Biology subjects

Braun, T.

Publications and source records attributed to Braun, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Cell-specific FUS loss enhances ageing-associated transcriptional elongation in lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is an age-related, lethal lung disease affecting over 3 million people worldwide. During aging, impairment of diverse molecular processes compromises cellular homeostasis. Eukaryotic cells adapt to this ageing-associated stress by large-scale transcriptome changes affecting the expression of genes involved in signaling, protein homeostasis, cell plasticity and immune response. The molecular mechanisms regulating extensive transcriptome rearrangements leading to senescence in specific cell types of IPF lungs remain sparsely investigated. Here we uncover fundamental molecular mechanisms underlying cellular senescence by integrative analysis of multi-omics studies and functional experiments monitoring fibrotic and senescence hallmarks in primary lung fibroblasts from control and IPF patients. Loss-of-function of the DNA/RNA binding protein fused in sarcoma (FUS) in control fibroblasts enhanced fibrotic and senescence hallmarks resembling IPF fibroblasts. Dissecting the molecular mechanisms, reduced FUS levels in IPF fibroblasts abolished R-loop formation at transcription start sites (TSS), leading to increased RNA polymerase II (Pol II) recruitment and its phosphorylation at serine 2, resulting in enhanced translocation speed of elongating Pol II and increased expression of fibrosis- and senescence-related genes. Summarizing, FUS maintains low Pol II processivity in control fibroblasts by stabilizing R-loops at TSS, acting as a gatekeeper of fibrotic and age-associated transcriptional programs.

molecular biology↗

bioRxiv2026

CryoConvNeXt enables robust identification of low-abundance molecular species in experimental cryo-EM data

Rare but biologically important molecular species are easily lost when reconstruction-based cryo-EM classification is applied to heterogeneous samples dominated by more abundant particles. We developed CryoConvNeXt, a deep-learning classifier that combines cyclic equivariance with adaptive frequency filtering. It is trained on simulated projections and adapted to experimental data by self-training, an unsupervised domain adaptation technique where the model acts as its own annotator. We tested CryoConvNeXt on cryo-EM data collected for this study from controlled binary and ternary mixtures of Catalase, Apoferritin, and HSP60. Manual particle curation provided reference labels for benchmarks with class ratios from 1:1 to 1:16. CryoConvNeXt retained minority-species recall across this range. In contrast, cryoSPARC's recall collapsed in four of six pairwise conditions, as early as 1:4 when Catalase was the minority species. By recovering low-abundance species that conventional classification fails to recover reliably, CryoConvNeXt takes an important step towards solving a central problem in quantitative visual proteomics: measuring the molecular composition of complex biological samples.

biophysics↗

bioRxiv2026

A novel role for Oxaloacetate Decarboxylase FAHD1 in cardiomyocyte maturation

Mitochondrial metabolism undergoes dramatic reprogramming during postnatal cardiac maturation, yet the enzymatic regulators that ensure continuity of TCA cycle flux in this period remain incompletely defined. FAHD1 is a mitochondrial oxaloacetate decarboxylase (ODx) with proposed roles in modulating the activity of Complex II of the electron transport chain (ETC), but its physiological relevance in vivo has remained unclear. Here, we identify FAHD1 as a critical regulator of mitochondrial function with strong impact on cardiomyocyte (CM) maturation. Using a germline Fahd1-knockout (KO) mouse model, we show that Fahd1 deficiency impairs Complex II respiration, reduces pyruvate levels, and induces a compensatory metabolic shift toward glycolysis and anabolic biosynthesis. Loss of FAHD1 disrupts sarcomere organization, delays the fetal-to-adult myosin isoform switch, and leads to left ventricle systolic dysfunction and cardiomyocyte hypertrophy. These findings highlight FAHD1 as a mitochondrial gatekeeper and potential target for modulating cardiac development and disease.

cell biology↗

bioRxiv2026

Affinity-tag-based microfluidic protein isolation enables high-resolution Cryo-EM from minimal starting material

