Biology subjects

Lim, B.

Publications and source records attributed to Lim, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Insulators As Dynamic, Tunable Regulators of Enhancer-Promoter Coordination in Living Drosophila Embryos

The three-dimensional organization of the genome enables enhancers and promoters to interact across vast distances and direct transcription. Yet whether architectural elements, such as insulators, serve as rigid, passive barriers or as dynamic, active organizers of this communication remains unclear. Here, using single-cell, live imaging of a Drosophila transgene in which a single enhancer regulates two equidistant promoters, we confirm that the enhancer engages both promoters simultaneously and show that coordinated bursting is intrinsically more productive than uncoordinated activity. Flanking this system with insulators increases coordinated bursting frequency and transcriptional output, indicating that insulator-mediated looping promotes multi-way enhancer-promoter interaction. Further, bidirectionally-paired, homotypic insulators produce stronger coordination than unidirectional pairs. Inserting an intermediate insulator to generate competing loop configurations, together with two-state promoter modeling, we show that these chromatin loops are highly dynamic. This work reframes insulators as active, tunable regulators that shape the frequency, coordination, and productivity of enhancer-promoter communication.

molecular biology↗

bioRxiv2026

Integrated genomics and transcriptomics reveal mechanisms of extreme dietary adaptation in vampire bats

Vampire bats are the only tetrapods that feed exclusively on blood. To uncover the molecular basis of this extreme dietary specialization, we generated six new reference genomes, including genomes of all three vampire bat species, and integrated comparative analyses of gene sequence evolution (selection signatures, duplications, and losses) with transcriptomic data from six major organs to identify shifts in gene expression. Our integrative analyses reveal sequence or expression changes in 150 genes that illuminate the genetic mechanisms underlying sanguivory. Through comparative analyses and experiments, we show that the enlarged vampire bat stomach has increased connective tissue content enabling extreme expansion, is pH-neutral, and exhibits reduced mucus production, together providing molecular insights into its shift from a digestive to an absorptive organ for water, electrolytes, and vitamins. We further uncover pathway-level molecular changes underlying altered gastrointestinal motility; trypsin-dependent protein digestion; upregulated amino acid catabolism with key aspects diverging from other mammals; impaired dietary fat digestion counterbalanced by increased fatty acid synthesis; defective sugar metabolism and natural insulin deficiency; enhanced heme iron absorption; and adult splenic erythropoiesis. Together, these findings reveal the molecular adaptations that enable one of the most extreme dietary transitions among vertebrates.

evolutionary biology↗

bioRxiv2026

An IL-34-IGF-1 inflammatory axis fuels KRAS-mutant lung cancer progression

Macrophages are innate immune cells of embryonic or adult origin with tissue specific roles in homeostasis, disease surveillance, and wound repair that can be co-opted to promote tumor growth and spread1-11. An understanding of the specific roles of macrophage subsets in lung tumor initiation and progression could promote new therapeutic approaches for this deadly disease. Here, we show that KRASG12D mutations in lung epithelium drive proliferation of resident, embryonically-derived alveolar macrophages, which then promote tumor cell proliferation and protection from ferroptosis, leading to tumor progression. Using genetically engineered mouse models of mutant KRASG12D non-small cell lung cancer12,13, we found that alveolar macrophages accumulate by proliferation in response to tumor cell-secreted IL-34, recapitulating events observed in late embryonic lung development. Tumor alveolar macrophages in turn drive IGF-1-dependent tumor cell proliferation. Neutralization or deletion of IL-34 suppresses IGF-1 expression, reduces macrophage and tumor cell proliferation and inhibits tumor progression. High IL34 and IGF1 correlate with poor survival in KRASG12D/V lung adenocarcinomas and in other solid tumors, indicating that bi-directional proliferative signaling between resident macrophages and tumor cells can drive human lung tumor progression. These studies identify resident macrophage-tumor cell interactions as key interception points for lung cancer therapy.

cancer biology↗

bioRxiv2026

Circulating miR-10b-5p drives Kawasaki vasculitis through endothelial reprogramming and nominates CXCL8 as an early diagnostic biomarker

