Biology subjects

Hu, M.

Publications and source records attributed to Hu, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Evolution of immune infiltration and immune triad formation across early melanoma progression

Tumor-infiltrating lymphocytes (TIL) in melanoma are a clinically important prognostic marker and are routinely used to inform patient care. However, when immune recognition of transformed melanocytes begins and how it evolves during early melanoma progression is not well understood. Using spatial transcriptomics and a unique cohort of patient-matched lesions representing multiple stages of early evolution, we investigate T cells, dendritic cells (DCs) and multi-cell immune "triads" during melanoma initiation. We find that T cell and DC density, triad density, and triad proximity to melanoma cells are elevated in regions of Stage I invasive melanoma as compared to Stage 0 in situ lesions and nevi. Further, we find that triad abundance correlates with both T cell and DC density, but is not predicted by clustering patterns of these cell types over larger spatial scales. We conclude that immune recognition of melanoma and engagement with the lesion significantly increases with progression from Stage 0 to Stage I disease. Further, using nevi that are depth-matched in the dermis to Stage I invasive lesions, we demonstrate this increased immune recognition is not a consequence of dermal invasion alone. This points to a paradigm where immune engagement of invasive melanoma reflects recognition of melanoma cell-intrinsic changes that emerge in Stage I disease.

cancer biology↗

bioRxiv2026

Multi-omics fine-mapping uncovers a pleiotropic intestinal enhancer variant linking PMAIP1 regulation to pig growth and fatness

Genetic loci influencing multiple complex traits often remain difficult to interpret because the underlying functional variants, target genes and tissue contexts are unresolved. Here, we investigated the regulatory basis of a shared signal for average daily gain (ADG) and backfat thickness (BF) in Yorkshire pigs. By integrating GWAS in 7,972 pigs, targeted capture sequencing, LD-based fine-mapping, porcine epigenomic annotations, three-dimensional chromatin interaction maps and functional assays, we narrowed the shared SSC1 signal to a 1.59-Mb interval and functionally prioritized rs334769214 (T>G) as the leading candidate variant. The T>G substitution produced marked allele-specific differences in enhancer activity and nuclear-protein binding, while transcription-factor co-transfection assays identified FOXL1-responsive, allele-dependent regulatory activity. Chromatin interaction and CRISPRa analyses supported a regulatory connection between the rs334769214-containing enhancer and PMAIP1, and PMAIP1 overexpression promoted apoptosis in porcine intestinal epithelial cells. In the capture-sequenced subset, the G allele was associated with higher ADG and BF, consistent with a relative loss-of-activation model. Together, these findings delineate a candidate FOXL1-enhancer-PMAIP1 regulatory pathway and demonstrate how multi-omics fine-mapping can advance a shared quantitative-trait locus toward a testable molecular mechanism underlying variation in growth and fatness.

genetics↗

bioRxiv2026

Base-mismatched crRNA in CRISPR Enabled Universal One-pot Rapid Nucleic Acid Detection

CRISPR-Cas12a-based diagnostics are promising for field detection yet are often constrained by two step workflows or laborious optimization of one-pot reactions. Herein, we present a universal one-pot RPA/CRISPR-Cas12a strategy using base-mismatched crRNA in the seed region (positions 1-6). Introducing of single-nucleotide mismatches (complementary or error) into crRNA suppresses cis-cleavage activity, enabling amplification and detection in a single tube without PAM re-design or costly modified crRNA. Molecular dynamics (MD) simulations indicated that moderate potential energy (PE) variation correlates with improved screening performance, providing guidance for the selection of effective mismatches. This strategy successfully converted established two-step assays for Burkholderia pseudomallei and Brucella into one-pot formats, achieving detection of targets at 10 copies/L within 30 min with good specificity. Lyophilized pellets supported field testing on lithium-ion battery-powered devices, and experiments with simulated soil samples confirm the robustness of the approach. Together, this cost-effective, adaptable strategy can streamline RPA-CRISPR diagnostics for infectious disease surveillance.

