Biology subjects

Montoya, A.

Publications and source records attributed to Montoya, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A Neuronally Active Bacterial Siderophore Manipulates Host Behavior and Longevity

Aging populations face a growing burden of neurodegenerative disease and behavioral decline. The microbiota is increasingly implicated in host physiology, highlighting an urgent need to understand how gut microbes modulate brain function and late life health. Here, we develop a high-throughput microbe-host screening platform to systematically dissect how bacterial genes shape organismal behavior. Using the Escherichia coli Keio library of 3,985 distinct non-essential single-gene deletion mutants in combination with Caenorhabditis elegans, we quantitatively profiled 768 behavioral features per bacterial clone and uncovered a coherent module of bacterial genes involved in iron homeostasis that robustly reprogrammed host behavior. Bacterial omics analyses revealed that alterations in iron homeostasis amplify siderophore production in a TCA cycle-dependent manner, and loss of enterobactin biosynthesis abolished the bacteria-induced changes in worm movement. Transcriptional, molecular and functional studies demonstrated that bacterial enterobactin activates host neuromuscular responses to shape both lifespan and healthspan in a manner dependent on the mitochondrial unfolded protein response. Thermal proteome profiling identified mitochondrial glutaryl-CoA dehydrogenase and the electron-transfer flavoproteins A and B as enterobactin-binding targets that control mitochondrial function. Finally, genetic manipulation of neuronal mitochondrial and cytosolic reactive oxygen species via superoxide dismutase and catalase revealed that redox state modulates enterobactin-driven changes in movement and lifespan through the secretin-family GPCR neuropeptide receptor PDFR-1. Together, these findings establish microbial enterobactin as a non-canonical neurotransmitter regulating neuronal mitochondrial function, behavior and aging, defining a gut bacteria-brain axis with broad implications for maintaining late-life health.

animal behavior and cognition↗

bioRxiv2026

Restoring Antibiotic Sensitivity via Metabolic Pathway Manipulation in Pseudomonas aeruginosa

Stopping antimicrobial multidrug resistance (AMR) is essential to maintain the efficacy of current treatments and sustain modern medicine, but current control relies on a coordinated One Health approach involving stewardship, infection prevention and other control measures, without direct actionable mechanisms against resistance itself. Here, integrating proteomics and metabolomics with high-throughput Caenorhabditis elegans infection models, we show that unique synergy of biotin biosynthesis inhibition with metformin uncouples virulence from bacterial viability shutting down major Pseudomonas aeruginosa (PA) virulence mechanisms that impair host survival. Under directed evolution induced by central carbon metabolic stress, laboratory and clinical PA strain isolates acquire mutations in the MexAB-OprM multidrug efflux pump, leading to reversal of the AMR phenotype and restored susceptibility to multiple legacy and last-resort antibiotics such as meropenem. Our findings reveal a previously unexploited vulnerability in PA, its co-dependence on biotin biosynthesis and energy metabolism for virulence and resistance. This work establishes a conceptual framework in which metabolic co-targeting can abolish pathogenesis and drive reversal of antimicrobial resistance. It lays the foundation for first-in-class, non-antibiotic PA-selective anti-virulence and resistance-reversing therapies, key to extending the lifesaving duration of existing antibiotics.

microbiology↗

bioRxiv2026

Distinct Spatial Immune Architectures in Tumor and Tumor-Adjacent Tissues of Early-Stage Non-Small Cell Lung Cancer

