Biology subjects

Wang, J.

Publications and source records attributed to Wang, J..

At least 379 records · Page 21Linked to original sources

bioRxiv2026

PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

Metastasis remains the leading cause of cancer mortality, yet effective therapies are limited. While therapeutic responses are influenced by organ-specific immune microenvironments, strategies to pharmacologically modulate these niches remain poorly defined. Here, using the clinical-stage inhibitor ABBV-CLS-484 (AC484) as a chemical probe, we demonstrate that systemic PTPN1/2 inhibition remodels the pulmonary myeloid landscape, specifically activating alveolar macrophages (AMs) toward a tumoricidal state. Integrated single-cell/spatial transcriptomics and functional assays reveal that AC484 promotes AM accumulation within metastatic lesions, elevates their IFN{gamma} production and responsiveness, and enhances their tumor-killing activity. Depletion of AMs diminishes the anti-metastatic efficacy of AC484. Mechanistically, inhibiting PTPN1/2 by AC484 amplifies IFN{gamma}-STAT1 signaling in AMs, while disrupting this pathway impairs their tumor control capability. These findings delineate a distinct innate immune axis where PTPN1/2 acts as a molecular "brake" on AM activation, suggesting that pharmacologically unleashing tissue-resident macrophages offers a therapeutic strategy to overcome metastatic progression, particularly in microenvironments where adaptive immunity is insufficient. SignificanceAMs are potent anti-metastatic immune effectors functionally constrained by PTPN1/2. Pharmacologically inhibiting PTPN1/2 amplifies their IFN{gamma}-STAT1 signaling and tumoricidal activity, establishing a strategy to reactivate tissue-resident immunity against metastasis.

immunology↗

bioRxiv2026

BARTsc identifies key transcriptional regulators from single-cell omics data

Inference of transcriptional regulatory mechanisms from single-cell (sc) omics data, such as scRNA-seq, scATAC-seq, and scMultiome, remains an important problem in single-cell biology and functional genomics. Most existing methods for predicting functional transcriptional regulators (TRs) from single-cell data rely on co-expression between regulator and target genes and/or sequence motif enrichment, holding inherent limitations. Here, we present BARTsc, a computational method that accurately predicts functional TRs from clustered single-cell omics data by leveraging a large collection of public ChIP-seq profiles. BARTsc implements a novel framework to infer a cis-regulatory profile from differential genomic features from either unimodal (RNA or ATAC) or bimodal (scMultiome) single-cell profiling data and identify TRs whose binding profiles most associate with the cis-regulatory profile. BARTsc can quantify TR activity across cell clusters and predict key regulators for each cell cluster. We demonstrate that BARTsc can successfully identify active TRs in each cell type and cell-type-defining key regulators across diverse biological systems, including mouse cortex, human peripheral blood mononuclear cells (PBMCs), and human pancreatic ductal adenocarcinoma (PDAC). Using a generative-AI-assisted, literature-supported collection of cell-type key regulators as benchmarks, we show that BARTsc consistently outperforms existing state-of-the-art methods. We apply BARTsc to identify critical regulators in PDAC, including NEFLA, a novel PDAC key regulator, and validate its function in pancreatic tumor proliferation by experiments. As a robust and versatile computational method, BARTsc provides deeper insights into cell-type-specific regulatory programs, facilitating the discovery of key regulators across diverse biological systems.

bioinformatics↗

bioRxiv2026

Tipping Points and Emergent Phases of Species Diversity in Mutualistic Ecological Networks under Global Warming

Global warming poses a severe threat to ecosystem resilience and biodiversity, potentially driving species loss, declining diversity, and ecosystem collapse. Accurately predicting tipping points of ecological resilience under global warming remains a fundamental challenge. Here, we develop a theoretical framework to identify climate-induced tipping points in mutualistic ecological networks, capturing critical transitions under warming scenarios. We show that rising temperatures, increasing intraspecific competition, and deteriorating mutualistic interaction can drive ecosystems through three distinct dynamical phases of species diversity: stable full coexistence, stable partial coexistence, and total extinction. Notably, ecosystems with low species abundances are particularly vulnerable, facing heightened risks of diversity loss and extinction. Moreover, the warming rate modulates these transitions, as slower warming grants endangered species a window of time within which to adjust and attenuate the loss of resilience. Applying this framework to assess various emission mitigation pathways, we find that "pollute first, clean up later" scenarios pose a threat to biodiversity, leading to irreversible biodiversity losses even after subsequent emission reductions. Our findings offer critical insights into the timing and mechanisms of ecological tipping points under climate change and underscore the urgency of proactive mitigation strategies.

