Biology subjects

Stanton, B.

Publications and source records attributed to Stanton, B..

11 recordsLinked to original sources

bioRxiv2026

Polymicrobial extracellular vesicles reduce the innate immune response of human cystic fibrosis bronchial epithelial cells

Chronic antibiotic-resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a pulmotype detected in [~]34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence, biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Elexacaftor/Tezacaftor/Ivacaftor (ETI), a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited ETI CFTR Cl- currents by CF pHBEC. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF. IMPORTANCECystic fibrosis (CF) is a genetic disease characterized by chronic polymicrobial lung infections that, if untreated, are one of the primary causes of death in CF. Elexacaftor/Tezacaftor/Ivacaftor (ETI) has many positive clinical outcomes, but it does not eliminate chronic polymicrobial lung infections or inflammation. Using a new biologically relevant co-culture model, we have demonstrated that bacteria secrete vesicles (bEVs) that contain proteins and RNAs. We observed that these RNA-loaded bEVs are predicted to promote the pathogenesis of chronic CF lung infections by enhancing bacterial virulence and biofilm formation, as well as upregulating the pro-inflammatory response in lung cells. ETI does not ameliorate the response of lung cells to bEVs. Our research will facilitate the development of more effective approaches to eliminate infection and inflammation in CF and other lung diseases characterized by chronic polymicrobial infections and excessive inflammation.

microbiology↗

bioRxiv2026

Hat1 Orchestrates Heterochromatin Inheritance by Regulating Localization of H3K9 Methyltransferases

Many regions of heterochromatin associate with the nuclear periphery and are known as Lamin-associated domains (LADs). Histone acetyltransferase 1 (Hat1) is a highly conserved enzyme which acetylates newly synthesized histones H4 on lysines 5 and 12 prior to their deposition on chromatin. Hat1 is required to preserve chromatin accessibility within a subset of LADs called Hat1-dependent accessibility domains (HADs). Here we profile a diverse set of histone modifications in Hat1 KO and WT immortalized mouse embryonic fibroblasts (iMEFs) and find that Hat1 regulates diverse aspects of the structure of HADs and non-HAD LADs (nhLADS). In HADs, these changes include the conversion of H3K9me2 to H3K9me3. Analysis of H3K9-specific histone methyltransferases (HMTs) shows that that Suv39h1 and Suv39h2 have distinct localization patterns, where only Suv39h2 localizes to LADs. G9a only localizes to LADs in regions enriched for H3K9me2. We find that Hat1 loss results in a redistribution of these HMTs in both HADs and nh LADs. There is a decrease in the levels of G9a with a concomitant increase in Suv39h2. These results suggest Hat1 functions to restrain the formation of a more strongly heterochromatic state and highlight a role for Hat1 as an essential regulator of heterochromatin inheritance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/713225v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@9f6238org.highwire.dtl.DTLVardef@1e95415org.highwire.dtl.DTLVardef@18f3e0aorg.highwire.dtl.DTLVardef@1322426_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

MIR17HG Expression Is Transcriptionally Regulated by PAX3::FOXO1 and MYCN 1 and is Necessary for Oncogenic Activity in Fusion-Positive Rhabdomyosarcoma

Alveolar rhabdomyosarcoma (RMS), an aggressive pediatric soft tissue cancer, is driven by the oncogenic fusion transcription factor PAX3::FOXO1 (P3F) or PAX7::FOXO1. In a subset of fusion-positive (FP)-RMS cases, amplification of the MIR17HG locus leads to overexpression of the miR-17-92 cluster of microRNAs (miRNAs). However, miR-17-92 is also highly expressed in FP-RMS tumors lacking this amplification, suggesting alternative regulatory mechanisms. Here, we show that P3F and MYCN cooperatively drive miR-17-92 expression in FP-RMS. CRISPR/Cas9-mediated knockout of P3F or MYCN in FP-RMS cell lines substantially reduced miR-17-92 expression. Using a human myoblast line or low P3F FP-RMS variant with inducible P3F or MYCN expression, P3F or MYCN alone induces minimal to low miR-17-92 expression whereas introduction of both MYCN and P3F leads to robust activation of the miR-17-92 cluster and acquisition of oncogenic phenotypes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed a P3F binding motif located 1.84 Mb upstream of the MIR17HG promoter. CRISPR-mediated deletion of this region in the myoblast system resulted in marked reduction of miR-17-92 expression and impaired oncogenic transformation. Functional inhibition of mature miRNAs of this cluster in FP-RMS cells using miRNA-sponge constructs suppressed proliferation and transformation. In the myoblast model system, transduction studies with exogenous miR-17-92 or miRNA-sponge expression constructs indicated that miR-17-92 is necessary but not sufficient for oncogenic transformation. Together, these findings establish a cooperative transcriptional axis in FP-RMS involving P3F and MYCN that activates MIR17HG through a distal regulatory element, thereby contributing to oncogenic behavior and uncovering a novel mechanistic vulnerability.