BackgroundKawasaki disease (KD) is an acute systemic vasculitis of unknown etiology and the leading cause of acquired heart disease in children. Coronary artery aneurysm formation represents its most serious complication, yet no specific biomarker exists for early diagnosis at emergency department (ED) presentation. We sought to define the molecular mechanisms driving KD vasculitis and to identify a clinically actionable early diagnostic marker. MethodsWhole microRNA sequencing was performed on buffy coat specimens from KD patients and febrile controls presenting to the ED. Disease-associated miRNA candidates were validated in two independent murine KD-like vasculitis models: Lactobacillus casei cell wall extract (LCWE; n=12-20) and Candida albicans water-soluble fraction (CAWS; n=12). The functional role of miR-10b-5p was assessed by antagomir-mediated inhibition in the LCWE model. Transcriptomic and chromatin-level reprogramming were characterized by single-cell RNA sequencing (scRNA-seq), bulk RNA-seq, and assay for transposase-accessible chromatin with sequencing (ATAC-seq) in human coronary artery endothelial cells (HCAECs). Transcription factor binding was validated by in vitro binding assays and chromatin immunoprecipitation. Serum chemokine (C-X-C motif) ligand 8 (CXCL8) was quantified by enzyme-linked immunosorbent assay. ResultsForty miRNAs were upregulated in KD patients and fourteen miRNAs were prioritized for further analysis. miR-10b-5p was consistently elevated across both vasculitis models, and its antagomir-mediated inhibition attenuated aortic root inflammation in vivo. In HCAECs, miR-10b-5p suppressed its direct targets marker of proliferation Ki-67 (MKI67)/Chromobox5 (CBX5), driving a cell-state transition from proliferative/metabolic programs toward a pro-inflammatory phenotype. Consistently, analyses of bulk RNA-seq supported cell-cycle arrest and altered chromatin remodeling. ATAC-seq revealed increased chromatin accessibility at the CXCL8 and Matrix metalloproteinases 10 (MMP10) promoters, and motif analysis identified CCAAT/enhancer-binding protein alpha (CEBPA) as the key transcriptional activator; CEBPA knockdown abrogated miR-10b-5p-induced CXCL8 and MMP10 upregulation. Serum CXCL8 was markedly elevated in KD patients relative to febrile controls at first ED presentation, prior to definitive diagnosis, and declined significantly at 8-week follow-up. ConclusionsWe define a miR-10b-5p-MKI67/CBX5-CEBPA-CXCL8 axis as a mechanistic driver of endothelial reprogramming and neutrophil-recruiting inflammation in KD. Serum CXCL8 emerges as a candidate early diagnostic biomarker that may facilitate timely KD recognition at ED presentation.

molecular biology↗

bioRxiv2025

The type VI secretion system governs strain maintenance in a wild mammalian gut microbiome

Bacteria inhabiting the mammalian gut coexist in dense communities where contact-dependent antagonism mechanisms are widespread. The type VI secretion system (T6SS) is an interbacterial toxin delivery pathway prevalent among gut Bacteroidales, yet its function in naturally evolved microbiomes remains poorly defined. Here, we examine the role of the T6SS in Bacteroides within a physiologically relevant gut community derived from wild mice (the WildR microbiome). Using newly developed genetic tools and a strategy for functional replacement of strains within the WildR community, we demonstrate that the WildR isolate B. acidifaciens employs a T6SS to antagonize co-resident Bacteroidales. We also show that loss of T6SS function compromises the long-term maintenance of B. acidifaciens in the community but not its initial colonization, establishing the system as a determinant of strain persistence. The T6SS we identified resides on an integrative and conjugative element (ICE). ICE-seq, a targeted sequencing approach, reveals that the T6SS-ICE is distributed among select Bacteroidales and Muribaculaceae species in the WildR microbiome, between which it appears to be recently exchanged. We also show that transfer of the T6SS-ICE to WildR isolate Phocaeicola vulgatus confers transient colonization benefits in mice, but is linked to eventual population decline. Our findings demonstrate that the T6SS can stabilize the presence of specific strains within a complex, co-evolved gut microbiome, yet its value is context dependent and constrained by the ecological and physiological landscape of the host community.

microbiology↗

bioRxiv2025

Succinate Dehydrogenase-Deficient Cancer Cells Have Increased Susceptibility to Ym155 Induced DNA Damage

