The folding of the genome in the 3D nuclear space is fundamental for regulating all DNA-related processes. The association of the genome with the nuclear lamina into lamina-associated domains (LADs) represents the earliest feature of nuclear organization during development. Here, we performed a gain-of-function screen in mouse embryos to obtain mechanistic insights. We find that perturbations impacting histone H3 modifications, heterochromatin, and histone content are crucial for the establishment of nuclear architecture in zygotes and/or 2-cell-stage embryos. Notably, some perturbations exerted differential effects on zygotes versus 2-cell-stage embryos. Moreover, embryos with disrupted LADs can rebuild nuclear architecture at the 2-cell stage, indicating that the initial establishment of LADs in zygotes might be dispensable for early development. Our findings provide valuable insights into the functional interplay between chromatin and structural components of the nucleus that guide genome-lamina interactions during the earliest developmental stages.

Type 1 immunity mediates host defense through pathogen elimination, but whether this pathway also impacts tissue function is unknown. Here, we demonstrate that rapid induction of interferon γ (IFNγ) signaling coordinates a multicellular response that is critical to limit tissue damage and maintain gut motility following infection of mice with a tissue-invasive helminth. IFNγ production is initiated by antigen-independent activation of lamina propria CD8+ T cells following MyD88-dependent recognition of the microbiota during helminth-induced barrier invasion. IFNγ acted directly on intestinal stromal cells to recruit neutrophils that limited parasite-induced tissue injury. IFNγ sensing also limited the expansion of smooth muscle actin-expressing cells to prevent pathological gut dysmotility. Importantly, this tissue-protective response did not impact parasite burden, indicating that IFNγ supports a disease tolerance defense strategy. Our results have important implications for managing the pathophysiological sequelae of post-infectious gut dysfunction and chronic inflammatory diseases associated with stromal remodeling.

Rice tillering is an important agronomic trait regulated by plant genetic and environmental factors. However, the role and mechanism of the root microbiota in modulating rice tillering have not been explored. Here, we examined the root microbiota composition and tiller numbers of 182 genome-sequenced rice varieties grown under field conditions and uncovered a significant correlation between root microbiota composition and rice tiller number. Using cultivated bacterial isolates, we demonstrated that various members of the root microbiota can regulate rice tillering in both laboratory and field conditions. Genetic, biochemical, and structural analyses revealed that cyclo(Leu-Pro), produced by the tiller-inhibiting bacterium Exiguobacterium R2567, activates the rice strigolactone (SL) signaling pathway by binding to the SL receptor OsD14, thus regulating tillering. The present work provides insight into how the root microbiota regulates key agronomic traits and offers a promising strategy for optimizing crop growth by harnessing the root microbiota in sustainable agriculture.

The prefrontal cortex (PFC) is crucial for economic decision-making. However, how PFC value representations facilitate flexible decisions remains unknown. We reframe economic decision-making as a navigation process through a cognitive map of choice values. We found rhesus macaques represented choices as navigation trajectories in a value space using a grid-like code. This occurred in ventromedial PFC (vmPFC) local field potential theta frequency across two datasets. vmPFC neurons deployed the same grid-like code and encoded chosen value. However, both signals depended on theta phase: occurring on theta troughs but on separate theta cycles. Finally, we found sharp-wave ripples-a key signature of planning and flexible behavior-in vmPFC. Thus, vmPFC utilizes cognitive map-based computations to organize and compare values, suggesting an alternative architecture for economic choice in PFC.

Epithelial tissues serve as the first line of host against bacterial infections. The self-organization of epithelial tissues continuously adapts to the architecture and mechanics of microenvironments, thereby dynamically impacting the initial niche of infections. However, the mechanism by which tissue geometry regulates bacterial infection remains poorly understood. Here, we showed geometry-guided infection patterns of bacteria in epithelial tissues using bioengineering strategies. We discovered that cellular traction forces play a crucial role in the regulation of bacterial invasive sites and marginal infection patterns in epithelial monolayers through triggering co-localization of mechanosensitive ion channel protein Piezo1 with bacteria. Further, we developed precise mechanobiology-based strategies to potentiate the antibacterial efficacy in animal models of wound and intestinal infection. Our findings demonstrate that tissue geometry exerts a key impact on mediating spatiotemporal infections of bacteria, which has important implications for the discovery and development of alternative strategies against bacterial infections.

