Condensates regulate transcription by selectively compartmentalizing biomolecules, yet the rules of specificity and their relationship to function remain enigmatic. To identify rules linked to function, we leverage the genetic selection bias of condensate-promoting oncofusions. Focusing on the three most frequent oncofusions driving translocation renal cell carcinoma, we find that they promote the formation of condensates that activate transcription by gain-of-function RNA polymerase II partitioning through a shared signature of elevated π and π-interacting residues and depletion of aliphatic residues. This signature is shared among a broad set of DNA-binding oncofusions associated with diverse cancers. We find that this signature is necessary and sufficient for RNA polymerase II partitioning, gene activation, and cancer cell phenotypes. Our results reveal that dysregulated condensate specificity is a shared molecular mechanism of diverse oncofusions, highlighting the functional role of condensate composition and the power of disease genetics in investigating relationships between condensate specificity and function.

Lysosomes maintain an acidic pH of 4.5-5.0, optimal for macromolecular degradation. Whereas proton influx is produced by a V-type H+ ATPase, proton efflux is mediated by a fast H+ leak through TMEM175 channels, as well as an unidentified slow pathway. A candidate screen on an orphan lysosome membrane protein (OLMP) library enabled us to discover that SLC7A11, the protein target of the ferroptosis-inducing compound erastin, mediates a slow lysosomal H+ leak through downward flux of cystine and glutamate, two H+ equivalents with uniquely large but opposite concentration gradients across lysosomal membranes. SLC7A11 deficiency or inhibition caused lysosomal over-acidification, reduced degradation, accumulation of storage materials, and ferroptosis, as well as facilitated α-synuclein aggregation in neurons. Correction of abnormal lysosomal acidity restored lysosome homeostasis and prevented ferroptosis. These studies have revealed an unconventional H+ transport conduit that is integral to lysosomal flux of protonatable metabolites to regulate lysosome function, ferroptosis, and Parkinson's disease (PD) pathology.

The lateral habenula (LHb) neurons and astrocytes have been strongly implicated in depression etiology, but it was not clear how the two dynamically interact during depression onset. Here, using multi-brain-region calcium photometry recording in freely moving mice, we discover that stress induces a most rapid astrocytic calcium rise and a bimodal neuronal response in the LHb. LHb astrocytic calcium requires the α1A-adrenergic receptor and depends on a recurrent neural network between the LHb and locus coeruleus (LC). Through the gliotransmitter glutamate and ATP/adenosine, LHb astrocytes mediate the second-wave LHb neuronal activation and norepinephrine (NE) release. Activation or inhibition of LHb astrocytic calcium signaling facilitates or prevents stress-induced depressive-like behaviors, respectively. These results identify a stress-induced positive feedback loop in the LHb-LC axis, with astrocytes being a critical signaling relay. The identification of this prominent neuron-glia interaction may shed light on stress management and depression prevention.

In humans, selective and promiscuous interactions between 46 secreted chemokine ligands and 23 cell surface chemokine receptors of the G-protein-coupled receptor (GPCR) family form a complex network to coordinate cell migration. While chemokines and their GPCRs each share common structural scaffolds, the molecular principles driving selectivity and promiscuity remain elusive. Here, we identify conserved, semi-conserved, and variable determinants (i.e., recognition elements) that are encoded and decoded by chemokines and their receptors to mediate interactions. Selectivity and promiscuity emerge from an ensemble of generalized ("public/conserved") and specific ("private/variable") determinants distributed among structured and unstructured protein regions, with ligands and receptors recognizing these determinants combinatorially. We employ these principles to engineer a viral chemokine with altered GPCR coupling preferences and provide a web resource to facilitate sequence-structure-function studies and protein design efforts for developing immuno-therapeutics and cell therapies.

Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here, we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic splicing machinery mutations. We utilized feature-barcoded peptide-major histocompatibility complex (MHC) dextramers to isolate neoantigen-reactive T cell receptors (TCRs) from healthy donors, patients with active myeloid malignancy, and following curative allogeneic stem cell transplant. Neoantigen-reactive CD8+ T cells were present in the blood of patients with active cancer and had a distinct phenotype from virus-reactive T cells with evidence of impaired cytotoxic function. T cells engineered with TCRs recognizing SRSF2 mutant-induced neoantigens arising from mis-splicing events in CLK3 and RHOT2 resulted in specific recognition and cytotoxicity of SRSF2-mutant leukemia. These data identify recurrent RNA mis-splicing events as sources of actionable public neoantigens in myeloid leukemias and provide proof of concept for genetically redirecting T cells to recognize these targets.

Leptin acts in the brain to suppress appetite, yet the responsible neurocircuitries underlying leptin's anorectic effect are incompletely defined. Prepronociceptin (PNOC)-expressing neurons mediate diet-induced hyperphagia and weight gain in mice. Here, we show that leptin regulates appetite and body weight via PNOC neurons, and that loss of leptin receptor (Lepr) expression in PNOC-expressing neurons in the arcuate nucleus of the hypothalamus (ARC) causes hyperphagia and obesity. Restoring Lepr expression in PNOC neurons on a Lepr-null obese background substantially reduces body weight. Lepr inactivation in PNOC neurons increases neuropeptide Y (Npy) expression in a subset of hypothalamic PNOC neurons that do not express agouti-related peptide (Agrp). Selective chemogenetic activation of PNOC/NPY neurons promotes feeding to the same extent as activating all PNOCARC neurons, and overexpression of Npy in PNOCARC neurons promotes hyperphagia and obesity. Thus, we introduce PNOC/NPYARC neurons as an additional critical mediator of leptin action and as a promising target for obesity therapeutics.

Virtually all individuals aged 65 or older develop at least early pathology of Alzheimer's disease (AD), yet most lack disease-causing mutations in APP, PSEN, or MAPT, and many do not carry the APOE4 risk allele. This raises questions about AD development in the general population. Although transcriptional dysregulation has not traditionally been a hallmark of AD, recent studies reveal significant epigenomic changes in late-onset AD (LOAD) patients. We show that altered expression of the LOAD biomarker phosphoglycerate dehydrogenase (PHGDH) modulates AD pathology in mice and human brain organoids independent of its enzymatic activity. PHGDH has an uncharacterized role in transcriptional regulation, promoting the transcription of inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKa) and high-mobility group box 1 (HMGB1) in astrocytes, which suppress autophagy and accelerate amyloid pathology. A blood-brain-barrier-permeable small-molecule inhibitor targeting PHGDH's transcriptional function reduces amyloid pathology and improves AD-related behavioral deficits. These findings highlight transcriptional regulation in LOAD and suggest therapeutic strategies beyond targeting familial mutations.

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.