The hereditary pheochromocytoma and paraganglioma (hPPGL) syndrome is caused by inherited mutations in Succinate Dehydrogenase genes (SDHx). Affected individuals are predisposed to developing pheochromocytomas (Pheo), paragangliomas (PGL), renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GIST). Notably, tumors with succinate dehydrogenase subunit B (SDHB) deficiency demonstrate increased metastatic risk, for which treatments remain palliative. Hence, discovering novel therapeutic avenues to improve the prognosis for SDHB-cancer patients is an urgent need. Here we employed human SDHB-deficient UOK269 RCC cells (SDHB-KO) and isogenic SDHB-reconstituted control cells (SDHB-WT) to discover SDH-dependent mitochondria-directed cytotoxic agents. Given the reduced ATP-generating capacity of SDHB-KO cells, we hypothesized they would be uniquely sensitive to futile cycle induction with mitochondrial ionophores (2,4-Dinitrophenol (2-DNP), BAM15, Niclosamide, Nitazoxanide). Indeed, these compounds exhibited preferential cytotoxicity toward SDHB-KO cells. However, the chemotherapeutic compound Ym155 demonstrated the most potent and dramatic (five-fold) preferential cytotoxicity towards SDHB-KO cells. Importantly, the SDH-dependent cytotoxicity of Ym155 was validated in both primary human pheochromocytoma cells and mouse pheochromocytoma (MPC) cells. Furthermore, because few SDH-deficient cell lines are available, we buttressed our findings in additional relevant cell lines by modeling SDH-deficiency using chemical SDH enzyme inhibition with 3-nitropropionic acid (3-NPA). We observed a persistent cooperativity between SDH-deficiency and Ym155 cytotoxicity across multiple cell lineages and disease models. Mechanistically, Ym155-induced cytotoxicity was independent of its primary target, Survivin. Instead, SDH-deficiency sensitized cells to Ym155-induced DNA damage. Strikingly, the phenotype of SDH-deficient Ym155 sensitivity was recapitulated by inhibition of the histone demethylase KDM4, a downstream consequence of SDH deficiency. Thus, the accumulation of succinate in SDH-deficient tumors inhibits KDM4 activity, impairs DNA repair and yields enhanced susceptibility to Ym155-induced reactive oxygen species (ROS) generation. The identified intrinsic susceptibilities of SDHB-deficient cancers has the potential to be therapeutically leveraged.

cancer biology↗

bioRxiv2025

Ancient DNA from the Toronto subway deer adds to the extinction list of North America's Ice Age megafauna

The late Pleistocene was a time of global megafaunal extinctions that were particularly severe in North America. The continent lost many mammal taxa, but the validity of several remain ambiguous, including a high proportion of Cervidae taxa. Torontoceros hypogaeus is represented by a single specimen (ROMM75974) discovered in 1976 during excavation work for the Toronto subway in Canada. The species was described based on its unique antler morphology, but the variable nature of that trait and the species near absence in the fossil record leads to uncertainty concerning its systematic relationships. We used ancient DNA to clarify the taxonomic relationship and evolutionary history of T. hypogaeus. We performed mitochondrial and whole genome analyses with related cervids and showed that ROMM75974 has a close affinity, but relatively high divergence from the Odocoileus sister species. While some ambiguity remains, ROMM75974 could represent a distinct Odocoileus species to be included in the list of extinct North American taxa. This unique population was likely adapted to open landscape which was rapidly replaced with dense woodland in this region at the end of the Pleistocene, highlighting the role of climate change in the extinction of megafauna biodiversity at the end of the ice age.

paleontology↗

bioRxiv2025

Programmable multiplexed phosphosignaling networks in bacteria

Natural bacterial two-component systems use crosstalk between histidine kinases to integrate multiple environmental cues, enabling complex multi-signal decisions. In comparison, most synthetic bacterial phosphosignaling circuits remain confined to single-input designs. Here, we establish a bottom-up framework for phosphosignaling circuits using two histidine kinases (2HKs) that converge on a shared response regulator (RR). As a proof of concept, we built a synthetic 2HK-1RR circuit in Escherichia coli that consists of a blue-light sensor (YF1) and an indole-3-aldehyde sensor, integrating opto- and chemo-signals through their shared RR. By using distinct dimerization tags on each HK, tuning HK activities, and reversing HK response modes, we program a spectrum of logic behaviors. These include ternary gates (an inverse count gate, a count gate, and two context gates), as well as a binary NAND gate. This work establishes fundamental phosphosignaling circuit design rules based on two-component systems that expand the scope of synthetic gene circuit design and pave the way towards complex, multi-input environmental biosensors and programmable therapeutics.