Cryo-EM is central to high-resolution structure determination, but conventional sample preparation consumes substantial amounts of protein and can destabilize sensitive complexes. Microfluidic approaches can miniaturize and accelerate these workflows while retaining the particle numbers required for single-particle analysis. Here, we present a generalized microfluidic isolation strategy that captures proteins directly from cell lysates or in vitro translation reactions using genetically encoded tags. The platform supports both affinitybased (ALFA-nanobody) and covalent (SpyTag3/SpyCatcher3) capture. Specificity derives from two sequential, orthogonal steps: (i) tag-mediated capture and concentration of the target protein on beads, and (ii) photoelution from the bead surface. By confining the sample to nanoliter volumes and minimizing transport, the approach suppresses non-specific carryover. Importantly, the isolation workflow is coupled to grid preparation, enabling preparation of three cryo-EM or negative-stain grids from a 25 nL eluate. Using this workflow, we isolated E. coli ferritin A, {beta}-galactosidase, and P. aeruginosa VgrG1 from cell lysate, obtaining reconstructions at 1.9-2.6 [A] resolution with B-factors comparable to optimized conventional workflows. We further isolated ferritin A from an in vitro translation reaction, yielding a 2.04 [A] reconstruction. A standardized 50 {micro}L starting volume enabled more than 16 independent microfluidic isolations. Tag-based microfluidic isolation thus provides a broadly applicable route to cryo-EM sample preparation, reducing sample volumes more than 1000-fold, reducing preparation times more than 3-fold for lysate-based workflows and up to 10-fold when combined with cell-free expression, and enabling high-throughput structural screening directly from in vitro translation reactions. Significance StatementCryo-electron microscopy (cryo-EM) enables protein structure determination at atomic resolution, but conventional sample preparation requires large amounts of purified protein and laborious workflows that can damage fragile complexes. We present a microfluidic platform that captures tagged proteins directly from minute amounts of crude lysates or cell-free synthesis reactions and deposits them onto cryo-EM grids in a single integrated workflow. Using this approach, we determine three protein structures at high resolution. By reducing sample volumes by roughly three orders of magnitude, the platform expands cryo-EM to proteins inaccessible to conventional workflows and provides a foundation for automated, high-throughput structural analysis.

molecular biology↗

bioRxiv2026

Reg3β removes aged neutrophils after myocardial infarction

AO_SCPLOWBSTRACTC_SCPLOWRecruitment and resolution of immune cell accumulation after myocardial infarction (MI) is critical for effective wound healing and prevention of maladaptive remodeling. Numerous signals are known to recruit neutrophils, critical drivers of inflammation, but knowledge about signals containing and resolving their accumulation is limited. We discovered that Regenerating-islet derived protein 3 beta (REG3{beta}) limits the persistence of neutrophils after MI, thereby promoting resolution of inflammation. REG3{beta} selectively binds to aged and hyperactive neutrophils and induces rapid cell death, enabling clearance via macrophage-mediated efferocytosis. Selective binding of REG3{beta} is achieved by interaction with paucimannosylated proteins that translocate from azurophile granules to the plasma membrane in an activation- and age-dependent manner. Endocytotic uptake and accumulation of REG3{beta} in lysosomes initiates programmed cell death of neutrophils via lyosomal membrane permeabilization and release of cathepsins. Our work establishes REG3{beta} as a local immune checkpoint essential for neutrophil resolution and cardiac repair.

immunology↗

bioRxiv2025

Structure-function relationship of alpha-synuclein fibrillar polymorphs derived from distinct synucleinopathies

The aggregation of the protein alpha-synuclein (Syn) is a common feature of multiple neurodegenerative diseases collectively called synucleinopathies, for which the pathobiology is not well understood. The different phenotypic characteristics of the synucleinopathies Parkinsons disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA) have been proposed to originate from the distinct structures adopted by Syn in its amyloid forms. Here, using covalent labeling and limited proteolysis coupled to mass spectrometry (LiP-MS) in vitro and in situ within neuronal cells and directly in native patient brain homogenates, we show that pathogenic Syn from distinct synucleinopathies (PD, DLB and MSA) are structurally different. Further, we found that fibrillar structural differences are associated with different fibril interactomes and neuronal responses. We discovered disease-specific ubiquitination patterns and turnover profiles for pathogenic Syn species, detected molecular pathways responding specifically to the uptake of different Syn fibrillar polymorphs, and identified a subset of the involved proteins as candidate direct interactors of Syn. In particular, components of the Ubiquitin-proteasomal System (UPS), including E3 ubiquitin ligases, chaperones, and Deubiquitinating proteins, showed disease/polymorph-specific interaction patterns, possibly accounting for different resistance of patient-derived Syn fibrils to degradation. Genetic modulation with CRISPR-based tools showed that members of the UPS degradation pathway (three E3 ligases: UBE3A, TRIM25, HUWE1 and the AAA+ ATPase VCP) reduced Syn inclusions, in a strain-specific manner. LiP-MS also identified sets of proteins with altered protease susceptibility in postmortem brain homogenates of PD, DLB, and MSA patients. These sets were largely disease-specific and included proteins altered in cells treated with fibrils derived from patients with the matching disease. Our findings provide insight into cellular processes involved in the accumulation and turnover of Syn pathogenic aggregates in PD, DLB and MSA in a disease specific manner and constitutes a resource of potential novel drug targets in these synucleinopathies.