The canonical complement-mediated lysis of mature red blood cells (RBCs) leads to severe pathogenesis. However, inhibition strategies targeting complement are not always as efficient as expected, indicating that unknown mechanisms are awaiting elucidation. In this study, we investigate the intracellular events in mature RBCs following complement activation. The collected evidence demonstrates that complement-induced hemolysis is a caspase-8-dependent programmed RBC death. Furthermore, short NLRP3 (miniNLRP3) fragments in RBCs are identified to engage in the assembly of NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC)-caspase-8 complex. Activated caspase-8 directly induces the proteolysis of β-spectrin, thereby disrupting the skeletal network of the RBC membrane, a process we refer to as spectosis. Spectosis signaling is also activated in autoimmune hemolytic anemia or paroxysmal nocturnal hemoglobinuria, and the inhibition of spectosis significantly reduced complement-induced hemolysis. These findings reveal a programmed death cascade in mature RBCs, which may have important implications for the treatment of hemolytic disorders.

Passive diffusion does not explain why many drugs are too large and/or too polar for rule-breaking membrane penetration, such as proteolysis-targeting chimeras (PROTACs, generally of a molecular weight > 800 Da). Here, using biotinylated chemical-probe-based target fishing and genetic knockdown/knockin approaches, we discovered that the membrane cluster of differentiation 36 (CD36) binds to and facilitates the uptake and efficacy of diverse PROTACs (e.g., SIM1-Me, MZ1, and clinical ARV-110) and large and/or polar small-molecule drugs (e.g., rapalink-1, rapamycin, navitoclax, birinapant, tubacin, and doxorubicin) via the CD36-mediated early endosome antigen 1 (EEA1)/Ras-related protein 5A (Rab5) endosomal cascade in vitro and/or in vivo. We then devised a novel chemical endocytic medicinal chemistry strategy to improve binding of PROTACs to CD36 using structural modifications via the prodrug approach, markedly enhancing PROTAC anti-tumor efficacy through spontaneously augmenting permeability and solubility.

Human blood vessel organoids (hBVOs) have emerged as a system to model human vascular development and disease. Here, we use single-cell multi-omics together with genetic and signaling pathway perturbations to reconstruct hBVO development. Mesodermal progenitors bifurcate into endothelial and mural fates in vitro, and xenografted BVOs acquire definitive arteriovenous endothelial cell specification. We infer a gene regulatory network and use single-cell genetic perturbations to identify transcription factors (TFs) and receptors involved in cell fate specification, including a role for MECOM in endothelial and mural specification. We assess the potential of BVOs to generate organotypic states, identify TFs lacking expression in hBVOs, and find that induced LEF1 overexpression increases brain vasculature specificity. Finally, we map vascular disease-associated genes to hBVO cell states and analyze an hBVO model of diabetes. Altogether, we provide a comprehensive cell state atlas of hBVO development and illuminate the power and limitation of hBVOs for translational research.

Major progress has been made in elucidating the molecular, cellular, and supracellular mechanisms underlying aging. This has spurred the birth of geroscience, which aims to identify actionable hallmarks of aging. Aging can be viewed as a process that is promoted by overactivation of gerogenes, i.e., genes and molecular pathways that favor biological aging, and alternatively slowed down by gerosuppressors, much as cancers are caused by the activation of oncogenes and prevented by tumor suppressors. Such gerogenes and gerosuppressors are often associated with age-related diseases in human population studies but also offer targets for modeling age-related diseases in animal models and treating or preventing such diseases in humans. Gerogenes and gerosuppressors interact with environmental, behavioral, and psychological risk factors to determine the heterogeneous trajectory of biological aging and disease manifestation. New molecular profiling technologies enable the characterization of gerogenic and gerosuppressive pathways, which serve as biomarkers of aging, hence inaugurating the era of precision geromedicine. It is anticipated that, pending results from randomized clinical trials and regulatory approval, gerotherapeutics will be tailored to each person based on their genetic profile, high-dimensional omics-based biomarkers of aging, clinical and digital biomarkers of aging, psychosocial profile, and past or present exposures.

Regulatory DNA provides a platform for transcription factor binding to encode cell-type-specific patterns of gene expression. However, the effects and programmability of regulatory DNA sequences remain difficult to map or predict. Here, we develop variant effects from flow-sorting experiments with CRISPR targeting screens (Variant-EFFECTS) to introduce hundreds of designed edits to endogenous regulatory DNA and quantify their effects on gene expression. We systematically dissect and reprogram 3 regulatory elements for 2 genes in 2 cell types. These data reveal endogenous binding sites with effects specific to genomic context, transcription factor motifs with cell-type-specific activities, and limitations of computational models for predicting the effect sizes of variants. We identify small edits that can tune gene expression over a large dynamic range, suggesting new possibilities for prime-editing-based therapeutics targeting regulatory DNA. Variant-EFFECTS provides a generalizable tool to dissect regulatory DNA and to identify genome editing reagents that tune gene expression in an endogenous context.