synthetic biology↗

bioRxiv2025

Enhancer Placement Impacts Transcriptional Dynamics in Drosophila Embryos

The functional role of enhancer-promoter configurations in transcription regulation remains poorly understood, despite the wide range of linear genomic distance and relative enhancer positioning observed in endogenous contexts. While canonical models suggest that enhancers operate independently of genomic position, technical limitations have obscured insights on transcriptional kinetics. Here, we employ single-cell MS2/MCP-based live imaging in Drosophila embryos to systematically analyze transcriptional dynamics driven by sub-10kb enhancer-promoter arrangements. Kinetic analyses reveal that while linear enhancer-promoter distance moderately tunes transcriptional output, downstream enhancer positioning reduces mRNA output by 70%. Each configuration modulates distinct transcriptional parameters: linear distance governs initiation kinetics, while relative enhancer positioning dictates transcriptional stability. These effects are consistent across varied enhancer and reporter sequences, revealing configuration-dependent cis-regulatory element arrangement as an intrinsic mechanism for transcriptional fine-tuning. This work challenges the paradigm of configuration-independent enhancer function and establishes a framework to dissect the interplay between genome architecture and trans-acting factors.

molecular biology↗

bioRxiv2025

Complementary cortical and thalamic contributions to cell-type-specific striatal activity dynamics during movement

Coordinated motor behavior emerges from information flow across brain regions. How long-range inputs drive cell-type-specific activity within motor circuits remains unclear. The dorsolateral striatum (DLS) contains direct- and indirect-pathway medium spiny neurons (dMSNs and iMSNs) with distinct roles in movement control. In mice performing skilled locomotion, we recorded from dMSNs, iMSNs, and their cortical and thalamic inputs identified by monosynaptic rabies tracing. An RNN classifier and clustering analysis revealed functionally heterogeneous subpopulations in each population, with dMSNs preferentially activated at movement onset and offset, and iMSNs during execution. Cortical and thalamic inputs were preferentially activated during onset/offset and execution, respectively, though dMSN- and iMSN-projecting neurons in each region showed similar patterns. Locomotion phase-specific rhythmic activity was found in a subset of thalamic dMSN-projecting neurons and dMSNs. Cortex or thalamus inactivation reduced MSN activity. These findings suggest that corticostriatal and thalamostriatal inputs convey complementary motor signals via shared and cell-type-specific pathways.

neuroscience↗

bioRxiv2025

Parkinson's disease-vulnerable and -resilient dopamine neurons display opposite responses to excitatory input

Dopamine (DA) neurons of the substantia nigra (SN) are essential for motor control and selectively degenerate in Parkinsons disease (PD). However, DA neurons are molecularly heterogeneous, with some showing greater vulnerability and others resilience. Here, we show that the DA subtype marker Anxa1, identified in mice, labels PD-vulnerable DA neurons in human SN. Using mice, we found that excitatory inputs from subthalamic (STN) and pedunculopontine (PPN) nuclei evoked frequency-dependent excitation in SN GABA neurons, but complex multiphasic DA neuron responses, suggesting heterogeneous DA subtype responses. Indeed, excitatory inputs evoked differential DA responses in striatal subregions, an increase in caudal striatum, but inhibition followed by rebound in dorsolateral striatum. Additionally, PD-resilient Vglut2+ DA neurons were excited by STN/PPN input, while vulnerable Anxa1+ DA neurons were inhibited. These findings demonstrate that DA subtypes are embedded in distinct functional networks, suggesting that some therapeutic interventions may differentially impact vulnerable and resilient DA subtypes.

neuroscience↗

bioRxiv2025

Decoding Plasticity Regulators and Transition Trajectories in Glioblastoma with Single-cell Multiomics

Glioblastoma (GB) is one of the most lethal human cancers, marked by profound intratumoral heterogeneity and near-universal treatment resistance. Cellular plasticity, the capacity of cancer cells to transition between phenotypic states, drives GB progression and resistance. However, the regulatory logic that permits or restricts specific state transitions remains poorly understood. Here, we integrated single-nucleus RNA and chromatin accessibility multi-ome profiles from over one million cells across primary IDH-wildtype GBs and developed scDORI, a scalable deep-learning framework to infer enhancer-driven gene regulatory networks (eGRNs) at single-cell resolution. Our analysis revealed a structured hierarchy of GB cell states governed by distinct regulatory programs, with marked variability in epigenetic plasticity that enables or constrains transitions. Neuronal-like tumor cells emerge as a low plasticity state that deploys active repression, in contrast to more permissive progenitor-like and astrocytic states. We identified the neuronal-like state-specific repressor MYT1L as a key regulator that silences master transcription factors of alternative states. MYT1L gain-of-function in patient-derived GB cells reduced chromatin accessibility, induced neuronal-like identity, and restricted proliferation and invasion in vivo, whereas loss-of-function reactivated plasticity and accelerated malignant features. Our findings delineate the epigenetic architecture and associated transcriptional master regulators that shape GB state trajectories, and establish safeguard repressors such as MYT1L as potential therapeutic targets to constrain malignant plasticity.