systems biology↗

bioRxiv2025

Alpha-synuclein fibrils induce budding of mitochondrial-derived vesicles

-synuclein (-syn) aggregation is a hallmark of synucleinopathies, a class of neurodegenerative disorders such as Parkinsons disease (PD). Several lines of evidence indicate the involvement of mitochondria in the disease pathology. Despite extensive study, the link between -syn aggregation and mechanisms of mitochondrial toxicity remains not fully understood. Using high-resolution imaging with electron microscopy, we examined cells exposed to -syn fibrils vs control cells with a focus on mitochondria. We found that upon exposure to -syn fibrils, mitochondria increase in size, cristae structure gets defects, and mitochondria enhance the budding of mitochondrial-derived vesicles (MDVs). MDV formation reflects an evolutionarily conserved mechanism reminiscent of bacterial outer membrane vesicle (OMV) biogenesis. Structural proteomics analysis by mass spectrometry corroborates this microscopy observation by identifying changes in multiple proteins that regulate cristae structure, MDV formation, and trafficking. Our results provide a new link between -syn and mitochondria and identify novel pathways responding to -syn aggregates, particularly that -synuclein directly triggers MDV generation. The processes we detected could be of interest for diagnostics and potential therapeutic interventions.

neuroscience↗

bioRxiv2025

Endothelial expression of ZBTB16 protects against cardiac aging

Background and aimAging significantly increases the risk of cardiovascular diseases, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms mediating EC-dependent cardiac aging and identifies a critical role of Zinc finger and BTB domain-containing protein 16 (ZBTB16). MethodsChromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses were performed on aged hearts to identify age-related regulatory changes. Functional studies using genetic models, assessed cardiac aging phenotypes. In vitro assays examined EC senescence and secretory profiles, while co-culture experiments analyzed the impact of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested the potential for ZBTB16 to mitigate aging-associated dysfunction. ResultsAged hearts exhibited decreased chromatin accessibility and expression of the transcription factor ZBTB16 in both human and mice. Loss of ZBTB16 in young mice, including Zbtb16 haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. Supernatants from ZBTB16-deficient ECs activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified key downstream targets of ZBTB16, including nuclear receptor-interacting protein 1 (NRIP1). ZBTB16 suppressed NRIP1 expression, limiting fibroblast activation and pro-fibrotic signaling. ConclusionsZBTB16 is a key regulator of endothelial function, maintaining vascular niche homeostasis and mitigating aging-associated cardiac dysfunction. Its loss promotes EC senescence and pro-fibrotic signaling, contributing to diastolic dysfunction. Overexpression of ZBTB16 presents a potential therapeutic strategy for preserving cardiac function during aging. These findings establish a novel role for ZBTB16 in endothelial aging and cardiovascular disease prevention. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/681100v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a56e2org.highwire.dtl.DTLVardef@13e23fforg.highwire.dtl.DTLVardef@ad681eorg.highwire.dtl.DTLVardef@8eca8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Redirection of SARS-CoV-2 to phagocytes by intranasal sACE2-Fc as a universal decoy confers complete prophylactic protection

The rapid evolution of SARS-CoV-2 and other respiratory RNA viruses limits the success of current vaccines and antibody-based therapies. Engineered decoy receptors based on soluble angiotensin-converting enzyme 2 (sACE2) offer promising alternatives. Clinical-grade recombinant sACE2 inhibits SARS-CoV-2 replication in vitro but shows limited clinical success. This study reports an optimized sACE2 mutant fused to human IgG1 Fc (B5-D3), which redirects virus-decoy complexes to lysosomal degradation in macrophages. Intranasal prophylactic delivery of B5-D3 confers complete protection in SARS-CoV-2-infected K18-hACE2 mice. Abrogation of Fc effector functions compromises antiviral protection, indicating that Fc-mediated uptake of virus-decoy complexes is critical. Transcriptomic analysis suggests that B5-D3 induces early immune activation in lungs of infected mice. Bio-distribution and flow cytometry reveal selective targeting of airway phagocytes. In vitro assays confirm lysosomal degradation of virus-decoy complexes by macrophages without productive infection. These findings reveal a distinct antiviral mechanism via phagocytic clearance, supporting refined regimens for decoy treatments against SARS-CoV-2 and potentially other respiratory viruses.