cancer biology↗

bioRxiv2025

Outer Membrane Vesicles Secreted by Bacteroides fragilis Inhibit CFTR Chloride Secretion by Human Colon Organoids

The goals of this study were to develop a model to study host pathogen interactions in primary human colon organoids and to test the hypothesis that Bacteroides fragilis toxin (BFT-2) secreted in outer membrane vesicles (OMVs) modulates mucosal immunity and CFTR Cl- secretion. Since Bacteroides species reside in mucus, OMVs are likely to represent a mechanism of communication between Bacteroides and the host. Two strains of Bacteroides were studied, Enterotoxigenic Bacteroides fragilis (ETBF), which produces BFT-2, and the non-toxigenic Bacteroides fragilis strain NCTC 9343 (NTBF) that does not produce BFT-2. We also utilized two additional strains of Bacteroides fragilis: one in which bft-2 was knocked out (ETBF {Delta}bft), and one that was engineered to contain bft-2 (NTBF+bft). We report that Bacteroides fragilis OMVs reduced CFTR Cl- secretion but had no effect on tight junction or cell adhesion proteins, transepithelial resistance (TER) or cytokine secretion by primary human colon organoids. NTBF OMVs containing BFT-2 were more effective in reducing CFTR Cl- secretion than NTBF lacking BFT-2. We conclude that OMVs secreted by Bacteroides can be an important mechanism of host pathogen interactions in the colon by reducing CFTR Cl- secretion.

microbiology↗

bioRxiv2024

Differential Regulation of Large-scale Chromosome Conformations in Osteoblasts and Osteosarcoma

Correct chromosome organization in the cell nucleus is essential for genome function. However, dynamics and regulations of large-scale chromosomal conformations beyond single compartments at larger than ten megabases in vivo in single cells remain largely unknown. Here we use CRISPR-Sirius, a high-resolution and high-sensitivity real-time imaging technique, to directly visualize distinctions in large-scale chromosomal conformations between live osteosarcoma (OS) cells and osteoblasts, suggesting extensive chromatin reorganization during cell transformation. A surprising discovery is that chromosome 19 long arm is primarily extended in osteoblasts and maintained by H3K27me3. Extended chromosome conformation has been reported in fly and mouse but not in human cells yet. However, in OS cells, chromosome 19 primarily folded into collapsed conformations, which reshape in minutes and are regulated by the chromosome architectural proteins CTCF and cohesin in the presence of H3K27ac. Changes in chromosome conformations by knocking down the cohesin subunit RAD21 resulted in altered gene expression, including proto-oncogenes. Transcription inhibition by a small molecule inhibitor did not have detectable effects on large-scale chromosome conformation, suggesting that local transcription events have limited effects on large-scale chromosomal architecture. Our results provide unique insights into the complex regulatory mechanisms of endogenous large-scale chromosome organization in normal and transformed osteogenic tissues. One-sentence summaryRegulations of chromosome territory organization in normal and cancer bone cells visualized by CRISPR-based live-cell imaging.

cell biology↗

bioRxiv2024

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection. New and noteworthyThe experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa, which reduced the OMV-LPS induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF. Graphical abstractThe anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. (B) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa. 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/578737v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@d9d5e7org.highwire.dtl.DTLVardef@16a5715org.highwire.dtl.DTLVardef@7b35ddorg.highwire.dtl.DTLVardef@10d4dc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