Background: Lung cancer remains the leading cause of cancer-related deaths in the United States with over 124,000 estimated deaths for 2026. Previous studies have found that tumor-adjacent lung tissues may provide additional insight into the immune microenvironment of early-stage NSCLC. Methods: Multiplex immunofluorescence (mIF) imaging was performed on 192 tissues from 101 early-stage non-small cell lung cancer patients including 91 matched tumor-adjacent pairs, using three mIF panels. Spatial analyses were performed to identify distinctions between tissues and identify associations with clinical and genomic features. Results: Tumor tissue showed significantly higher densities of T cells, macrophages, B cells, and memory/regulatory populations than adjacent tissue (p<0.001). Tumors exhibited greater spatial heterogeneity, with higher prevalence of spatial patterning (47.7% vs. 31.2%) and more consistently localized organization, whereas adjacent tissue showed stronger individual-cell clustering. Pairwise colocalization (Ripley's L-cross) revealed selective spatial segregation in tumors (including B-cells from memory/regulatory cells and among cytotoxic T-cell subsets) and reduced immune proximity to malignant cells relative to the strong immune-epithelial association in adjacent tissue. Spatial features were linked to genomic features or patient outcomes: tumor neoantigen burden exhibited a positive association with CD3+ T cells in the tumor, while colocalization between CD45RO+CD57+GZMB+ cells and CD57+GZMB+ cells was negatively associated with overall survival outside the tumor. Conclusion: Tumor and adjacent tissues harbor distinct spatial immune architectures, with spatial features displaying associations with genomic features or patient outcomes. These findings highlight immune microenvironment reorganization and the importance of incorporating spatial context from both compartments into risk assessment in NSCLC.

immunology↗

bioRxiv2026

Chromatin is dispensable for bacterial life

Inside cells, DNA is intimately associated with proteins, forming chromatin. The protein constituents of chromatin vary across the tree of life: histones are the principal building blocks of chromatin in eukaryotes and many archaea, whereas bacteria typically encode a collection of nucleoid-associated proteins (NAPs) that wrap, bend, bridge or coat the DNA. Although chromatin proteins appear to be a universal feature of cellular life, DNA-templated processes such as transcription, replication, and DNA repair can take place in vitro in the absence of chromatin, raising the possibility that cellular systems might exist -- or could be built -- that lack chromatin proteins. To explore this possibility, the molecular consequences and potential systemic adjustments required for life without chromatin, we serially deleted the nine most abundant NAPs from E. coli (hupA , hupB , ihfA , ihfB , hns , stpA , fis , dps , lrp), resulting in a strain ({Delta}NAP9) that lacks its native chromatin. Using an array of different techniques, we document change -- and sometimes surprising lack thereof -- in compaction, composition and 3D architecture of the nucleoid, supercoiling, prophage activity, growth, viability, and genetic make-up of {Delta}NAP9. Most notably, we find that {Delta}NAP9 exhibits global dysregulation of gene expression, marked by a striking homogenization of transcriptional output across the genome that is reminiscent of the effects of histone depletion in eukaryotic cells. Our results reinforce the notion that chromatin plays a key role in compartmentalizing the use of genomic information, enabling both the localized suppression of selfish elements and dynamic reprogramming of genome activity in response to environmental change. At the same time, the successful construction of {Delta}NAP9 demonstrates that bacterial cells can carry out basic cellular functions in the absence of co-evolved chromatin proteins, highlighting the potential for radical (re-)engineering of prokaryotic chromatin and systems of gene expression.

microbiology↗

bioRxiv2026

HER2 mutation-derived neoantigens in NSCLC as actionable targets for TCR therapy

HER2 mutations are oncogenic drivers in 1-6% of non-small cell lung cancers (NSCLC), but therapeutic resistance limits the durability of current HER2-targeted treatments. Here, we identify T-cell receptors (TCRs) targeting recurrent HER2 hotspot mutations as a potential immunotherapeutic strategy for HER2-mutant NSCLC. Using neoepitope prediction and antigen-specific T-cell enrichment, we isolated HLA-A*02:01restricted TCRs recognizing HER2 A775insYVMA, S310F, and G776delinsVC mutations, collectively covering approximately 60% of HER2-mutant NSCLC. These TCRs selectively recognized mutant HER2 epitopes without detectable wild-type reactivity and some displayed cross-recognition of related hotspot variants, expanding the spectrum of targetable tumors. The G776delinsVC-specific TCR also exhibited co-receptorindependent activity showcased by its ability to activate CD4+ T cells. Importantly, timelapse single-cell flow cytometry analyses demonstrated that TCR-engineered T cells repeatedly reacquired activated polyfunctional states following serial antigen stimulation, while serial tumor rechallenge assays confirmed sustained cytotoxic activity across multiple rounds of tumor killing. These findings identify recurrent HER2 mutations as shared immunotherapeutic targets and provide a foundation for the development of TCR-based therapies for HER2-mutant NSCLC.

