Nature

Gene expression during mammalian development is orchestrated by non-coding cis-regulatory DNA elements (CREs) such as distal enhancers1-3. Despite their fundamental importance, and notwithstanding recent progress in predictive modeling4-9, many high-level properties of enhancer grammar remain unresolved. How does the length of an autonomously active CRE constrain its activity? How robust are CREs to mutations or rearrangements of transcription factor binding sites (TFBSs)? And how much epistasis exists among these sites? As predictive models solely trained on endogenous CREs are unlikely to resolve these questions10, we subjected several endogenous CREs to intensive sequence-level perturbation. Specifically, we assayed >35,000 variants of 5 parietal endoderm enhancers, with variants organized into four perturbation classes, designed to probe: (i) the functional sufficiency of sub-fragments via dense multi-size tiling, (ii) local epistasis via multi-hit saturation mutagenesis, (iii) activity-size tradeoffs via model-guided compaction, or (iv) functional resilience via sequence derivatization anchored on key TFBSs, including random deposition, reconstitution, and synthetic thripsis. This multi-scale dissection revealed rich phenomena. Sub-tiling uncovered sharp non-additivity between activity and fragment size, highlighting strongly synergistic TFBS clusters. Compaction showed that natural CREs lie far from the activity-size Pareto front, and that model-guided deletions can yield shorter yet stronger elements. Mutational scanning exposed a spectrum of CRE robustness, from tolerant to fragile, together with rare but consequential epistasis between individual TFBSs. Finally, TFBS-anchored derivatization demonstrated that background sequence can influence activity on par with TFBS arrangement. Strikingly, a substantial fraction of CRE derivatives exceeded the activity of their endogenous progenitors. Taken together, these results reveal both soft and stiff directions in regulatory sequence space, advancing a quantitative phenomenology of how enhancer sequences encode function and robustness.

Defense-associated reverse transcriptases (DRTs) are widespread in bacteria1,2, but how multi-domain DRTs containing RT and additional catalytic activities coordinate antiviral defense remains unclear. Here we show that DRT7, which contains both reverse transcriptase (RT) and primase-polymerase (PP) domains, provides broad-spectrum anti-phage immunity through abortive infection and can be activated by a phage-encoded putative transcriptional regulator. Upon activation, DRT7 synthesizes long, protein-primed, palindromic poly(A)/poly(T)-rich duplex-like DNA. Cryo-electron microscopy structures reveal that RT initiates protein-primed, protein-templated, sequence-specific poly(T) synthesis through an arginine-rich recognition pocket without requiring a complementary nucleic acid template, thereby converting DRT7 from an inactive closed dimer to an active open dimer. The RT-produced poly(T) then serves as both primer and template for PP-mediated poly(A) extension, with iterative handoff between RT and PP generating palindromic, alternating poly(A)/poly(T) ssDNA tracts that assemble into fold-back duplex-like DNA. These findings uncover an unexpected antiviral strategy based on protein self-templating, sequence-specific duplex-like DNA synthesis and reveal how coupling RTs with additional catalytic activities expands the functional scope of nucleic acid synthesis pathways.

Sensorimotor associative learning enables animals to adaptively link sensory cues with motor actions, a process that is critical for survival and everyday behavior. While Hebbian mechanisms explain associations formed through temporally overlapping neural activity, a fundamental challenge arises when sensory stimuli and motor responses are separated by a delay, because sensory and motor neurons are rarely coactive. Here, we identify the rostro-lateral posterior parietal cortex (PPC-rl) as a cortical hub that bridges tactile stimuli and temporally delayed licking actions during sensorimotor associative learning. Using cortex-wide calcium imaging with single-cell resolution to track [~]16,000 neurons simultaneously across sensory, motor, and association cortices, we find that PPC-rl uniquely exhibits sustained neural activity during the temporal delay early in learning, a signature that diminishes with expertise. Optogenetic silencing of this activity slows learning without impairing sensorimotor execution in expert mice. Learning strengthens the coupling of population dynamics within and between somatosensory and motor cortices. PPC-rl mediates this process by amplifying a low-dimensional communication subspace that synchronizes co-fluctuations across the somatosensory and motor cortices to facilitate linking. This PPC-rl dependent co-fluctuation dissolves post learning, underscoring PPC-rls role in bridging sensation to distal action. A biologically plausible network indicates that Hebbian plasticity with an eligibility trace gated by reward, PPC-rl persistent activity and PPC-rl dependent sensorimotor subspace communication synergize to support delayed association. Together, our findings uncover a PPC-rl based circuit mechanism that maintains temporal continuity to guide associative learning when sensory and motor events are separated in time.