immunology↗

bioRxiv2025

Erlotinib induces 3D genome rearrangements in lung cancer cells activating tumor suppressor genes through FOXA2-bound Epromoters

Non-small cell lung cancer (NSCLC) is the most frequent lung cancer (LC). While erlotinib is an epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI) used in the treatment of NSCLC, it remains unclear how this FDA-approved drug affects the genome. We performed integrative multi-omics studies in human pulmonary carcinoma cells to elucidate the epigenetic mechanisms induced by erlotinib. We identified 746 genes (including 34 tumor suppressor genes, TSG) that were upregulated after treatment with erlotinib or gefitinib (another EGFR-TKI). Interestingly, 45% of the upregulated genes (including 24 TSG) were in broad domains of the euchromatin histone mark H3K4me3, and 63% (including 26 TSG) exhibited reduced levels of the heterochromatin histone mark H3K27me3 after erlotinib treament. Further, H3K27ac-specific chromosome conformation capture-based methods revealed that erlotinib significantly increased number and length of chromatin loops between promoters of upregulated genes and active enhancers. We also detected augmented chromatin accessibility after erlotinib treatment at the promoters of upregulated genes, which correlated with binding of the transcription activator FOXA2. Remarkably, we identified gene clusters that seem to be upregulated by promoters with enhancer activity (Epromoters) enriched with FOXA2. The clinical relevance of our findings was confirmed by data from The Cancer Genome Atlas, showing significantly improved survival outcomes in LC patients with high levels of FOXA2 and/or the 34 TSG found upregulated by erlotinib. Our results establish 3D genome rearrangements as molecular mechanism mediating EGFR-TKI effects in NSCLC cells, supporting the design of more specific therapies for NSCLC targeting different chromatin features.

cancer biology↗

bioRxiv2025

Targeting Runx1 protects against heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a public health problem and an elusive illness for which there are few treatment options. HFpEF is a systemic condition with a broad phenotype including diastolic dysfunction, pulmonary oedema, exercise intolerance, and left ventricular (LV) hypertrophy, collectively resulting in enhanced morbidity and mortality. Master-regulator transcription factor RUNX1 has recently been identified as a mediator of pathological changes in many cardiac diseases, however its role in HFpEF was unknown. Here we show that inhibition of Runx1 limits adverse cardiac remodelling in a clinically relevant mouse model of HFpEF. Cardiomyocyte-specific tamoxifen-inducible Runx1-deficient mice with HFpEF are protected, with preservation of diastolic function, and attenuation of pulmonary oedema, exercise intolerance, and hypertrophy. Furthermore, targeting Runx1 in HFpEF by using gene transfer or small molecule inhibitors improves diastolic function, both in female and male mice. Overall, our research enhances our understanding of RUNX1 in cardiac disease and demonstrates a novel translational target for the treatment of HFpEF. Keywords: Heart failure with preserved ejection fraction, metabolic heart failure, diastolic dysfunction, hypertrophy, pulmonary oedema, exercise intolerance CLINICAL PERSPECTIVEHeart failure (HF) is a leading cause of death world-wide and traditionally divided into different subtypes according to cardiac ejection fraction (EF). In contrast to HF with reduced EF (HFrEF), there are limited treatment options for HF with preserved EF which is of considerable concern given that HFpEF is projected to become the dominant HF subtype in the future 1. RUNX1 has been demonstrated to play an important role in the development of many cardiac and non-cardiac diseases. As a result, the potential for RUNX1 inhibitors as therapeutic agents across various conditions has become increasingly evident. In this study we established the therapeutic potential of targeting RUNX1 in the context of HFpEF. Targeting RUNX1 in cardiomyocytes markedly attenuates the development of the HFpEF phenotype and therefore this novel translational therapeutic target has great potential to address one of the biggest challenges in cardiac research.

physiology↗

bioRxiv2024

The Initiator Caspase Dronc Drives Compensatory Proliferation of Apoptosis-Resistant Cells During Epithelial Tissue Regeneration After Ionizing Radiation