ecology↗

bioRxiv2026

Discovery and dynamic pharmacology of μ-opioid receptor positive allosteric modulators

Opioid agonists such as morphine and fentanyl exert analgesic effects by binding and activating the {micro}-opioid receptor ({micro}OR), yet agonism of the {micro}OR causes a slate of serious side effects. {micro}OR-mediated addiction and respiratory depression are the major causes of the current opioid overdose crisis, largely driven by the explosion in illicit use of fentanyl, a potent opioid receptor full agonist. Given these serious side effects (and high resulting societal cost), molecules that act as analgesics with distinct mechanisms of action are of great interest. Positive allosteric modulators (PAMs) of the {micro}OR have the potential to avoid many off-target side effects of conventional opioid orthosteric agonists by enhancing the signaling properties of natural opioid peptide systems. We used a DNA-encoded chemical library screening approach to selectively discover active-state-specific {micro}OR PAMs. Two out of 3 selected prospective PAMs displayed the anticipated enhancement in agonist activity. The most effective of these compounds enhanced the activity of all orthosteric opioid agonists tested, including the native opioid peptide met-enkephalin. Little is known about the underlying dynamic basis of allosteric modulation of Family A GPCRs like the {micro}OR. To that end, we used single-molecule fluorescence resonance energy transfer experiments to detail the impact that our novel {micro}OR PAM has on the dynamic activation behavior of a key region on the intracellular face of the receptor. Our results here provide both a new chemical scaffold that acts as a {micro}OR PAM and detailed pharmacological and dynamic insights into its mechanism of action.

biophysics↗

bioRxiv2026

Cell Type Architecture and Positional Gene Gradients in an Adult Animal at Subcellular Resolution

The spatial organization of cell types and gene expression underlies tissue architecture and organismal physiology. However, resolving complete cellular and molecular landscapes within a three-dimensional context remains challenging, particularly in organisms with high cellular heterogeneity and complex geometry. Here, we developed a semi-supervised workflow to reconstruct high-resolution three-dimensional spatial transcriptomic models of the planarian Schmidtea mediterranea at single-cell resolution. Planarians are basal bilaterians capable of regenerating body structures after injury. Our reconstruction maps the distribution of cell types and spatially patterned gene expression across the intact organism and identifies 119 candidate positional control genes (PCGs). These genes are expressed across muscle, neural, and epidermal populations. Functional perturbation of selected candidates, including pitx3, ptpn11, pi4ka, upf3b, and cul1, revealed roles in regeneration following injury. In addition, we identify intestinal cells as prominent components of the microenvironment surrounding adult tgs1 pluripotent stem cells (neoblasts). Together, these results demonstrate the utility of three-dimensional spatial transcriptomic reconstruction for mapping cellular architecture and positional gene regulation in complex adult organisms.

developmental biology↗

bioRxiv2026

Distinct amygdalar pathways to frontopolar cortex and medial temporal lobe support temporal memory for emotional events