AO_SCPLOWBSTRACTC_SCPLOWThe biology that governs progression and therapeutic response in autoimmune disease is organized in affected tissue, but direct molecular readout of that biology requires invasive biopsy and is rarely repeated during clinical trials or routine care. Using paired blood-skin single-cell RNA-sequencing from a systemic sclerosis (SSc) cohort of 74 individuals (57 patients and 17 matched controls, 192,809 cells across 53 annotated cell states), we show that peripheral blood carries a recoverable projection of tissue-resident molecular state. Across 63 pathways scored in both compartments, 43 same-pathway blood-skin associations reach FDR < 0.05; at cell-type resolution, 212 cross-compartment associations survive residualization for disease status and sex. Per-patient classifiers recover tissue-defined molecular states out of fold with AUCs between 0.62 and 0.79, with the strongest recoveries on fibroblast subtype programs that have no direct circulating analog: fibroblast COMP at 0.79, COCH at 0.75, MYOC2 at 0.74, POSTN at 0.74. Tissue programs route through different blood compartments at different representational levels: fibroblast programs resolve through T-cell, Treg, monocyte and B-cell axes at compositional and distributional levels, while interferon resolves through expression state across multiple cell types. Within SSc alone, a cross-validated partial least squares model learns a shared blood-skin latent axis at r = 0.486 (permutation p = 0.006); the induced patient ranking recovers tissue-interferon-high patients at 86% precision at the top-20% screening threshold against a 50% base rate. A paired multiview autoencoder, trained on module-level dependency structure under contrastive alignment, paired reconstruction, neighborhood preservation and tissue-target supervision, learns a shared latent geometry in which blood-only projections land in the same tissue-state region as their matched tissue samples and supports recovery of held-out tissue targets above simpler baselines and above two permutation null families. These results map the empirical geometry of cross-compartment inference in autoimmune disease and position peripheral blood as a substrate for tissue-state inference at trial and clinical scale.

Cis-regulatory elements (CREs) such as enhancers play a central role in orchestrating mammalian development, yet how they have gained, lost, maintained or changed function over the course of mammalian evolution remains poorly understood. To address this gap, we densely mapped the functional evolution of five mouse developmental enhancers by testing orthologous sequences from 480 extant and ancestrally reconstructed mammalian genomes (Zoonomia1, Cactus2) with massively parallel reporter assays (MPRAs). This phylogenetic dissection revealed diverse modes of evolution, from lineage-restricted activity to deep functional conservation despite extensive sequence divergence. To pinpoint causal changes, we developed a model-driven reconstitution strategy that uses deep learning-based predictions of chromatin accessibility to re-introduce a succession of mutations into ancestral orthologs; this revealed critical transcription factor binding site (TFBS) changes and pervasive context-dependent epistasis, including instances where mutational effects were strongly contingent on the order of their introduction. When we extended this strategy to tune the activity of extant orthologs, we found that ablation of enhancer function required as few as one to seven mutations, whereas enhancement was constrained by element-specific activity ceilings--a striking asymmetry in the predictability of model-guided enhancer editing. Together, these results shed light on how the plasticity of mammalian enhancers intersects with their evolution, and advance a framework for reprogramming the activity of endogenous CREs at nucleotide resolution.

Selecting a diet containing all essential amino acids (EAAs) is critical for health. Following EAA deprivation, animals can select a nutritiously complete food source; however, the underlying mechanisms in vertebrates remain unclear. In mice, we show that leucine deficiency activates hypothalamic agouti-related protein (AgRP) neurons, which project to the paraventricular thalamus (PVT) via {gamma}-aminobutyric acid and are required for EAA deficiency-induced leucine appetite in mice. Furthermore, the peripheral tongue amino acid sensor general control nonderepressive-2 (GCN2) mediates acute EAA appetite via AgRP neurons. Together, these findings identify a tongue-AgRP-PVT circuit underlying EAA appetite, which is important for the rapid and accurate selection of essential nutrients.