molecular biology↗

bioRxiv2026

Stressed alga-free coral cells can initiate coral bleaching

Coral bleaching is driving the rapid decline of coral reefs worldwide, yet a fundamental question remains unresolved: does the bleaching cascade begin in the coral host or in its intracellular algal symbionts, Symbiodiniaceae? Resolving where this process starts is critical for understanding how symbiosis collapses and where early intervention might be effective. Here we use transient stress to create a temporal window between the early cellular response and subsequent symbiosis breakdown. Across transient menthol and thermal stress in soft and stony corals, bleaching developed after the stress had ended. In Xenia sp., single-cell transcriptomics revealed a broad host response before detectable symbiont loss, including coordinated redox changes consistent with conditions favouring reactive oxygen species (ROS) accumulation. Cell-resolved imaging showed that alga-free coral cells were the first to become ROS-positive, whereas Symbiodiniaceae showed no detectable ROS signal. This early host response was transmissible. Conditioned medium from stressed cultures induced the emergence of ROS-positive host cells and subsequent symbiosis breakdown in unstressed recipients. Purified ROS-positive alga-free coral cells alone were sufficient to trigger symbiosis breakdown in recipient cultures without direct stress exposure. Suppressing the early emergence of ROS-positive coral cells markedly reduced later bleaching, whereas intervention after stress was substantially less effective. These findings identify a host-initiated, intercellularly propagating route to coral bleaching and reveal an early host response that can determine the subsequent fate of the symbiosis.

cell biology↗

bioRxiv2026

Cell Type-Specific Structural and Functional Signatures of APOE4, Age and Sex in the Mouse Anterior Olfactory Nucleus

As the strongest genetic risk factor for late-onset Alzheimer's disease (AD), the human APOE {varepsilon}4 allele alters neuronal physiology before overt pathology. However, how aging and sex modify these effects in the anterior olfactory nucleus (AON), an early AD-affected brain region, remains unclear. Here, we performed whole-cell recordings and post hoc morphological reconstructions of pyramidal cells (PCs) and interneurons (INs) in acute AON slices from adult and aged humanized APOE3 (E3) and APOE4 (E4) knock-in mice of both sexes with identification of context-dependent remodeling across structural, intrinsic, and synaptic domains. In PCs, morphology was broadly susceptible to APOE genotype x age x sex and their interactions. Functionally, aged E4 PCs exhibited a more depolarized resting membrane potential than aged E3 PCs. Additionally, afterhyperpolarization amplitude displayed a marked age x genotype interaction, reversing the direction of the E4 - E3 difference from adulthood to aging. Excitatory synaptic event frequency showed a complex genotype and age and sex interaction, whereas inhibitory synaptic event amplitude was elevated in E4 mice. In contrast, INs displayed far more restricted alterations. Morphological changes were limited to dendritic length, which was modulated by age and sex specifically in E4 mice. Physiologically, genotype effects were confined to action potential amplitude, while aging primarily altered membrane properties and excitatory synaptic inputs. Together, these findings demonstrate that APOE genotype cell-specifically shapes AON neuronal properties through interactions with age and sex, suggesting that APOE4 establishes altered cellular states that influence how this circuit responds to subsequent disease-related stress.

neuroscience↗

bioRxiv2026

Protein expression is amplified by stabilizing mRNA using gene-specific antisense oligonucleotides

Effective and safe strategies to restore gene expression in haploinsufficiency disorders or to augment protective protein signaling have long been attractive but challenging therapeutic goals. Here, we demonstrate the potential of antisense oligonucleotides (ASOs) to upregulate gene targets through mRNA stabilization. The 3 untranslated regions (UTRs) of mRNAs regulate stability via activating and repressive elements. One prominent destabilizing cis-element found in ~30% of transcripts is the Pumilio RNA-binding protein recognition element (PRE), which can be targeted to enable gene-specific protein upregulation. A scan for PREs highlights hundreds of disease-relevant transcripts that may be targetable via ASO-mediated gene-specific blocking of Pumilio binding. We show that masking specific PREs with ASOs produces sustained protein upregulation through mRNA stabilization using multiple reporter and endogenous gene targets. Using PRE-masking ASOs, we neutralized PRE-mediated destabilization of TANK-binding kinase-1 (TBK1) mRNA and restored TBK1 protein expression in participant-derived fibroblasts with TBK1 haploinsufficiency, causal for amyotrophic lateral sclerosis. Additionally, by targeting PRE sites in the myoprotective gene follistatin (FST), we achieved effective FST mRNA upregulation in tested mouse tissues. These findings suggest that ASO strategies that neutralize destabilizing 3UTR sequences like PREs may provide a powerful approach to upregulate haploinsufficiency-related genes or enhance protective protein levels for therapeutic benefit.