cancer biology↗

bioRxiv2025

Regulation of Transcriptional Bursting and Spatial Patterning in Early Drosophila Embryo Development

Nascent RNA synthesis often occurs in periods of high transcriptional activity, interspersed with basal or no activity periods. This phenomenon, known as transcriptional bursting, drives high intercellular variability in gene expression levels. How do key patterning genes in early Drosophila melanogaster embryos overcome this variability to establish precise spatial patterns for tissue development? To address this question, we study single cell transcriptional activity from MS2-based live imaging data of four transgenic constructs (rho, Kr, sna shadow, sna proximal enhancers) and the endogenous eve gene. We developed an algorithm to infer promoter states in hundreds of cells within the embryo using transcriptional activity data. Results show that while mean transcription levels exhibit spatial gradients, the burst duration and interburst timing remain surprisingly invariant across the embryo and different constructs. The time between consecutive bursts was consistent with a memoryless exponential distribution, whereas the burst duration exhibited tighter control with lesser stochastic variations. Our analysis identified two regulatory mechanisms for gene expression gradients: (1) similar burst-timing statistics across genes, with the activity time (the time from the first to the last burst) being modulated to regulate distinct expression levels; (2) for the same gene with different enhancers (sna shadow and sna proximal), we observed changes in the mean burst duration and variability of the inter-burst timing. This study provides a comprehensive approach to analyzing transcriptional bursting kinetics, revealing activity time as a major regulator of spatiotemporal expression patterning in early embryonic development.

systems biology↗

bioRxiv2025

Ischemic Injury Drives Tumor Growth via Accelerated Hematopoietic Aging

BackgroundPatients with peripheral artery disease have increased risk of cancer development. Aging-associated changes in hematopoietic stem and progenitor cells (HSPCs), including inflammation and increased myelopoiesis, are implicated in both cardiovascular disease (CVD) and cancer, but their contributions to CVD-driven tumor progression are unclear. ObjectivesTo study cancer growth following peripheral ischemia and consequent changes within the HSPC bone marrow compartment to uncover mechanisms through which altered hematopoiesis promotes oncogenesis. MethodsMammary cancer cell (E0771) growth was monitored in C57BL/6J mice after hind limb ischemia (HLI) or sham surgery. The tumor immune microenvironment, circulatory immune cells, and HSPC compartment were assessed by flow cytometry. Next-generation single-cell RNA and ATAC sequencing of HSPCs was performed to assess transcriptomic and epigenetic changes. The functional impact on tumor progression and persistence of ischemia-induced epigenetic reprogramming of HSPCs and their myeloid progeny was examined by bone marrow transplantation. ResultsPeripheral ischemia increased monocyte and neutrophil output at the expense of lymphocytes, driven by a shift toward CD150hi myeloid-biased hematopoietic stem cells (HSCs). This was associated with accelerated breast cancer growth and increased accumulation of tumoral immunosuppressive regulatory T cells and monocytes. Increased myelopoiesis was also supported by multiomic analyses showing HLI-induced transcriptional and epigenetic enrichment for inflammatory (NLRP3 inflammasome) and aging-associated (Neogenin-1, Thrombospondin-1) signatures in subsets of monocyte/dendritic progenitors. HLI-accelerated tumor growth and myeloid-skewing was transmissible via bone marrow transplantation, indicating long-term reprogramming of innate immune responses. ConclusionsPeripheral ischemia promotes inflammaging of HSCs and long-lasting alterations to anti-tumoral immunity, accelerating breast tumor growth.

immunology↗

bioRxiv2025

MYC and AP-1 oncogenes synergistically bind enhancers to rewire transcription

The transcription factor c-MYC (MYC) is deregulated in about 70% of human cancers. Through de novo motif discovery analysis on published MYC ChIP-seq datasets from cancer cell lines, we found cell-type specific co-enrichment of the TRE motifs (AP-1 binding sites), alongside MYCs canonical EBOX motif. TRE motifs serve as indirect MYC binding sites in synergy with AP-1, predominantly at enhancers rather than promoters. At elevated MYC levels, as seen in cancers, MYC preferentially occupies TRE sites over EBOX sites at enhancers. Integration of ChIP-seq and RNA-seq data revealed TRE enhancer binding sites are frequently associated with MYC-mediated transcriptional repression. Gene Ontology analysis showed that MYC utilizes TRE sites to transcriptionally rewire cells, modulating cancer hallmarks like proliferation, apoptosis, and cell adhesion. These molecular insights into how increased MYC levels alter its DNA binding preference and gene regulation could inform new therapeutic strategies targeting cancer-specific MYC functions and its co-regulators.