Understanding behavioral differences between experimental groups and quantifying action structure in behavioral experiments remains challenging. Currently, most approaches rely on pose estimation followed by downstream classification, resulting in assay specific pipelines with substantial annotation requirements. Here we present SingleBehavior Lab (SBL), a framework for modeling behavior across experimental contexts using a standardized graphical interface. SBL leverages spatiotemporal embeddings from large video foundation models and combines them with lightweight contrastive adapters, a multi-head attention pooling (MAP) module and a temporal decoder to enable behavior sequencing and task-specific refinement. The framework supports few-shot learning, allowing small models trained on pretrained embeddings to improve action segmentation and classification with limited labeled data, without fine-tuning the underlying video model. In parallel, a large segmentation model with motion-aware memory is used to extract object-centered representations that, together with shared spatiotemporal embeddings, enable unsupervised clustering of behavioral states and analysis of their structure, including cluster prioritization, transition dynamics and attention-based interpretability. Across multiple assays and species, SBL supports identification of group-level differences and rare behaviors, and provides a basis for integrating behavioral representations across experimental contexts.

The superior colliculus integrates retinal input to drive rapid, adaptive visual behavior, yet how the functional diversity of retinal ganglion cell types is represented in superior colliculus remains poorly understood. Using chronic two-photon calcium imaging of retinal ganglion cell axonal boutons in awake mice, we recorded over 200,000 boutons across superficial superior colliculus layers -- a scale that enabled systematic comparison with large-scale ex vivo retinal datasets. This revealed that the superior colliculus receives a near-complete sampling of retinal ganglion cell functional diversity. Functionally distinct response types were organized in systematic laminar gradients: not only response properties such as direction selectivity and contrast suppression, but retinal response types themselves varied systematically with depth. To probe how this organized input encodes natural scenes, we trained a "digital twin" deep network model on natural movie responses and validated its generalization to parametric stimuli, including cell type identification. Leveraging this model to generate predicted responses to looming stimuli, we identify a discrete subset of retinal response types tuned for collision detection at low angular thresholds -- a specialization embedded within a broader, non-specialized retinal population. The digital twin is made publicly available as a community resource. Together, these findings provide a comprehensive functional map of retinal drive to the superior colliculus and an in silico platform for linking retinal cell types to behaviorally relevant superior colliculus computations.

All cells remodel their membranes to divide. The highly conserved ESCRT-III system forms contractile polymers which, through direct interactions with membrane lipids, remodel membranes across the tree of life. In exploring how ESCRT-III divides the chemically and structurally unique archaeal membrane, we reveal that the homologue CdvB1 is required for the establishment of a distinct membrane domain within the division bridge of Sulfolobus acidocaldarius, associated with an accumulation of membrane-spanning inositol phosphate lipids. We show that CdvB1 associates with phosphoinositides in vitro and that this interaction aids cytokinesis in vivo. Together, we suggest that although eukaryotes inherited their membrane lipids from bacteria during eukaryogenesis, key features of the ESCRT-III:membrane interface that allow these polymers to bind, organise, and remodel eukaryotic membranes, may originate in archaea.

The synaptic vesicle (SV) cycle is the fastest membrane trafficking and protein sorting process in biology. It underlies neuronal communication and cognition, yet synaptic function declines during normal aging, increasing vulnerability to neurologic disease. How the SV cycle is maintained across the lifespan of a complex organism remains unclear. Here, we used wild-type mice (C57BL/6J) to define the age- and sex-stratified molecular landscape of SVs and identified apolipoprotein E (APOE) as an abundant presynaptic protein further enriched in aged female samples. Super-resolution imaging, cell-type selective expression, and protease protection assays demonstrate that APOE originates from astroglia and associates with the cytosolic face of SVs. Using iGluSnFR and pHluorin optophysiology, we find that both decreased and increased APOE levels impair neurotransmission during stimulus trains. Together, these findings place APOE at the synapse and establish it as a cell-nonautonomous regulator of the SV cycle.