molecular biology↗

bioRxiv2026

Mural-VISTA: A tool for mural cell-vessel interaction assessment and multiscale single-cell topo-morphological analysis

Three-dimensional (3D) mural cell morphology is heterogeneous and coupled to vessel geometry, however, measurements from two-dimensional (2D) maximum intensity projections (MIP) obscure overlapping processes and cell-vessel contacts. Accordingly, we developed Mural-VISTA, a semi-automated Python workflow for mural cell-vessel interaction and single-cell topo-morphology analysis of reconstructed surface meshes. This workflow integrates mesh pretreatment, interactive centerline extraction, hierarchical segmentation of cell soma, main axis and secondary processes (branches), and extraction of 36 multiscale (cell process segment level, process level, and whole cell level) topo-morphological and vessel-referenced metrics. Mural-VISTA identified morphological changes in pericytes and vascular smooth muscle cells (vSMCs) with altered RhoA activity. Constitutive active RhoA (RhoA CA) over-expression reduced branch complexity and increased process alignment in both cell types, while increased whole-cell and branch solidity only in vSMCs. Dominant negative RhoA (RhoA DN) over-expression increased branch abundance and reduced branch solidity in pericytes but not vSMCs, suggesting cell-type specific effect of reduced RhoA activity. In conclusion, Mural-VISTA enables quantitative 3D profiling of mural cell architecture and its spatial relationship with the vessel.

bioinformatics↗

bioRxiv2026

DPF3a couples H3K36me2-dependent chromatin remodeling to genome architecture during myogenesis

Chromatin remodelers are generally thought to regulate gene expression through nucleosome mobilization and modulation of local chromatin accessibility. Whether these complexes can instead control transcription through higher-order chromatin organization remains poorly understood. Here we identify the BAF subunit DPF3a as an essential regulator of skeletal muscle regeneration and myogenic differentiation. DPF3a functions through association with the H3K36me2/3 reader HRP2 and preferentially localizes to a distinctive chromatin state characterized by focal depressions within broad H3K36me2 domains. Biochemical reconstitution demonstrates that H3K36 methylation directly enhances remodeling activity of the DPF3a-containing cBAF complex. Unexpectedly, despite profound transcriptional defects, loss of DPF3a produces minimal changes in local chromatin accessibility. Instead, DPF3a is required for long-range chromatin looping associated with activation of myogenic genes. Together, our findings uncover a non-canonical mechanism whereby a chromatin remodeler regulates transcription primarily through three-dimensional genome organization rather than local accessibility control, and establish histone modification-guided chromatin remodeling as a key principle in gene regulation.

molecular biology↗

bioRxiv2026

Isoflurane and surgery aggravate APOE4-dependent lipid dysregulation and neural dysfunction, leading to neurological impairment in male mice

PurposePerioperative exposure to the volatile anesthetic isoflurane (ISO) has been associated with cognitive and olfactory deficits and may increase the risk of Alzheimers disease (AD). Apolipoprotein E4 (APOE4), the strongest genetic risk factor for AD, contributes to disease pathogenesis through disrupted lipid homeostasis. However, whether and how isoflurane interacts with APOE genotype to influence neurological vulnerability remains unclear. MethodsYoung adult, presymptomatic humanized APOE4 and APOE3 knock-in mice underwent laparotomy under 2 h of isoflurane anesthesia. Microglia and astrocytes were isolated from the olfactory bulb (OB) and hippocampus (HI) by magnetic-activated cell sorting. Lipid composition, transcriptional responses, and functional outcomes were assessed using lipidomic, bulk RNA-seq, and longitudinal behavioral testing. In vivo and ex vivo electrophysiological recordings evaluated neuronal excitability and synaptic transmission in both regions. ResultsBy day 7 post-anesthesia, cell type-specific lipidomic profiling of both OB and HI revealed more pronounced lipid perturbations in microglia and astrocytes from APOE4/ISO mice than from APOE3 mice, characterized by elevated free fatty acids, increased lipid peroxidation, triglyceride depletion, and reduced hippocampal hexosylceramides and cardiolipins. Electrophysiological recordings showed greater olfactory circuit dysfunction in APOE4/ISO mice, accompanied by persistent odor memory deficits, transient olfactory sensitivity loss, early motor coordination impairments, and delayed cognitive deficits. RNA sequencing of the OB identified downregulated lipid metabolism and atherosclerosis-related pathways. ConclusionThese findings establish a mechanistic link between APOE4-dependent glial lipid dysregulation, olfactory circuit dysfunction, and delayed cognitive impairment following isoflurane anesthesia and surgery, highlighting lipid homeostasis as a potential therapeutic target.