immunology↗

bioRxiv2026

A Bactericidal Phospholipase from Archaea

Archaea kill bacteria, at least on occasion. The molecular underpinnings of these lethal interactions are barely understood. Here, we describe cinquedea, an /{beta} hydrolase secreted by the halophilic archaeon Haloferax larsenii s5a-1. Cinquedea exhibits bactericidal activity in the nanomolar range, killing halophilic Pontibacillus bacteria. Bacterial death is accompanied by gross morphological abnormalities, indicative of severe damage to the cell envelope. We predict, and confirm in vitro, that cinquedea is a phospholipase, with structural similarities to a phospholipase A1 enzyme isolated from hornet venom. Exposing lipids extracted from a cinquedea-sensitive Pontibacillus strain to the enzyme leads to accumulation of lysophosphatidylglycerol, a cleavage product of phospholipase A activity. This is consistent with direct activity of cinquedea against the Pontibacillus membrane, which we show is chiefly composed of phosphatidylglycerol. Considered alongside recent findings that some archaea encode bactericidal peptidoglycan hydrolases, these results suggest that archaea can kill bacteria in mechanistically diverse ways. Our work provides a template for future experimental discovery and characterization of bactericidal proteins of archaeal origin and reinforces an emerging view that archaea represent a substantial reservoir for the discovery of new antibacterial compounds.

microbiology↗

bioRxiv2026

Mechanisms of MCM2-7 helicase activation and initial DNA melting at near base-pair resolution

During eukaryotic DNA replication initiation, inactive MCM2-7 double-hexamers assembled at replication origins must be converted into two active CMG helicases, yet how this transition is coupled to origin DNA unwinding in vivo remains unclear. Here, we identify a DNA-bound intermediate with an extended genomic footprint that forms during helicase activation. Genome-wide mapping of initial strand separation reveals that DNA unwinding initiates near the N-terminal interface of opposing MCM2-7 hexamers. At these sites, the origin DNA exhibits a conserved AT-rich/GC-rich/AT-rich sequence architecture on which the helicase complex is centred, consistent with a role in promoting DNA extrusion following double-hexamer separation. We further show that restricting hexamer rotation and splitting delays release of the Cdc45-loading factor Sld3, demonstrating that mechanical transitions during helicase activation are tightly coupled to complex disassembly. Finally, we provide in vivo evidence that single-stranded DNA is ejected through a specialised DNA exit gate at the Mcm2/5 interface during helicase activation, which is dispensable for ongoing DNA synthesis. Together, these findings establish a mechanistic framework for how replication origins are remodelled to initiate DNA replication and reveal key intermediates and DNA transactions during helicase activation.

molecular biology↗

bioRxiv2026

Discovering Novel Therapeutic VHHs for Emerging Viruses: Perspectives from VEEV Selection Strategies

Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (VHHs). These proteins can neutralize or sequester viral particles, preventing or reducing infection. However, due to the evolution of viruses, there is a need to isolate new antibodies and direct their binding to particular epitopes on the virus. In vitro selections offer a promising pathway for the selection of therapeutic antibodies, but as we demonstrate, the choice of a target for these selections is key to obtaining the desired viral binding characteristics. Here we report four novel "human" VHHs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These VHHs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these VHHs. Though non-neutralizing, when immobilized, these VHHs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these VHHs to perform viral "sponging". The selection strategies we report may have applications to further antibody developments against other viruses. Significance/ImportanceAlphaviruses, and in particular Venezuelan equine encephalitis virus (VEEV), are recognized for their ability to cause severe disease and for their potential to be used as a biothreat. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. This study reports on a novel antibody selection pipeline to produce antibody fragments against VEEV. Antibodies produced via this method showed strong affinity and high specificity to the VEEV E2 glycoprotein in multiple conformations. Additionally, while not neutralizing, the antibody fragments described were shown to be effective as "viral sponges", having the ability to bind, sequester, and remove VEEV virions from solution, which represents a novel therapeutic approach.