Growing evidence suggests that emotion shapes temporal aspects of memory--such as remembering when an event occurred--yet the neural bases of these effects remain unclear. Prior work indicates that temporal-context representations in memory are supported by the function of medial temporal lobe (MTL) and prefrontal regions with privileged access to amygdala-originated affective signals, which may mediate emotional influences on temporal memory--including hippocampus, perirhinal cortex, and lateral frontopolar cortex (FPl). To test whether these candidate pathways shape temporal memory for emotional events, participants encoded trial-unique negative and positive images in the scanner, followed by a surprise temporal memory task. We found that more negative stimuli were associated with greater amygdala activation and lower temporal-memory error for when emotional images occurred. Replicating and extending prior work, higher stimulus-evoked multivoxel pattern similarity in the hippocampus (between trials) and FPl (within and between trials) was associated with higher temporal memory errors. Notably, greater amygdala responding to emotional images predicted higher between-trial pattern similarity in the hippocampus, but lower within- and between-trial similarity in FPl. Amygdala engagement was also correlated with greater perirhinal activation, which in turn predicted stronger item memory and a recency bias in temporal memory estimates. Collectively, these findings reveal distinct--and opposing--modulatory effects of amygdala-FPl and amygdala-MTL pathways on temporal memory, with the potential to sharpen and blur memory for when emotional events occurred. Significance statementEmotional events influence how we remember time. Prior work implicates function of the lateral frontal pole (FPl) and medial temporal lobe (MTL) regions--including hippocampus and perirhinal cortex--in temporal memory. Yet, whether and how these areas support temporal memory for emotional events remains unclear. Using trial-unique emotional images, we found that negative-emotional valence was associated with stronger amygdala activation and temporal-memory accuracy. Higher pattern similarity in hippocampus and FPl predicted worse temporal memory. Notably, amygdala activation predicted higher between-trial hippocampal pattern similarity but lower within-trial FPl similarity. Perirhinal engagement, which correlated with amygdala activation and item memory, predicted a recency bias in temporal-memory judgments. Together, these findings reveal distinct amygdala-MTL and amygdala-prefrontal pathways through which emotion shapes temporal memory.

neuroscience↗

bioRxiv2026

Super-resolution single-cell spatial atlas of plant de novo regeneration

Plant de novo regeneration, a capacity absent in animals, relies on stem-cell-niche formation rather than pre-existing reservoirs. Growth hormones such as auxin and cytokinin, together with numerous genetic regulators, participate in regeneration, but the architectural principles coordinating extensive cellular reprogramming at the individual-cell scale remain elusive. Using super-resolution multimodal spatial transcriptomics to 1.16 million cells, we tracked tomato regeneration across temporal intervals from wounding to organogenesis. Datasets are available at http://www.single-cell-spatial.com.

cell biology↗

bioRxiv2026

Experimental Time Points Guided Transcriptomic Velocity Inference

Time-series single-cell RNA sequencing enables longitudinal tracking of biological processes, yet cellular trajectory reconstruction informed by experimental time remains challenging. Existing trajectory inference methods either perform de novo reconstruction without leveraging experimental time points, or prioritize transitions between time points while paying less attention to intra-time-point dynamics. To reconcile experimental time points with local precision, we present CellDyc, a semi-supervised learning framework that leverages experimental time-point supervision to reconstruct transcriptomic velocities and recover an intrinsic gene-embedded time. CellDyc consistently outperforms existing approaches in reconstructing cellular trajectories across development, disease, and reprogramming contexts. Biologically, CellDyc provides novel insights, such as resolving temporal heterogeneity in erythroid maturation and quantitatively demonstrating that the immunosuppressive environment delays monocyte differentiation in glioblastoma. CellDyc integrates seamlessly with downstream tools like CellRank and remains robust even when only inferred temporal information is available. Collectively, CellDyc offers a rigorous, data-driven solution for deciphering time-resolved cellular dynamics.

bioinformatics↗

bioRxiv2026

ATLAS: Graph-based 3D RNA Motif Library Incorporating non-Watson-Crick Interactions

The structures of RNA exhibit recurrent patterns, which are defined as motifs. Motifs are crucial for the biological functions of RNA molecules. The 3D RNA motif library includes 3D RNA structures, providing a resource for motif-based RNA 3D structure prediction and design. We built the Advanced Template Library for Assembly and Structure (ATLAS) that features graph representations of RNA structures, atomic 3D structures from the Protein Data Bank (PDB), and an isomorphism graph searching algorithm. ATLAS includes nucleotide-level graph representations of RNA motifs and corresponding PDB IDs from the PDB and 3D atomic structures retrieved from the PDB. We provide the web service to search for and download RNA motifs and user-defined structures, supporting non-WC interactions. Graph representations include two types of graphs: motifs with non-Watson-Crick (non-WC) and without non-WC. Compared to existing motif libraries, ATLAS includes graph representations with non-Watson-Crick interactions and pseudoknots and integrates the latest RNA structures from the PDB. We also develop a measure of similarity between RNAs based on shared motifs. We proposed a physics-inspired, time-dependent mechanistic model for RNA evolution, whose steady-state solution fits the global distribution of RNA structural similarity.

biophysics↗

bioRxiv2026

4D Single-Cell Spatial Transcriptomics Reveals Dynamic Morphogenetic Gradients and Regenerative Domains in Planarians