The discovery and collection of the enigmatic Golden Orb by the NOAA Ship Okeanos Explorer and ROV Deep Discover in deep Alaskan waters during 2023 has yielded substantial interest by the scientific and public communities alike. Initial field identifications of the specimen collected at 3,250 meters depth ranged from an egg mass to sponge to microbial biofilm. Here we characterize the biology and ecology of the Golden Orb, as well as other specimens of similar appearance identified since the collection of the original material. Through an integrative taxonomic approach including morphological analysis and genomic characterization of the Golden Orb, we identified the presence of cnidocytes of the spirocyst type (restricted to Hexacorallia), as well as metazoan DNA, from which we were able to derive complete mitochondrial genomes and Ultra Conserved Elements. These results indicate that the Golden Orb and a similar specimen from deep equatorial waters represent remnant cuticles belonging to the geographically widespread deep-sea anemone ally Relicanthus daphneae. We also document the presence of cuticle from a collected specimen of R. daphneae from the Southern Ocean and in situ photographic evidence of similar cuticles beneath living individuals. These findings underscore the extent to which the biodiversity and organismal biology of obscure deep sea fauna broadly remain unresolved and highlight the value of whole-specimen collections and rigorous taxonomic follow-up in telepresence-enabled ocean exploration.

Pathogenic coronaviruses profoundly rewire host cell metabolism to support viral replication, yet whether these metabolic alterations expose shared and actionable vulnerabilities remains unclear. By integrating transcriptomic profiles from cells infected with SARS-CoV, SARS-CoV-2, and MERS-CoV with genome-scale metabolic models, we identify conserved and virus-specific metabolic perturbations affecting mitochondrial transport, nucleotide biosynthesis, fatty acid metabolism, and redox balance. Despite distinct transcriptional responses, all three viruses converge on a limited set of metabolic reactions whose flux ranges deviate strongly from healthy states. Using a network-based predictive framework, we systematically identify gene-pair perturbations that restore perturbed reaction fluxes toward non-infected metabolic states. Predicted rescue mechanisms reveal shared metabolic dependencies across coronaviruses, as well as time-dependent virus-specific vulnerabilities, and nominate druggable host targets. Notably, several top predictions align with independent experimental and clinical evidence, including metabolic interventions shown to reduce viral replication or disease severity in COVID-19 patients. Together, our results define conserved metabolic rescue pathways in coronavirus infection and provide a general strategy for identifying host-directed therapeutic opportunities from transcriptomic data. HighlightsO_LICoronaviruses converge on shared metabolic vulnerabilities in host cells C_LIO_LINiTRO predicts gene pairs that rescue viral-induced metabolic states C_LIO_LIMitochondrial transport emerges as a key pan-coronavirus target C_LIO_LITop predictions validated by clinical trials and in vitro evidence C_LI eTOC BlurbDohai et al. develop NiTRO, a network-based algorithm that integrates coronavirus-induced transcriptomic changes with genome-scale metabolic models to identify gene-pair perturbations capable of rescuing infected metabolic states. The approach reveals shared and virus-specific druggable metabolic vulnerabilities, with top predictions corroborated by clinical evidence.

Germline pathogenic variants in BRCA1 and BRCA2 confer disproportionately elevated cancer risks in breast and ovarian tissues, yet the basis for this tissue specificity remains incompletely understood. Here, we integrate bulk-tumor aneuploidy analysis across 340,824 cancer cases from three independent cohorts (TCGA, ICGC PCAWG, and FoundationCore) with single-cell whole-genome sequencing from two independent studies to investigate whether tissue-specific patterns of chromosomal deletion contribute to this phenomenon. We find that breast and ovarian cancers are consistently enriched for deletions of chromosome arms 17q and 13q--harboring the BRCA1 and BRCA2 genes, respectively--relative to other solid tumor types, and that mutational timing analysis independently places these deletions among the earliest somatic events in these cancers. Phylogenetic reconstruction of single-cell data reveals that in pre-malignant breast tissue from germline BRCA1/2 carriers, chr17q and chr13q deletions appear as localized subclonal events within small clades against a largely diploid background. In established malignancies, these same deletions are found within dominant clonal lineages accompanied by widespread genomic instability--consistent with clonal sweeps originating from early deletion events. These findings suggest that breast and ovarian cellular environments confer a selective advantage for chr17q and chr13q deletions, providing a mechanism that may contribute to the tissue-specific cancer risk observed in gBRCA1/2 carriers.