immunology↗

bioRxiv2026

A protein language model unveils the E. coli pangenome functional landscape regulating host proteostasis

Understanding how bacterial diversity at strain level resolution shapes host physiology is a central challenge in microbiome research. The vast, functionally unknown genetic diversity within a species pangenome makes it difficult to connect genes to function and their impact on host physiology. Here, we explore how the functional landscape of the Escherichia coli pangenome impacts transcriptional responses in Caenorhabditis elegans and show that traditional gene-centric methods fail to provide significant functional associations with the host. Thus, we developed a pangenome framework that leverages the protein language model ProtT5 and generates unique strain embeddings representing the functional potential of each 9,558 E. coli isolate. Stratification of the pangenome into distinct functional guilds aligned with key host processes such as cell division, metabolism and proteostasis. Further, we identify a critical interplay between the extensive network of bacterial chaperones and proteases in regulating host proteostasis. We find that the bacterial chaperone DNAK/HSP70 and protease ClpX fine-tune the host ubiquitin-proteasome system by controlling propionate and vitamin B12 availability. These findings reveal a conserved co-proteostasis mechanism as a key phenomenon modulating host-microbe interactions through metabolic communication. Our pangenome-to-phenotype approach offers a powerful strategy to decode bacterial pangenome functional diversity, directly linking microbial genomic variation to host physiological outcomes.

systems biology↗

bioRxiv2025

Wee1 opposes APC/C(Cdh1) activity to promote S-phase entry

Wee1 phosphorylates and inhibits CDK activity to inhibit mitotic entry and establish a G2 DNA damage checkpoint. Consequently, Wee1 inhibitors are in clinical trials, developed to be synthetically lethal in TP53 mutant tumours that become reliant on a Wee1-mediated DNA damage checkpoint. However, Wee1 inhibitors have efficacy in TP53 wild-type tumours and many trials have been terminated due to high levels of toxic side-effects, suggesting that Wee1 has unknown functions. Here, we show that Wee1 promotes cell cycle re-entry from quiescence (G0) by opposing the activity of the E3 ubiquitin ligase, APC/CCdh1. Wee1 phosphorylates Cdh1 (FZR1) at key residues that mediate the interaction between Cdh1 and APC/C. Cells with loss-of-function of Wee1 during G0/G1 have delayed S-phase entry, an impaired G1/S transition, abnormal S-phase accumulation of the CDK inhibitor p21 and enter a p21-dependent G2 arrest. Reduced expression of APC/CCdh1 or p21 renders cells more sensitive to acute Wee1 inhibition and both pathways are downregulated in acquired Wee1 inhibitor resistance. Our study reveals a new cell cycle control mechanism that has implications for how Wee1 inhibitors should be used in the clinic.

cell biology↗

bioRxiv2025

Multimodal imaging reveals a lysosomal drug reservoir that drives heterogeneous distribution of PARP inhibitors

For all drugs, effective target engagement requires sufficient intracellular concentrations of drug to be reached, but whether tumour heterogeneity impacts drug distribution and efficacy is poorly studied. PARP inhibitors have transformed treatment of high-grade serous ovarian carcinoma (HGSOC), but resistance remains a clinical hurdle in this highly heterogeneous tumour type. We developed a patient-derived explant multi-modal imaging pipeline, which demonstrated that cell-intrinsic PARP inhibitor accumulation is highly variable, both between patients and within tumours. Spatial transcriptomics revealed enrichment of apoptotic and lysosomal signatures in high-drug regions. Rucaparib, an intrinsically fluorescent PARP inhibitor, accumulates heterogeneously at the single-cell level, with rucaparib-high cells demonstrating increased drug response relative to rucaparib low. Mechanistically, lysosomal sequestration creates a rucaparib reservoir that determines drug levels in the nucleus. Perturbation of lysosomal content altered intracellular levels of weak base PARP inhibitors rucaparib and niraparib, but not olaparib. Together these data suggest that lysosomes act as a reservoir for a subset of PARP inhibitor drugs to improve drug response.