Regeneration relies on precise spatiotemporal gene expression and cellular responses to establish tissue identity and body patterning. Using high-resolution Stereo-seq (715 nm) on 353 sections from 16 whole animals at 8 regeneration timepoints, we constructed a 4D spatiotemporal transcriptomic map of planarian regeneration. Our analysis captured 36 refined cell types from 3,508,004 segmented cells, enabling genome-wide transcriptional imputation of gene expression dynamics across body axes at cellular, tissue, and organismal scales. We identified dynamic positional gradients and distinct spatially distributed cell types during regeneration, including an injury-induced Anterior Regenerative Zone (ARZ). The ARZ exhibited enriched positional signals in epidermal, muscle, and neural cells and was regulated by Mediator 8, which is crucial for polarity remodeling and blastema formation. This study provides a comprehensive spatial molecular and cellular map of regenerative processes, highlighting injury-induced spatial domains and key regulatory factors in planarian regeneration. We also provide an interactive web portal, offering a valuable resource for exploring and analyzing regeneration mechanisms in a spatiotemporal context.

developmental biology↗

bioRxiv2026

Potassium-Selective Nanoelectrode Arrays for Single-Cell Profiling of human iPSC-Derived Cardiomyocytes

Potassium ion (K) dynamics are central to cardiac electrophysiology, with early disruptions in K flux often preceding arrhythmia and contractile dysfunction. However, current sensing technologies, such as patch-clamp, Microelectrode arrays (MEAs), and fluorescent indicators, either lack chemical specificity for K or are unsuitable for long-term, single-cell analysis. Conventional ion-selective electrodes (ISEs), while more selective, are limited by bulk-phase design and poor spatial resolution. To address these limitations, we present KINESIS (K-Ion Nano-Electrode Selective Interface System), a nanofabricated, cell-compliant platform that enables direct, label-free potentiometric measurement of K gradients with subcellular precision. KINESIS features high-aspect-ratio nanopillars coated with a valinomycin-based K recognition membrane, forming a stable, non-invasive interface with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). This architecture allows localized, Nernstian sensing of K efflux or depletion without disrupting cell membranes. Pharmacological validation shows distinct potential shifts in response to caffeine and ouabain. KINESIS thus offers a highly selective, spatially resolved approach for studying K handling in cardiotoxicity screening and patient-specific disease modeling.

pharmacology and toxicology↗

bioRxiv2026

A PERK/FOXO1 AXIS LINKS DNA DAMAGE TO FIBROBLAST SURVIVAL IN DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS

ObjectiveDiffuse cutaneous systemic sclerosis (dcSSc) is a life-limiting fibrotic disease. We and others have shown that dcSSc fibroblasts accumulate numerous somatic mutations associated with senescence-like features; however, the mechanism(s) enabling their survival remain unclear. MethodsSkin biopsies were obtained from lesional tissues from dcSSc (n=10), dcSSc treated with autologous hematopoietic stem cell transplantation (ASCT, n=8) or 7 age/sex-matched healthy controls. Primary dermal fibroblasts were generated from biopsies. Spatial RNA sequencing, immunoblotting, confocal microscopy, and functional assays were used to mechanistically delineate signaling pathways linking DNA-damage with fibroblast survival. ResultsdcSSc fibroblasts demonstrated increased pH2AX DNA double-strand-break foci yet remained apoptosis resistant. These cells displayed features of metabolic-stress remodeling, including mitochondrial hyperpolarization, increased reactive oxygen species production, and enhanced mitochondrial biogenesis. Spatial transcriptomics and subsequent biochemical analyses identified activation of a PERK/ATF4/FOXO1 axis, characterized by PERK phosphorylation, selective ATF4 translation, FOXO1 nuclear translocation, and induction of downstream antioxidant and metabolic programs. In contrast, fibroblasts from post-ASCT patients exhibited normalization of DNA-damage markers and mitochondrial parameters without ATF4/FOXO1 activation. Pharmacologic inhibition of either PERK or FOXO1 selectively restored mitochondrial-dependent apoptosis in dcSSc fibroblasts, demonstrating that this axis is required for their survival following extensive genomic injury. ConclusiondcSSc fibroblasts persist despite substantial genomic injury by engaging a PERK/ATF4/FOXO1 metabolic-adaptation program that suppresses mitochondrial-dependent apoptosis. This survival axis is not present after ASCT. Targeting PERK or FOXO1 restores apoptosis selectively in dcSSc fibroblasts, highlighting its potential use as a therapeutic target for eliminating pathogenic senescence-like fibroblasts in dcSSc. HighlightsO_LIBoth ex-vivo skin and in-vitro primary dermal fibroblasts derived from dcSSc patients have a higher frequency of intrinsic DNA damage signals and senescence-associated features; yet they evade mitochondrial-dependent apoptosis. C_LIO_LIPathogenic dcSSc fibroblasts rewire their metabolism, characterized by mitochondrial hyperpolarization and elevated ROS. C_LIO_LISpatial transcriptomics and functional analyses reveal a PERK/ATF4/FOXO1 stress-adaptation axis that drives fibroblast survival in dcSSc. C_LIO_LIThis maladaptive survival program characterized by increased genotoxic stress, and mitochondrial remodelling is absent in post-ASCT fibroblasts. C_LIO_LITargeting PERK or FOXO1 selectively sensitizes dcSSc fibroblasts to apoptosis revealing a potential promising therapeutic strategy in dcSSc. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/706443v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1b3f791org.highwire.dtl.DTLVardef@42548aorg.highwire.dtl.DTLVardef@bc4ce1org.highwire.dtl.DTLVardef@5b4d48_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Sub-stoichiometric Degradation is Dispensable for Potent PROTACs: A Case Study for Irreversible Covalent BTK Degraders