Successful chromosome segregation depends on robust kinetochore-microtubule attachments. The outer kinetochore load-bearers Ndc80 and Ska complexes functionally cooperate, but the molecular basis of their interaction remains elusive. Here, we combine cryo-EM and functional investigations of Ndc80:Ska on microtubules. Ndc80 forms longitudinal arrays along single protofilaments using two modules. The HEC1 N-terminal tail stabilizes interactions between microtubule-binding heads regulated by Aurora B. The HEC1 loop, away from microtubules, organizes Ndc80 coiled-coils into stacks matching the periodicity of tubulin subunits. SkaC binds to a previously unknown interface of Ndc80 as well as to microtubules, simultaneously stapling tubulin dimers longitudinally and neighboring protofilaments laterally. Our work demonstrates how several weak interactions of a small number of individual complexes are harnessed to generate a robust and regulated kinetochore coupler.

Widespread genomic sequencing efforts have characterized the molecular foundations of the different cancers. By combining these genomic data in a manner proportional to the population-level abundances of these different cancers, we estimate the overall abundances of each observed missense and nonsense mutation within the U.S. cancer patient population. We find BRAF V600E (5.2%) is the most common mutation in the cancer patient population, TP53 R175H (1.5%) is the most common tumor suppressor mutation, and APC R876X (0.4%) is the most common nonsense mutation. These values differ largely and significantly from what would be found in a typical pan-cancer analysis, where different cancer types are included out of proportion to population level incidence. We present the full ordered lists of population-level abundances for specific missense and nonsense mutations, and we demonstrate the value of these data by further analyzing high priority genes (e.g., TP53, KRAS, BRAF) and pathways (e.g., RTK/RAS, PI3K, and WNT/{beta}-catenin). Overall, this information is a resource that should benefit the basic science, translational, and clinical cancer research communities.

How conserved stress responses are across the plant kingdom remains poorly understood. Here, we present a kingdom-wide stress transcriptome atlas of 36 Viridiplantae species, from chlorophytes to angiosperms, across nine abiotic and biotic stresses. The atlas integrates reanalyzed public RNA-seq data with new in-house stress experiments on three species representing basal lineages, yielding 13.6 million differential expression calls from over 3,200 manually curated control-treatment comparisons. We find that ancient gene families respond broadly but moderately, while lineage-specific families respond narrowly but intensely, revealing a division of labor in stress gene deployment. Stress response conservation decays with phylogenetic distance yet remains detectable across more than 700 million years of divergence, with upregulated genes diverging faster than downregulated genes. Functional co-occurrence analysis uncovers a deeply conserved growth-defence tradeoff alongside stress-specific transcriptional rewiring. Conserved stress co-expression modules undergo regulatory subfunctionalization through duplication, with whole-genome duplicate pairs preferentially retained within modules. Finally, DNA and RNA foundation models predict stress responsiveness from sequence alone (auROC 0.755), suggesting a partially conserved cis-regulatory code underlying stress responses across the kingdom.

O_LICardiac rate and rhythm reveal how animals adapt physiologically to day-to-day challenges, with consequences for health and fitness. However, these data remain difficult to collect in wild animals, despite their relevance for individual health and fitness. C_LIO_LIHere, we present a system for collecting and transmitting long-term, fine-scaled physiological data in wild animals. We implanted Bluetooth-enabled cardiac and physiological monitor devices in three wild adult female baboons in the Amboseli ecosystem in Kenya and paired these devices with collars that enabled remote data downloads over long-range wide area network (LoRaWAN). C_LIO_LIThe system performed well over >10 months, providing the first long-term cardiac data in wild primates. The baboons showed strong circadian patterns in heart rate, heart rate variability, and activity. We also present data on one female who left her social group for unknown reasons; while alone she exhibited higher heart rate variability, lower activity, and evidence of disrupted sleep. C_LIO_LIIn sum, physiologgers paired with low-energy methods of remote data retrieval are powerful tools for investigating physiology in wild animals on timescales that extend over many months, with minimal disruption to their behavior. C_LI