genomics↗

bioRxiv2025

Heterogeneous Sox2 transcriptional dynamics mediate pluripotency maintenance in mESCs in response to LIF signaling perturbations

The LIF signaling pathway and its regulation of internal factors like Sox2 is crucial for maintaining self-renewal and pluripotency in mESCs. However, the direct impact of LIF signaling on Sox2 transcriptional dynamics at the single-cell level remains elusive. Here, we employ PP7/PCP-mediated live imaging to analyze the transcriptional dynamics of Sox2 under perturbation of the LIF signaling pathway at single-cell resolution. Removal of the LIF ligand or addition of a JAK inhibitor heterogeneously affects the cell population, reducing the number of Sox2-active cells, rather than completely abolishing Sox2 expression. Moreover, Sox2-active cells under LIF perturbation exhibit significant reductions in mRNA production per cell. This reduction is characterized by decreased size and frequency of transcriptional bursting, resulting in shorter duration of Sox2 activity. Notably, cells with reduced or absent Sox2 expression demonstrate a significant loss in pluripotency, indicating that a reduction in Sox2 transcription (rather than a complete loss) is sufficient to trigger the transition from embryonic to an early differentiated state. In LIF-perturbed cells with Sox2 expression reduced to about 50% of non-perturbed levels, we observe a binary behavior, with cells either retaining or losing pluripotency-associated traits. Lastly, we find Sox2 expression is transcriptionally inherited across cell cycles, with Sox2-active mother cells more likely to reactivate Sox2 after mitosis compared to Sox2-inactive cells. This robust transcriptional memory is observed independent of LIF signaling perturbation. Our findings provide new insights into the transcriptional regulation of Sox2, advancing our understanding of the quantitative thresholds of gene expression required for pluripotency maintenance and highlighting the power of single-cell approaches to unravel dynamic regulatory mechanisms.

molecular biology↗

bioRxiv2025

3D dynamics of trans enhancer-promoter interactions in living Drosophila embryos reveals spatiotemporal thresholds for transcription activation

While it is well acknowledged that specific enhancer-promoter interactions are essential for transcription, the spatiotemporal thresholds required for transcription initiation remain unclear. Here, we employed the phenomenon of transvection, where an enhancer activates the target gene in trans at the homologous position, to analyze the mechanism of long-range enhancer-promoter interactions. We combined the MS2- and PP7-based RNA labeling with the ParB/parS DNA labeling to simultaneously visualize active transcription and a target DNA locus in living Drosophila embryos. By quantifying the 3D enhancer-promoter distances in nuclei that exhibited active or inactive transcription, we found that sustained proximity between the enhancer and promoter is necessary and sufficient to initiate transcription. The enhancer-promoter proximity was established before transcription initiation and remained stable after the termination, suggesting the localization of an enhancer and the target promoters in a "transcription hub." When transvection occurred, the cis-linked reporter gene showed a delayed activation and reduced mRNA production, suggesting homologous promoter competition in the hub. Our DNA-labeled co-transvection assay sheds insights into the activation criteria for the long-range enhancer-promoter interactions and provides a stepping stone for future endeavors to understand the complex gene regulation in 3D space.

developmental biology↗

bioRxiv2025

Metabolically purified human stem cell-derived hepatocytes reveal distinct effects of Ebola and Lassa viruses

Ebola and Lassa viruses require biosafety-level-4 (BSL4) containment, infect the liver, and cause deadly hemorrhagic fevers. The cellular effects of these viruses, and whether different families of hemorrhagic-fever viruses elicit similar effects, remain fundamental questions in BSL4 virology. Here, we introduce a new metabolic selection approach to create nearly-pure hepatocytes from human pluripotent stem cells, killing non-liver cells by withholding essential nutrients. Unexpectedly, Ebola and Lassa exerted starkly different effects on human hepatocytes. Ebola infection activated the integrated stress response (ISR) and WNT pathways in hepatocytes in vitro and killed them, whereas Lassa did not. Within non-human primates, Ebola likewise infected hepatocytes and activated ISR signaling in vivo. In summary, we present a single-cell transcriptional and chromatin accessibility roadmap of human hepatocyte differentiation, purification, and viral infection.

cell biology↗