neuroscience↗

bioRxiv2026

Dynamic Nanoparticle Assembly-Based Biomedical Microrobots

Precise drug delivery within anatomically complex tissues demands systems capable of both active navigation and deep tissue access, properties that have remained difficult to reconcile in existing nanocarriers and microrobots. Here we introduce Dynabots, a dynamic microrobotic assembly constructed from multifunctional nanoparticles covalently linked by thermally cleavable molecular connectors. This nanoparticle-rich architecture enables the integration of magnetic, imaging, and therapeutic components while preserving a high content of functional material. Collective assembly imparts enhanced magnetic responsiveness and maneuverability, enabling controlled navigation through tortuous biological environments. Upon exposure to mild thermal stimuli, the assemblies undergo programmed disassembly, releasing individual nanoparticles that can diffuse through tissue for localized therapeutic action. We establish the programmable transitions, biocompatibility, and therapeutic efficacy of this process across in vitro and in vivo models, including real-time fluoroscopic guidance within anatomically realistic phantoms and live rodent and porcine systems. By integrating magnetic control, reconfigurable architecture, and stimulus-triggered disassembly, Dynabots unite navigational precision with tissue permeability, providing a versatile platform for adaptive and deep-tissue drug delivery.

bioengineering↗

bioRxiv2026

Sustained Delivery of a SARS-CoV-2 Subunit Vaccine in An Adjuvanted Hydrogel Depot Enhances Vaccine Responses in Nonhuman Primates

While natural infections expose the immune system for days to weeks of inflammation and antigen presentation, immunizations with conventional bolus vaccines often lead to rapid clearance of antigens and adjuvants. Prolonged exposure to vaccines using controlled delivery devices or repeated dosing regimens has been shown to enhance germinal center reactions, leading to improved humoral responses, including increased magnitude of antibody titers and enhanced neutralizing activity. Herein, we report the use of injectable polymer-nanoparticle (PNP) hydrogels as a vaccine depot technology for sustained delivery of the clinically relevant SARS-CoV-2 Hexapro subunit antigen and a toll-like receptor agonist adjuvant. In mice, we demonstrated that PNP hydrogel vaccines enhanced germinal center responses and antibody responses relative to bolus vaccination. In nonhuman primates, hydrogel vaccines induced enhanced and durable antibody responses against wildtype and variants of concern such as Omicron BA.5 compared to bolus vaccination. We report the first use of a biomaterials-based approach for sustained delivery of vaccines in nonhuman primates, further advancing toward clinical translation.

bioengineering↗

bioRxiv2026

Neuropeptide Y4 receptor activation delays autoimmune diabetes by reprogramming β-cell stress and immune tolerance