Proteolysis-targeting chimeras (PROTACs) represent a transformative therapeutic modality, yet the viability of covalent PROTACs remains debated, as irreversible binding seemingly contradicts the catalytic mechanism central to their function. Here, we develop and characterize PSIRC3, a highly potent covalent PROTAC for Brutons tyrosine kinase (BTK) that addresses this ambiguity. PSIRC3 induces potent and selective BTK degradation with a sub-nanomolar DC50 of 0.75 nM and a Dmax greater than 85%, while its non-covalent counterpart is completely inactive. This degradation activity is strictly dependent on covalent bond formation with the Cys481 residue, as evidenced by a total loss of efficacy against the C481S BTK mutant. PSIRC3 acts with remarkable speed, achieving maximum BTK degradation within 30 minutes, a kinetic profile linked to rapid cell permeation and efficient ternary complex formation. In vivo, a single administration of PSIRC3 leads to substantial BTK degradation in both PBMCs (>80%) and splenocytes (>50%). Computational modeling, parameterized with experimental data, reveals that degradation efficacy is governed by a delicate balance between E3 ligase and target protein affinities. Specifically, excessively high E3 affinity is detrimental by inducing a hook effect, while higher target affinity is generally beneficial. Our findings provide strong evidence that covalent engagement can drive potent and selective protein degradation, challenging the prevailing notion that catalytic turnover is indispensable for PROTAC efficacy. This work establishes a new benchmark for covalent degraders and opens new avenues for targeting previously intractable proteins.

biochemistry↗

bioRxiv2026

A novel uN2CpolyG Transgenic Mouse Model Recapitulates Multisystemic polyG Proteinopathy Pathology of Neuronal Intranuclear Inclusion Disease

Neuronal intranuclear inclusion disease (NIID) is a polyglycine disease that primarily affects the neuronal and neuromuscular systems. Here, we developed a novel transgenic mouse model that faithfully recapitulates the multisystemic impairments associated with polyG intranuclear inclusions. Our findings demonstrate that polyG expression induces neurodegeneration, behavioral deficits, and age-dependent accumulation of uN2CpolyG aggregates across multiple tissues.

animal behavior and cognition↗

bioRxiv2026

Elimination of intramuscular immunoglobulin accumulation alleviates Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disorder due to loss of dystrophin, a cytoskeletal protein critical for muscle integrity and functionality. Despite recent therapeutic advances, there remains a significant unmet need for more effective and accessible therapeutics. Here, we discovered an early accumulation of immunoglobulin G (IgG) in the sarcolemma, which exacerbates tissue inflammation and disease progression. The IgG accumulation primarily resulted from ectopic localization of Fc{gamma}R1, a high-affinity IgG Fc receptor, on dystrophin-deficient myofibers. In two independent murine models, eliminating IgG accumulation via B cell depletion provided sustained benefit to alleviate DMD progression. Our findings uncover a novel disease accelerator in DMD and demonstrate the potential to target this mechanism as therapeutics for broader population of patients with DMD.