The scarcity of expandable, functional human islet cells remains a major barrier to diabetes therapy. Here, we identify PROCR+ cells within adult human islets and establish a defined culture system to generate pancreatic islet organoids. These organoids self-organize into -, {beta}-, {delta}-, and PP cells at near-native ratios, exhibit regulated insulin and glucagon secretion, and support exponential in vitro expansion. Single-cell transcriptomics reveals a unique progenitor-like cell population that is transcriptionally primed for endocrine differentiation but shares molecular features with fetal trunk cells and endocrine progenitors. When transplanted, the organoids rapidly ameliorate hyperglycemia in diabetic mice. Importantly, in a non-human primate model, intraportal transplantation of these organoids reduced exogenous insulin requirements, restored glucose-stimulated C-peptide secretion, and achieved sustained glycemic control-representing a critical step toward clinical translation. This study provides a strategy for expanding human islet organoids, offering a scalable platform for diabetes treatment, disease modeling, and regenerative medicine.

Acinetobacter baumannii is a high-priority multidrug-resistant pathogen that survives within host cells by hijacking intracellular defense pathways. Here, we identify a previously unrecognized signaling axis linking bacterial invasion to host lysosomal regulation. We show that A. baumannii activates calcium-independent phospholipase A2 (iPLA2), leading to increased lysophosphatidylcholine (LPC) production and calcium influx through the ORAI1 channel, which together drive activation and nuclear translocation of the lysosomal transcription factor EB (TFEB). Pharmacological inhibition or genetic silencing of iPLA2 or ORAI1 markedly impaired TFEB activation and lysosomal biogenesis. Mechanistically, we demonstrate that this pathway is initiated by the outer membrane protein A (OmpA), which promotes bacterial invasion and enhances iPLA2 activity, LPC production, and downstream TFEB signaling. Despite induction of lysosomal biogenesis, A. baumannii persists intracellularly by producing ammonia and alkalinizing the lysosomal environment, thereby counteracting host antibacterial activity. In vivo, infection induces activation of HLH-30, the TFEB ortholog, in Caenorhabditis elegans in an OmpA-dependent manner. Together, our finding define an OmpA-iPLA2-LPC-ORAI1-TFEB signaling axis that coordinates host lipid and calcium signaling with lysosomal responses, while revealing a bacterial counterstrategy that promotes intracellular survival.

Metabolic remodeling is a hallmark of cardiomyopathy, yet which cell types bear the metabolic burden and how cell-type-specific contributions are disrupted remain unclear. Here, we developed a cell-type-resolved genome-scale metabolic flux inference pipeline optimized for post-mitotic cardiac tissue by maximizing ATP synthesis rather than biomass production and applied it to a single-nucleus transcriptomic atlas of human cardiomyopathies (78 donors, 869,449 nuclei). Metabolic impairment in dilated cardiomyopathy (DCM) was most profound in stromal cells, whereas myeloid cells exhibited opposing metabolic activation. DCM- associated impairment followed a genotype-dependent severity gradient from structural gene mutations to pathogenic variant-negative (PVneg) cases. PVneg hearts uniquely harbored 24 altered metabolic pathways not significant in any other genotype. These PVneg-specific signatures were independent of clinical severity, indicating a genotype-intrinsic metabolic program. Extending the analysis to arrhythmogenic cardiomyopathy and hypertrophic cardiomyopathy showed that ATP depletion is shared across cardiomyopathy subtypes, whereas metabolic remodeling differed across disease subtypes. Additionally, gene regulatory network analysis linked these alterations to broad transcription factor (TF) dysregulation and pervasive TF-metabolic coupling across all cell types. These findings redefine PVneg DCM as a metabolically distinct entity and reveal conserved stromal metabolic remodeling across cardiomyopathies, providing a framework for genotype-informed mechanistic stratification.