Type 1 diabetes (T1D) involves immune-mediated destruction of pancreatic {beta}-cells, yet current disease-modifying therapies mainly target immunity without enhancing {beta}-cell resilience. We show selective neuropeptide Y4 receptor (Y4R) agonism protects {beta}-cells while reshaping islet immunity across T1D models. Multi-modal localisation using cell sorting, qPCR, RNAscope and fluorescent ligand competition demonstrated predominant Y4R expression and functional accessibility on mouse and human {beta}-cells. Selective Y4R agonism was non-toxic and did not impair islet network integrity, Ca{superscript 2} dynamics, glucose-stimulated insulin secretion or systemic glucose tolerance. Y4R activation conferred cytoprotection against inflammatory cytokines, streptozotocin, lipotoxicity and ER stress, reducing caspase-3/7 activation and {beta}-cell loss whilst sustaining insulin release and promoting proliferation in both mouse and human islets. Bulk RNA-seq revealed a coordinated {beta}-cell resilience programme characterised by reinforced identity and insulin processing, KEAP1-NFE2L2-driven antioxidative and proteostatic activation, and suppression of EIF2 signalling and associated biosynthetic and ER stress pathways. Concurrently, Y4R agonism dampened pathogenic chemokine and cytokine networks, including CXCL10, CCL3/4/7 and IL-6, while preserving IL-2 and Foxp3 signals, thereby limiting CD8 T cell, CD4 T cell and macrophage chemotaxis toward cytokine-stressed islets. Reduced immune-cell recruitment was conserved in a fully human immune-islet system, where Y4R activation significantly attenuated IL-2-activated human PBMC migration and invasion toward cytokine-stressed human islets. In a stringent NY8.3 CD8 T cell adoptive-transfer model, systemic Y4R agonism significantly delayed diabetes onset. These data position Y4R as a {beta}-cell-centric therapeutic target coupling intrinsic resilience with local immune modulation, offering a complementary approach for {beta}-cell preservation in T1D and islet replacement therapies. Graphical abstractThe selective Y4 receptor agonist K22 binds {beta}-cell-enriched NPY4R in mouse and human islets, activates a {beta}-cell resilience programme that preserves insulin secretion under inflammatory and metabolic stress, and simultaneously dampens islet chemokine output, thereby limiting innate and adaptive immune-cell recruitment and delaying autoimmune diabetes onset. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/736290v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@7efd48org.highwire.dtl.DTLVardef@1268860org.highwire.dtl.DTLVardef@c38a81org.highwire.dtl.DTLVardef@1ba3e6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Function-guided design of active enzymes

Designing enzymes from functional descriptions remains challenging because catalytic activity is governed by sequence-structure-function relationships. Here we present EnzymeArt, a function-conditioned enzyme-design framework centred on a generative sequence model. EnzymeArt couples function-conditioned sequence generation with structure-guided refinement, annotation checks and substrate-aware computational prioritization to select candidates for synthesis and biochemical testing. Across alcohol dehydrogenase (ADH), malate dehydrogenase (MDH) and triacylglycerol lipase design campaigns, 57 of 60 synthesized designs showed crude-lysate activity above matched background controls. Purified representatives further showed quantitative steady-state catalytic activity. The best designed ADH reached kcat = 223.7 s-1 and exceeded a wild-type reference under matched conditions, an MDH reached kcat = 267.57 s-1 despite having only 33% sequence identity to its closest BLASTP hit, and a designed lipase hydrolysed both short- and long-chain triglycerides with apparent activity modestly above that of a commercial lipase reference. Together, these results establish a route for converting functional descriptions into experimentally validated enzyme designs with quantitative steady-state kinetic activity.

bioinformatics↗

bioRxiv2026

Conversion of a Viral Glycan Shield into a Binding Anchor via Causal-Driven Antibody Optimization

Therapeutic antibodies are challenged by rapidly evolving pathogens that exploit glycosylation to shield epitopes. SARS-CoV-2 JN.1 exemplifies this, escaping antibodies through the N354-linked glycan. However, targeting glycosylated epitopes remains vacant, as scarce and heterogeneous glycan structures render existing approaches ineffective. Here, we introduce the Antibody Evolution Nexus with Causal-Driven Simulation (AENCS), integrating molecular simulation with causal inference. Applying AENCS to restore S309 efficacy against JN.1, we identified ACC01, exhibiting [~]24-fold improved neutralization. With limited prior knowledge of the N354 glycosylation site, ACC01 stabilized this glycan conformation, facilitating the determination of its cryo-EM structure. Causal dissection revealed how this glycan shield is functionally inverted into a binding anchor through multi-layered interactions. This mechanistic conversion, combined with the conservation of N354 glycosylation, enabled ACC01 to maintain potent activity against the latest variant NB.1.8.1. Collectively, AENCS demonstrates causal-driven antibody engineering can illuminate cryptic glycosylated epitopes, providing viable paradigms for exploring this vacant frontier.

bioengineering↗

bioRxiv2026

Mural cell contractility regulates vessel diameter by controlling cell morphology and vessel coverage

Mural cells are key regulators of vascular architecture, yet how their contractility and morphology jointly influence vessel diameter in vivo remains poorly understood. Here, using high-resolution live imaging and single-cell morphometric analysis in zebrafish, we show that vascular smooth muscle cells (vSMCs) and pericytes undergo a developmental reduction in cell size through the progressive retraction of actin-rich primary and secondary processes during vascular remodelling. By specifically manipulating RhoA activity to alter mural cell contractility and shape, we uncover vessel-specific roles for mural cells in diameter regulation. We find that vSMC contractility is dispensable for the initial constriction of the dorsal aorta but is required to stabilise its diameter following vasoconstriction and to maintain vascular tone. In contrast, pericyte contractility is dispensable for both the constriction and stabilisation of intersegmental vessels. In the brain vasculature, vessel diameter is governed by the balance between contractile force and the extent of vessel coverage by vSMCs. Together, our findings redefine the role of mural cell contractility in the control of vessel constraint, demonstrating that morphology and vessel coverage, rather than contractility alone, are key determinants of vascular diameter in vivo.