Mycobacteriophage Bxb1 is a well-characterized virus of Mycobacterium smegmatis with double-stranded DNA and a long, flexible tail. Mycobacteriophages show considerable potential as therapies for Mycobacterium infections, but little is known about the structural details of these phages or how they bind to and traverse the complex Mycobacterium cell wall. Here, we report the complete structure and atomic model of phage Bxb1, including the arrangement of immunodominant domains of both the capsid and tail tube subunits, as well as the assembly of the protein subunits in the tail-tip complex. The structure contains protein assemblies with 3-, 5-, 6-, and 12-fold symmetries, which interact to satisfy several symmetry mismatches. Cryoelectron tomography of phage particles bound to M. smegmatis reveals the structural transitions that occur for free phage particles to bind to the cell surface and navigate through the cell wall to enable DNA transfer into the cytoplasm.

Microbiota-derived bile acids (BAs) are associated with host biology/disease, yet their causal effects remain largely undefined. Herein, we speculate that characterizing previously undefined microbiota-derived BAs would uncover previously unknown BA-sensing receptors and their biological functions. We integrated BA metabolomics and microbial genetics to functionally profile >200 putative microbiota BA metabolic genes. We identified 56 less-characterized BAs, many of which are detected in humans/mammals. Notably, a subset of these BAs are potent antagonists of the human androgen receptor (hAR). They inhibit AR-related gene expression and are human-relevant. As a proof-of-principle, we demonstrate that one of these BAs suppresses tumor progression and potentiates the efficacy of anti-PD-1 treatment in an AR-dependent manner. Our findings show that an approach combining bioinformatics, BA metabolomics, and microbial genetics can expand our knowledge of the microbiota metabolic potential and reveal an unexpected microbiota BA-AR interaction and its role in regulating host biology.

How developmental signals program gene expression in space and time is still poorly understood. Here, we addressed this question for the plant master regulator, auxin. Transcriptional responses to auxin rely on a large multigenic transcription factor family, the auxin response factors (ARFs). We deconvoluted the complexity of ARF-regulated transcription using auxin-inducible synthetic promoters built from cis-element pair configurations differentially bound by ARFs. We demonstrate using cellular systems that ARF transcriptional properties are not only intrinsic but also depend on the cis-element pair configurations they bind to, thus identifying a bi-layer ARF/cis-element transcriptional code. Auxin-inducible synthetic promoters were expressed differentially in planta showing at single-cell resolution how this bi-layer code patterns transcriptional responses to auxin. Combining cis-element pair configurations in synthetic promoters created distinct patterns, demonstrating the combinatorial power of the auxin bi-layer code in generating diverse gene expression patterns that are not simply a direct translation of auxin distribution.

Polyglutamine (polyQ) expansion is associated with pathogenic protein aggregation in neurodegenerative disorders. However, long polyQ tracts are also found in many transcription factors (TFs), such as FOXP2, a TF implicated in human speech. Here, we explore how FOXP2 and other glutamine-rich TFs avoid unscheduled assembly. Throughout interphase, DNA binding, irrespective of sequence specificity, has a solubilizing effect. During mitosis, multiple phosphorylation events promote FOXP2's eviction from chromatin and supplant the solubilizing function of DNA. Further, human-specific amino acid substitutions linked to the evolution of speech map to a mitotic phospho-patch, the "EVO patch," and reduce the propensity of the human FOXP2 to assemble. Fusing the pathogenic form of Huntingtin to either a DNA-binding domain, a phosphomimetic variant of this EVO patch, or a negatively charged peptide is sufficient to diminish assembly formation, suggesting that hijacking mechanisms governing solubility of glutamine-rich TFs may offer new strategies for treatment of polyQ expansion diseases.

Three-dimensional (3D) genome dynamics are crucial for cellular functions and disease. However, real-time, live-cell DNA visualization remains challenging, as existing methods are often confined to repetitive regions, suffer from low resolution, or require complex genome engineering. Here, we present Oligo-LiveFISH, a high-resolution, reagent-based platform for dynamically tracking non-repetitive genomic loci in diverse cell types, including primary cells. Oligo-LiveFISH utilizes fluorescent guide RNA (gRNA) oligo pools generated by computational design, in vitro transcription, and chemical labeling, delivered as ribonucleoproteins. Utilizing machine learning, we characterized the impact of gRNA design and chromatin features on imaging efficiency. Multi-color Oligo-LiveFISH achieved 20-nm spatial resolution and 50-ms temporal resolution in 3D, capturing real-time enhancer and promoter dynamics. Our measurements and dynamic modeling revealed two distinct modes of chromatin communication, and active transcription slows enhancer-promoter dynamics at endogenous genes like FOS. Oligo-LiveFISH offers a versatile platform for studying 3D genome dynamics and their links to cellular processes and disease.