immunology↗

bioRxiv2026

Lipid monounsaturation confers cold tolerance and improves tissue viability during hypothermic storage

The rapid loss of tissue viability during hypothermic storage frequently results in organ failure and high discard rates in transplantation. In contrast, ectothermic animals can tolerate prolonged exposure to low temperatures in their natural environments. Elucidating the mechanisms underlying this cold tolerance may therefore inform improved strategies for tissue preservation. Here, we investigate proteomic and metabolomic responses to cold exposure in the livers of two frog species from distinct habitats: the cold-sensitive African clawed frog (Xenopus laevis) and the cold-tolerant Northeastern Asian brown frog (Rana dybowskii). Cold exposure induced lipid mobilization in X. laevis, whereas it promoted phospholipid mono-unsaturation in R. dybowskii. Notably, treatment with monounsaturated fatty acids (MUFAs) or overexpression of stearoyl-CoA desaturase (SCD) reduced cell death during cold storage. Moreover, MUFA infusion significantly improved cell viability in mouse liver tissue under hypothermic conditions. Together, these findings suggest that lipid mono-unsaturation, an adaptive feature of cold-tolerant frogs, can be leveraged to enhance tissue preservation during cold storage.

biochemistry↗

bioRxiv2026

Repeat expansions in C9orf72 rewire the 3D chromatin landscape in ALS

Amyotrophic lateral sclerosis (ALS) is frequently driven by GGGGCC short tandem repeat (STR) expansions in C9orf72, yet the mechanisms by which these expansions lead to neurodegeneration remain incompletely understood. Here, we propose a novel mechanism involving higher-order chromatin architecture where C9orf72-STR expansions induce widespread, neuron-specific gains in chromatin loops that are closely linked to transcriptomic dysregulation in ALS. These ectopic loops colocalize with the genomic binding sites of C9orf72-STR RNAs and the architectural protein CTCF, supporting a model in which RNA-DNA interactions promote aberrant loop formation. Together, our findings demonstrate how C9orf72-STR expansions remodel the neuronal genome and disrupt gene expression, uncovering an RNA-driven mechanism of chromatin reorganization in C9-ALS that connects altered nuclear topology to gene dysregulation in neurodegeneration.

genomics↗

bioRxiv2026

Chlamydial Histones Control Developmental Fitness in the Next Infection Cycle

The unique chlamydial developmental cycle comprises three stages: primary differentiation of infectious elementary bodies (EBs) into reticulate bodies (RBs), RB replication, and secondary differentiation into progeny EBs. Extensive chromosome remodeling during RB-to-EB differentiation is thought to be mediated by the histones HctA and HctB. Here, we used an inducible CRISPR interference system to repress hctA, hctB, or both genes during development in Chlamydia trachomatis. Surprisingly, repression of either histone gene alone or in combination caused only modest reductions in EB yield and did not prevent nucleoid condensation during the parental developmental cycle. In contrast, when progeny EBs generated under histone-repressing conditions were used to initiate secondary infections in the absence of inducer, histone deficiency during EB maturation profoundly impaired fitness in the next infection cycle. Secondary cultures initiated with HctA-deficient EBs exhibited a delayed onset of genome replication, consistent with inefficient primary EB-to-RB differentiation, whereas combined repression of hctA and hctB caused both delayed genome replication and persistently reduced genome accumulation, indicative of defects in RB formation and subsequent growth. Repression of hctB alone did not measurably affect genome replication in secondary cultures. Together, these findings reveal a transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles. IMPORTANCEChlamydial histones HctA and HctB are unusual among bacterial chromatin-binding proteins in that they share sequence homology with mammalian histones and are developmentally regulated during the formation of infectious particles. Here, we show that reduced expression of HctA and HctB has only modest effects on genome condensation and EB production, consistent with partial functional redundancy between the two histones and suggesting that additional chromatin factors contribute to EB chromosome compaction. In contrast, deficiency of HctA and HctB during EB maturation has profound consequences in the next infection cycle, impairing primary EB-to-RB differentiation and subsequent RB growth. These findings reveal a previously unrecognized transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles.

microbiology↗