cancer biology↗

bioRxiv2025

Mitochondrial dysfunction underlies monocyte immune deficiency in patients with severe alcohol-related hepatitis

Severe alcohol-related hepatitis (sAH) is a life-threatening form of alcohol-related liver disease (ARLD) associated with a significant short-term mortality. Opportunistic infections due to impaired immune function are a major cause of patient mortality. Mitochondrial dysfunction within the liver is a well-recognised feature of ARLD and sAH. However, whether these hepatic mitochondrial defects extend to the immune system of sAH patients, underlying their immune dysfunction, remains unclear. Here, we demonstrate that sAH monocytes exhibit an increased content of inefficient, dysfunctional mitochondria. These changes were underpinned by abnormal mitochondrial cristae ultrastructure, which were associated with depletion of cristae structural proteins and alterations in cardiolipin profiles. Overall, our study uncovers novel structural and functional mitochondrial defects, which likely contribute to impaired monocyte immune function in sAH.

immunology↗

bioRxiv2024

In vivo functional profiling and structural characterisation of the human Glp1r A316T variant

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable. Variation in the human Glp1r gene might be directly linked to therapeutic responses. A naturally occurring missense variant, A316T, protects against T2D and cardiovascular disease. Here, we have generated and characterised a human Glp1r A316T mouse model. Human Glp1rA316T/A316T mice displayed lower fasting blood glucose versus wildtype littermates, even under metabolic stress, and exhibited alterations in islet cytoarchitecture and /{beta}-cell identity under a high-fat, high-sucrose diet. This was however associated with blunted responses to GLP-1RAs in vivo. Further investigations in rodent and human {beta}-cell models demonstrated that human Glp1r A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R-targeting therapies. TeaserThe Glp1r A316T missense variant displays improved glucose tolerance but impaired pharmacological incretin responses in vivo.

cell biology↗

bioRxiv2024

An inter-organelle contact between endosomal GLP-1R, ER VAP-B, and the mitochondrial AKAP SPHKAP triggers PKA-dependent MIC19 phosphorylation and β-cell mitochondrial remodelling

Glucagon-like peptide-1 receptor (GLP-1R) agonists (GLP-1RAs) ameliorate mitochondrial health by increasing mitochondrial turnover in metabolically relevant tissues. Mitochondrial adaptation to metabolic stress is crucial to maintain pancreatic {beta}-cell function and prevent type 2 diabetes (T2D) progression. While the GLP-1R is well-known to stimulate cAMP production leading to Protein Kinase A (PKA) and Exchange Protein Activated by cyclic AMP 2 (Epac2) activation, there is a lack of understanding of the molecular mechanisms linking GLP-1R signalling with mitochondrial and {beta}-cell functional adaptation. Here, we present a comprehensive study in {beta}-cell lines and primary islets that demonstrates that, following GLP-1RA stimulation, GLP-1R-positive endosomes associate with the endoplasmic reticulum (ER) membrane contact site (MCS) tether VAPB at ER-mitochondria MCSs (ERMCSs), where active GLP-1R engages with SPHKAP, an A-kinase anchoring protein (AKAP) previously linked to T2D and adiposity risk in genome-wide association studies (GWAS). The inter-organelle complex formed by endosomal GLP-1R, ER VAPB and SPHKAP triggers a pool of ERMCS-localised cAMP/PKA signalling via the formation of a PKA-RI biomolecular condensate which leads to changes in mitochondrial contact site and cristae organising system (MICOS) complex phosphorylation, mitochondrial remodelling, and {beta}-cell functional adaptation, with important consequences for the regulation of {beta}-cell insulin secretion and survival to stress.

cell biology↗

bioRxiv2024

PBK/TOPK mediates Ikaros, Aiolos and CTCF displacement from mitotic chromosomes and alters chromatin accessibility at selected C2H2-zinc finger protein binding sites