Ferroptosis is a form of cell death due to iron-induced lipid peroxidation. Ferroptosis suppressor protein 1 (FSP1) protects against this death by generating antioxidants, which requires nicotinamide adenine dinucleotide, reduced form (NADH) as a cofactor. We initially uncover that NADH exists at significant levels on cellular membranes and then find that this form of NADH is generated by aldehyde dehydrogenase 7A1 (ALDH7A1) to support FSP1 activity. ALDH7A1 activity also acts directly to decrease lipid peroxidation by consuming reactive aldehydes. Furthermore, ALDH7A1 promotes the membrane recruitment of FSP1, which is instigated by ferroptotic stress activating AMP-activated protein kinase (AMPK) to promote the membrane localization of ALDH7A1 that stabilizes FSP1 on membranes. These findings advance a fundamental understanding of NADH by revealing a previously unappreciated pool on cellular membranes, with the elucidation of its function providing a major understanding of how FSP1 acts and how an aldehyde dehydrogenase protects against ferroptosis.

We investigate the impact of germline variants on cancer patients' proteomes, encompassing 1,064 individuals across 10 cancer types. We introduced an approach, "precision peptidomics," mapping 337,469 coding germline variants onto peptides from patients' mass spectrometry data, revealing their potential impact on post-translational modifications, protein stability, allele-specific expression, and protein structure by leveraging the relevant protein databases. We identified rare pathogenic and common germline variants in cancer genes potentially affecting proteomic features, including variants altering protein abundance and structure and variants in kinases (ERBB2 and MAP2K2) impacting phosphorylation. Precision peptidome analysis predicted destabilizing events in signal-regulatory protein alpha (SIRPA) and glial fibrillary acid protein (GFAP), relevant to immunomodulation and glioblastoma diagnostics, respectively. Genome-wide association studies identified quantitative trait loci for gene expression and protein levels, spanning millions of SNPs and thousands of proteins. Polygenic risk scores correlated with distal effects from risk variants. Our findings emphasize the contribution of germline genetics to cancer heterogeneity and high-throughput precision peptidomics.

Integrator (INT) is a metazoan-specific complex that targets promoter-proximally paused RNA polymerase II (RNAPII) for termination, preventing immature RNAPII from entering gene bodies and functionally attenuating transcription of stress-responsive genes. Mutations in INT subunits are associated with many human diseases, including cancer, ciliopathies, and neurodevelopmental disorders, but how reduced INT activity contributes to disease is unknown. Here, we demonstrate that the loss of INT-mediated termination in human cells triggers the integrated stress response (ISR). INT depletion causes upregulation of short genes such as the ISR transcription factor activating transcription factor 3 (ATF3). Further, immature RNAPII that escapes into genes upon INT depletion is prone to premature termination, generating incomplete pre-mRNAs with retained introns. Retroelements within retained introns form double-stranded RNA (dsRNA) that is recognized by protein kinase R (PKR), which drives ATF4 activation and prolonged ISR. Critically, patient cells with INT mutations exhibit dsRNA accumulation and ISR activation, thereby implicating chronic ISR in diseases caused by INT deficiency.

Self-pollination in self-compatible plant species often occurs prior to flower opening. By tracking the temporal progress of pollination in Arabidopsis, we observed that pollen predominantly targets the lateral region of the stigma in unopened flowers. Notably, approximately 7 h after flower opening, flowers close, thereby pressing anthers toward the central region of the stigma for a second self-pollination. This two-step self-pollination results in a doubling of pollen deposition, which significantly increases the ovule-targeting ratio and improves fertility under pollen-limiting conditions, as evident in the anther-dehiscence-defective mutant myb108 and under environmental stress conditions. Analysis using gamete-interaction-defective mutants hap2/gcs1 and dmp8 dmp9 revealed that the timely separation of both pollination events promotes fertilization recovery efficiency. A similar two-step pollination was observed in two other self-pollinating but not in outcrossing Brassicaceae species. This mechanism represents a reproductive assurance strategy in predominantly self-pollinating annuals to maximize fertility under unfavorable conditions.