including the associated dynamics of protein residues that control selectivity and conduction rate. A single time series of one channel in action shows the orderly appearance of features observed in diverse homologs with diverse methods, arguing for deep conservation of the dynamics underlying the reaction coordinate in this protein family.

In a recently published article in Device, Saehyun Kim et al. report that selective excitation of bacteria can inhibit their proliferation in an antibiotic-free manner. We herein discuss the molecular and thermodynamic principles underlying this "selective excitability," which provides a new aspect to understand bacterial physiology.

As the brain transitions from wakefulness to sleep, processing of external information diminishes while restorative processes, such as glymphatic removal of waste products, are activated. Yet, it is not known what drives brain clearance during sleep. We here employed an array of technologies and identified tightly synchronized oscillations in norepinephrine, cerebral blood volume, and cerebrospinal fluid (CSF) as the strongest predictors of glymphatic clearance during NREM sleep. Optogenetic stimulation of the locus coeruleus induced anti-correlated changes in vasomotion and CSF signal. Furthermore, stimulation of arterial oscillations enhanced CSF inflow, demonstrating that vasomotion acts as a pump driving CSF into the brain. On the contrary, the sleep aid zolpidem suppressed norepinephrine oscillations and glymphatic flow, highlighting the critical role of norepinephrine-driven vascular dynamics in brain clearance. Thus, the micro-architectural organization of NREM sleep, driven by norepinephrine fluctuations and vascular dynamics, is a key determinant for glymphatic clearance.

Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extracellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. We determined cryogenic-electron microscopy (cryo-EM) structures of each receptor to understand the spatial arrangement of proton-sensing residues. Using deep mutational scanning (DMS), we determined the functional importance of every residue in GPR68 activation by generating ∼9,500 mutants and measuring their effects on signaling and surface expression. Constant-pH molecular dynamics simulations provided insights into the conformational landscape and protonation patterns of key residues. This unbiased approach revealed that, unlike other proton-sensitive channels and receptors, no single site is critical for proton recognition. Instead, a network of titratable residues extends from the extracellular surface to the transmembrane region, converging on canonical motifs to activate proton-sensing GPCRs. Our approach integrating structure, simulations, and unbiased functional interrogation provides a framework for understanding GPCR signaling complexity.

Animals have evolved pH-sensing membrane receptors, such as G-protein-coupled receptor 4 (GPR4), to monitor pH changes related to their physiology and generate adaptive reactions. However, the evolutionary trajectory and structural mechanism of proton sensing by GPR4 remain unresolved. Here, we observed a positive correlation between the optimal pH of GPR4 activity and the blood pH range across different species. By solving 7-cryoelectron microscopy (cryo-EM) structures of Xenopus tropicalis GPR4 (xtGPR4) and Mus musculus GPR4 (mmGPR4) under varying pH conditions, we identified that protonation of HECL2-45.47 and H7.36 enabled polar network establishment and tighter association between the extracellular loop 2 (ECL2) and 7 transmembrane (7TM) domain, as well as a conserved propagating path, which are common mechanisms underlying protonation-induced GPR4 activation across different species. Moreover, protonation of distinct extracellular HECL2-45.41 contributed to the more acidic optimal pH range of xtGPR4. Overall, our study revealed common and distinct mechanisms of proton sensing by GPR4, from a structural, functional, and evolutionary perspective.

Pyroptosis mediated by gasdermins (GSDMs) plays crucial roles in infection and inflammation. Pyroptosis triggers the release of inflammatory molecules, including damage-associated molecular patterns (DAMPs). However, the consequences of pyroptosis-especially beyond interleukin (IL)-1 cytokines and DAMPs-that govern inflammation are poorly defined. Here, we show intercellular propagation of pyroptosis from dying cells to bystander cells in vitro and in vivo. We identified extracellular vesicles (EVs) released by pyroptotic cells as the propagator of lytic death to naive cells, promoting inflammation. DNA-PAINT super-resolution and immunoelectron microscopy revealed GSDMD pore structures on EVs released by pyroptotic cells. Importantly, pyroptotic EVs transplant GSDMD pores on the plasma membrane of bystander cells and kill them. Overall, we demonstrate that cell-to-cell vesicular transplantation of GSDMD pores disseminates pyroptosis, revealing a domino-like effect governing disease-associated bystander cell death.

Zinc is an essential micronutrient that regulates a wide range of physiological processes, most often through zinc binding to protein cysteine residues. Despite being critical for modulation of protein function, the cysteine sites in the majority of the human proteome that are subject to zinc binding remain undefined. Here, we develop ZnCPT, a deep and quantitative mapping of the zinc-binding cysteine proteome. We define 6,173 zinc-binding cysteines, uncovering protein families across major domains of biology that are subject to constitutive or inducible zinc binding. ZnCPT enables systematic discovery of zinc-regulated structural, enzymatic, and allosteric functional domains. On this basis, we identify 52 cancer genetic dependencies subject to zinc binding and nominate malignancies sensitive to zinc-induced cytotoxicity. We discover a mechanism of zinc regulation over glutathione reductase (GSR), which drives cell death in GSR-dependent lung cancers. We provide ZnCPT as a resource for understanding mechanisms of zinc regulation of protein function.

Defining the subcellular distribution of all human proteins and their remodeling across cellular states remains a central goal in cell biology. Here, we present a high-resolution strategy to map subcellular organization using organelle immunocapture coupled to mass spectrometry. We apply this workflow to a cell-wide collection of membranous and membraneless compartments. A graph-based analysis assigns the subcellular localization of over 7,600 proteins, defines spatial networks, and uncovers interconnections between cellular compartments. Our approach can be deployed to comprehensively profile proteome remodeling during cellular perturbation. By characterizing the cellular landscape following HCoV-OC43 viral infection, we discover that many proteins are regulated by changes in their spatial distribution rather than by changes in abundance. Our results establish that proteome-wide analysis of subcellular remodeling provides key insights for elucidating cellular responses, uncovering an essential role for ferroptosis in OC43 infection. Our dataset can be explored at organelles.czbiohub.org.

The detection of molecular patterns associated with invading pathogens is a hallmark of innate immune systems. Prokaryotes deploy sophisticated host defense mechanisms in innate anti-phage immunity. Shedu is a single-component defense system comprising a putative nuclease SduA. Here, we report cryoelectron microscopy (cryo-EM) structures of apo- and double-stranded DNA (dsDNA)-bound tetrameric SduA assemblies, revealing that the N-terminal domains of SduA form a clamp that recognizes free DNA ends. End binding positions the DNA over the PD-(D/E)XK nuclease domain, resulting in dsDNA nicking at a fixed distance from the 5' end. The end-directed DNA nicking activity of Shedu prevents propagation of linear DNA in vivo. Finally, we show that phages escape Shedu immunity by suppressing their recombination-dependent DNA replication pathway. Taken together, these results define the antiviral mechanism of Shedu systems, underlining the paradigm that recognition of pathogen-specific nucleic acid structures is a conserved feature of innate immunity across all domains of life.