PBK/TOPK is a mitotic kinase implicated in haematological and non-haematological cancers. Here we show that the key haemopoietic regulators Ikaros and Aiolos require PBK-mediated phosphorylation to dissociate from chromosomes in mitosis. Eviction of Ikaros is rapidly reversed by addition of the PBK-inhibitor OTS514, revealing dynamic regulation by kinase and phosphatase activities. To identify more PBK targets, we analysed loss of mitotic phosphorylation events in Pbk-/-preB cells and performed proteomic comparisons on isolated mitotic chromosomes. Among a large pool of C2H2-zinc finger targets, PBK is essential for evicting the CCCTC-binding protein CTCF and zinc finger proteins encoded by Ikzf1, Ikzf3, Znf131 and Zbtb11. PBK-deficient cells were able to divide but showed altered chromatin accessibility and nucleosome positioning consistent with CTCF retention. Our studies reveal that PBK controls the dissociation of selected factors from condensing mitotic chromosomes and contributes to their compaction.

molecular biology↗

bioRxiv2024

Conserved helical motifs in the Ikaros IDR mediate NuRD interaction and transcriptional repression

The transcription factor IKZF1/Ikaros is essential for B cell development, and recurrently mutated in human B-ALL. Ikaros has been ascribed both activating and repressive functions via interactions with coactivator and corepressor complexes, but the relative abundance of Ikaros-associated coregulatory complexes and their contribution to Ikaros-mediated gene regulation are not well understood. To address this issue, we performed an unbiased identification of Ikaros-interacting proteins in pre-B cells, and found that Ikaros interacts overwhelmingly with corepressors and heterochromatin-associated proteins. Time-resolved analysis of transcription and chromatin state identified transcriptional repression as the immediate response to Ikaros induction. Transcriptional repression preceded transcriptional activation by several hours, and was accompanied by a rapid loss of chromatin accessibility and reduced levels of H3K27ac particularly at enhancers. Functional characterisation of intrinsically disordered regions in the Ikaros protein identified highly conserved helical motifs that mediate Ikaros association with the NuRD corepressor complex and contribute to the silencing of target genes in pre-B cells and antiproliferative functions of Ikaros in human B-ALL.

molecular biology↗

bioRxiv2023

Establishment of beta cell heterogeneity via differential CpG methylation at Nnat

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected hub cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. MethodsSingle cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "{beta}HI" cells and were diminished in db/db mice. Hub cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. Research in contextWhat is already known about this subject? - Neuronatin (Nnat/NNAT) is an imprinted gene in humans and mice and is required for glucose-stimulated insulin secretion in vivo - Pancreatic beta cells are functionally heterogeneous with specific highly-connected subpopulations known to coordinate islet wide Ca2+ dynamics - Functional subpopulations have been described in human beta cells and their distribution is altered in type 2 diabetes What is the key question? - Does NNAT mark a discrete subpopulation of functional beta cells and which epigenetic pathways coordinate its formation and maintenance? What are the new findings? - A subpopulation of NNAT+ beta cells is established prior to the first week of postnatal life in mice via de novo DNA methylation at the Nnat promoter - NNAT+ beta cells are transcriptionally highly differentiated and appear to be functionally specialised for insulin production, possibly corresponding to recently-described "{beta}HI" and "CD63hi" beta cells. NNAT is expressed in a subset of beta cells across the human islet, and its deficiency in human beta cells diminishes glucose-stimulated insulin secretion - NNAT+ cells are likelier to belong to the population of follower, rather than hub cells, consistent with a role in insulin production rather than glucose detection How might this impact on clinical practice in the foreseeable future? - Epigenome-modifying compounds may provide a way of enhancing beta cell function and the ensemble behaviour of the islet to stimulate insulin secretion

cell biology↗

bioRxiv2022

Loss of H3K9 tri-methylation alters chromosome compaction and transcription factor retention during mitosis

Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases (DNMTs) and Polycomb Repressor Complexes (PRCs), bind to chromosomes throughout mitosis and their depletion results in increased chromosome size. Here we show that enzymes that catalyse H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly however, mutants lacking H3K9me3 have unusually small and compact mitotic chromosomes that are associated with increased H3S10ph and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous Suv39h1, or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wildtype versus Suv39h1/Suv39h2 double-null ESCs revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis, and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.

molecular biology↗