Caspases, well-known for their role in executing apoptosis, also participate in various non-apoptotic processes. Despite this, their involvement in promoting compensatory proliferation - a key aspect of tissue regeneration following extensive cell death - has been a subject of ongoing ambiguity. In our study, we investigate compensatory proliferation in the Drosophila wing imaginal disc following ionizing radiation, a model epithelial tissue that has been a pioneering system for studying this regenerative response. Using a delayed genetic reporter to monitor the activity of the initiator caspase-2/9 ortholog, Dronc, we identified two populations of apoptosis-resistant epithelial cells involved in compensatory proliferation: those that activate Dronc (termed DARE cells) and those that do not (NARE cells). We show that DARE cells pass their apoptosis-resistance trait to their daughter cells, suggesting a molecular memory. We demonstrate that Dronc in DARE cells, but not the apoptosome adapter Dark and the effector caspases, promotes compensatory proliferation both within these cells and in NARE cells through a non-cell-autonomous mechanism. We found that Myo1D, an unconventional myosin interacting with Dronc, is essential for the survival of DARE cells by preventing the lethal activation of effector caspases and subsequent apoptosis. In contrast, Myo7A/Crinkled, another unconventional myosin that interacts with Dronc, promotes effector caspase activation in DARE cells. We demonstrate that the TNFR>JNK signaling pathway in DARE cells directly regulates their proliferation, which in turn influences NARE cell proliferation. Consequently, we show that maintaining proliferative homeostasis between DARE and NARE cells is vital for balanced tissue regeneration. Given the widespread use of ionizing irradiation in cancer treatment and prevention, our findings have potential implications for understanding treatment-resistant cells and cancer recurrence.

developmental biology↗

bioRxiv2024

Genomic evidence of Escherichia coli gut population diversity translocation in leukemia patients

Escherichia coli, a commensal species of the human gut, is an opportunistic pathogen which can reach extra-intestinal compartments, including the bloodstream and the bladder, among others. In non-immunosuppressed patients, purifying or neutral evolution of E. coli populations has been reported in the gut. Conversely, it has been suggested that when migrating to extra-intestinal compartments, E. coli genomes undergo diversifying selection as supported by strong evidence for adaptation. The level of genomic polymorphism and the size of the populations translocating from gut to extra-intestinal compartments is largely unknown. To gain insights in the pathophysiology of these translocations, we investigated the level of polymorphism and the evolutionary forces acting on the genomes of 77 E. coli isolated from various compartments in three immunosuppressed patients. Each patient had a unique strain which was a mutator in one case. In all instances, we observed that translocation encompasses the majority of the genomic diversity present in the gut. The same signature of selection, whether purifying or diversifying, and as anticipated, neutral for mutator isolates, was observed in both the gut and bloodstream. Additionally, we found a limited number of non-specific mutations among compartments for non-mutator isolates. In all cases, urine isolates were dominated by neutral selection. These findings indicate that substantial proportions of populations are undergoing translocation and that they present a complex compartment-specific pattern of selection at the patient level. ImportanceIt has been suggested that intra and extra-intestinal compartments differentially constrain the evolution of E. coli strains. Whether host particular conditions, such as immunosuppression, could affect the strain evolutionary trajectories remain understudied. We found that, in immunosuppressed patients, large fractions of E. coli gut populations are translocating with variable modifications of the signature of selection for commensal and pathogenic isolates according to the compartment and/or the patient. Such multiple site sampling should be performed in large cohorts of patients to get a better understanding of E. coli extra-intestinal diseases.

microbiology↗

bioRxiv2024

T-cell development and activation in humanized mice lacking mouse major histocompatibility complexes

Humanized mice transplanted with CD34+ hematopoietic progenitor cells (HPCs) are used to study human immune responses in vivo. However, the mismatch between the mouse major histocompatibility complexes (MHCs) and the human leukocyte antigens (HLAs) is not optimal for T-cell development and can trigger xenograft reactivity. We evaluated human T-cell development in NOD.Scid.Gamma mice lacking expression of MHC class I and II (NSG-DKO). Human leukocyte engraftment was detectable at 8 weeks post-transplantation. Human CD4+ and CD8+ T-cells were detectable in blood, thymus, bone marrow and spleen of humanized NSG-DKO mice for up to 20 weeks post-transplantation. Further, we evaluated the effects of lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN- and the human cytomegalovirus gB antigen. LV delivery promoted development and activation of human central memory, {beta} and {gamma}{delta} T-cells with amplifications of the T-cell repertoire. LV administration unleashed multiple reactome pathways such as type-I interferon responses, cell cycle and metabolic processes. In summary, development of human T-cells in humanized mice does not rely on mouse MHCs and can be boosted systemically via LV administration.