cell biology↗

bioRxiv2026

A red-emitting, genetically encoded indicator for two-photon voltage recording in vivo

Genetically encoded voltage indicators (GEVIs) enable minimally invasive, cell-type-specific optical measurements of neuronal membrane potential with millisecond temporal resolution. Red-shifted GEVIs are especially advantageous because they permit spectral multiplexing with complementary sensors and enable all-optical circuit interrogation in combination with blue-light-activated opsins. Despite these advantages, existing red GEVIs remain poorly suited for in vivo use due to limited performance under two-photon (2P) excitation, the predominant modality for deep-tissue imaging. Here, we introduce VADER1, a red GEVI that overcomes this limitation and enables reliable spike detection in vivo under 2P illumination. Under 2P excitation, VADER1 supports extended voltage imaging with both random-access and resonant-scanning microscopy, enables recordings from neurons as deep as cortical layer 5, and allows dual-color imaging with calcium indicators. By filling a critical spectral gap, VADER1 enables integrated optical measurements of fast electrical activity alongside other neural signals and establishes a foundation for two-photon all-optical electrophysiology.

neuroscience↗

bioRxiv2026

Molecular underpinning of Hydra viridissima-algal facultative symbiosis and vertical algal transmission

The symbiosis between algae and animals represents a relatively recent evolutionary innovation, and exemplified by species in the Cnidaria phylum. Cnidaria can harbor algae within a modified cellular organelle called the symbiosome in a process called endosymbiosis. This animal-algal symbiosis can be facultative or obligate. Algae acquisition occurs either by horizontal transmission, where free-swimming planula gain algae through feeding, or by algae deposition into the developing oocyte in vertical transmission[1-5]. Most studies focus on anthozoans that perform obligate endosymbiosis and transmit algae horizontally. How facultative endosymbiosis in combination with vertical algal transmission impacts the evolutionary adaptation between host and symbiont remains unclear. By studying Hydra viridissima, which performs facultative endosymbiosis and transmits its Chlorella algae vertically, we define different cell types and identify the endoderm cell lineage that gives rise to three major cell types hosting algae. Compared to obligate endosymbiosis[6, 7], Hydra viridissima algal host cells exhibit distinct features, including algal uptake, elevated oxidative phosphorylation and redox activities, and express genes that can provide ammonium for their algal symbionts. We further show when and how the developing Hydra oocytes may take up algae and where oocytes may obtain lipids. Since Hydra is amenable to genetic manipulations, our findings should enable mechanistic studies of how facultative endosymbiosis and vertical transmission evolve and adapt in a changing climate.

molecular biology↗

bioRxiv2026

Paternal folate deficiency reveals meiosis as a metabolic sensing window in the male germline

Folate-dependent one-carbon metabolism supplies methyl donors required for chromatin modification, yet how metabolic conditions shape epigenome establishment in the male germline remains poorly understood. Here, using a folate-deficient mouse model, we identify meiotic prophase I as a metabolically sensitive window in the male germline. By integrating in vivo germline analysis with bulk and single-cell transcriptomic and epigenomic profiling, we show that folate deficiency disturbs transcriptional programs in pachytene spermatocytes and preferentially perturbs CpG island (CGI)-associated promoters, which are characterized by bivalent H3K4me3 and H3K27me3 during meiosis. Consistent with this selective vulnerability, active chromatin marks, including H3K4me3 and H3K27ac, are markedly reduced at CGI-associated promoters under folate-deficient conditions. Notably, loci that later exhibit altered H3K4me3 enrichment in mature sperm show earlier chromatin perturbations during meiosis, suggesting that these sperm epigenomic alterations may originate during meiotic development. Together, these findings establish a mechanistic link between paternal folate deficiency and dynamic epigenomic remodeling of CGI-associated chromatin in the male germline.

developmental biology↗