immunology↗

bioRxiv2024

CXCL8 secreted by immature granulocytes inhibits wildtype hematopoiesis in chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrated early clonal dominance with few residual wildtype hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate. Our hypothesis is that therapeutic control of the inflammatory component in CMML could contribute to stepping down disease progression. The present study explores the contribution of immature granulocytes (iGRANs) to CMML progression. iGRANs can be detected and quantified in the peripheral blood of patients by spectral and conventional flow cytometry. Their accumulation is a potent and independent poor prognostic factor. These cells belong to the leukemic clone and behave as myeloid-derived suppressor cells. Bulk and single cell RNA sequencing revealed a pro-inflammatory status of iGRAN that secrete multiple cytokines of which CXCL8 at the highest level. This cytokine inhibits the proliferation of wildtype but not CMML hematopoietic stem and progenitor cells (HSPCs) in which CXCL8 receptors are epigenetically downregulated. CXCL8 receptor inhibitors and CXCL8 blockade restore wildtype HSPC proliferation, suggesting that relieving CXCL8 selective pressure on wildtype HSPCs is a potential strategy to slow CMML progression and restore some healthy hematopoiesis.

cancer biology↗

bioRxiv2024

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohns Disease

Background and AimsWe previously identified small molecules predicted to reverse an ileal gene signature for future Crohns Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). MethodsInduced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. ResultsiPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. ConclusionsETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

molecular biology↗

bioRxiv2023

3D genome organization during TGFB-induced transcription requires nuclear microRNA and G-quadruplexes

Studying the dynamics of three-dimensional (3D) chromatin structure is essential to understand biological processes in the cell nucleus. Recent publications based on integrative analysis of multi-omics studies have provided comprehensive and multilevel insights into 3D genome organization emphasizing its role during transcriptional regulation. While enhancers are regulatory elements that play a central role in the spatiotemporal control of gene expression, chromatin looping has been broadly accepted as a means for enhancer-promoter interactions allowing them to stablish cell-type-specific gene expression signatures. On the other hand, G-quadruplexes (G4s) are non-canonical DNA secondary structures that are both, enriched at promoters and related to increased gene expression. However, the role of G4s in promoter-distal regulatory elements, such as super-enhancers (SE), as well as in 3D genome organization and chromatin looping mediating long-range enhancer-promoter interactions has remained elusive. Here we show that mature microRNA 9 (miR-9) is enriched at promoters and SE of genes that are inducible by tissue growth factor beta 1 (TGFB1) signaling. Further, we found that nuclear miR-9 is required for chromatin features related to increased transcriptional activity, such as broad domains of the euchromatin histone mark H3K4me3 (histone 3 tri-methylated lysine 4) and G4s. Moreover, we show that nuclear miR-9 is required for promoter-super-enhancer looping. Our study places a nuclear microRNA in the same structural and functional context with G4s and promoter-enhancer interactions during 3D genome organization and transcriptional activation induced by TGFB1 signaling, a critical regulator of proliferation programs in cancer and fibrosis.

molecular biology↗

bioRxiv2023

A common gene signature of the right ventricle in failing rat and human hearts

The molecular mechanisms of progressive right heart failure are incompletely understood. We systematically compared rat models of pulmonary artery or aortic banding to identify the transcriptomic changes that occur over months in the failing right versus left ventricle. Detailed bioinformatics analyses of 181 RNAseq datasets from cardiomyocytes or whole heart samples from these models, led to the identification of gene signatures, protein, and transcription factor networks specific to ventricles, compensated or decompensated disease states and type of heart failure. RNA-FISH approaches confirmed PAB-mediated regulation of key genes and revealed striking, spatially heterogeneous mRNA expression in the heart. Intersection of rat PAB-specific gene sets with 95 transcriptome data sets from human patients with chronic thromboembolic pulmonary hypertension led to the identification of more than 50 genes whose expression levels strongly correlated with the severity of right heart disease. Together, these data define a conserved, differentially regulated genetic network that coordinates progressive right heart failure in rats and humans. HighlightsO_LISide-by-side comparisons of RV or LV transcriptomes in the slowly failing rat heart C_LIO_LIIdentification of RV-specific gene sets in heart hypertrophy versus heart failure C_LIO_LIIdentification of RV gene sets correlating with severity of human CTEPH C_LIO_LIDevelopment of a core gene signature characteristic for RV failure C_LI

genomics↗