The 3D chromatin landscape of rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric soft tissue cancer with a lack of precision therapy option for patients. We hypothesized that with a general paucity of known mutations in RMS, chromatin structural driving mechanisms are essential for tumor proliferation. Thus, we carried out high-depth in situ Hi-C in representative cell lines and patient-derived xenografts to understand chromatin architecture in each major RMS subtype. We report a comprehensive 3D chromatin structural analysis and characterization of fusion-positive (FP-RMS) and fusion-negative rhabdomyosarcoma (FN-RMS). We have generated spike-in in situ Hi-C chromatin interaction maps for the most common FP-RMS and FN-RMS cell lines, and compared our data with patient derived xenograft (PDX) models. In our studies we uncover common and distinct structural elements in large Mb-scale chromatin compartments, tumor-essential genes within variable topologically associating domains, and unique patterns of structural variation. Our comprehensive analysis provides high-depth chromatin interactivity maps for contextualizing gene regulation events identification of functionally critical chromatin domains in RMS. HIGHLIGHTSO_LIPAX3-FOXO1 and MYOD localize in both A- and B-compartments C_LIO_LIConserved mechanisms dictate CTCF orientation at TAD boundaries in RMS C_LIO_LIDifferential TADs in each RMS subtype encompass tumor-specific genes C_LIO_LINeo-TADs are formed from SV events in each subtype of RMS C_LIO_LIBoth major RMS subtypes have structural variation that is identifiable from Hi-C C_LIO_LIDistinct mechanisms can produce the major fusion alleles in rhabdomyosarcoma C_LIO_LIPAX3-FOXO1 and MYOD genomic binding is more enriched at regions with CNV C_LI

cancer biology↗

bioRxiv2022

High resolution mapping reveals hotspots and sex-biased recombination in Populus trichocarpa

Fine-scale meiotic recombination is fundamental to the outcome of natural and artificial selection. Here, dense genetic mapping and haplotype reconstruction were used to estimate recombination for a full factorial Populus trichocarpa cross of seven males and seven females. Genomes of the resulting 49 full-sib families (N=829 offspring) were re-sequenced, and high-fidelity biallelic SNP/INDELs and pedigree information were used to ascertain allelic phase and impute progeny genotypes to recover gametic haplotypes. The fourteen parental genetic maps contained 1820 SNP/INDELs on average that covered 376.7 Mb of physical length across 19 chromosomes. Comparison of parental and progeny haplotypes allowed fine-scale demarcation of cross-over (CO) regions, where 38,846 CO events in 1,658 gametes were observed. CO events were positively associated with gene density and negatively associated with GC content and long terminal repeats. One of the most striking findings was higher rates of COs in males in 8 out of 19 chromosomes. Regions with elevated male CO rates had lower gene density and GC content than windows showing no sex bias. High-resolution analysis identified 67 candidate CO hotspots spread throughout the genome. DNA sequence motifs enriched in these regions showed striking similarity to those of maize, Arabidopsis and wheat. These findings, and recombination estimates, will be useful for ongoing efforts to accelerate domestication of this and other biomass feedstocks, as well as future studies investigating broader questions related to evolutionary history, perennial development, phenology, wood formation, vegetative propagation, and dioecy that cannot be studied using annual plant model systems.

genetics↗

bioRxiv2022

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa

Lung infection by antibiotic resistant strains of Pseudomonas aeruginosa is a well-known concern for immunocompromised hosts including people with lung diseases such as cystic fibrosis. We have previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AEC) deliver miRNA let-7b-5p to P. aeruginosa where it suppresses biofilm formation and increases sensitivity to beta-lactam antibiotics. In this study we used RNA-seq to characterize the small RNA (sRNA) content of EVs secreted by AEC and demonstrate transfer of multiple distinct RNA fragments from EVs to P. aeruginosa. Bioinformatic predictions reveal that several sRNAs may target all three subunits of the fluoroquinolone efflux pump MexHI-OpmD, an effect predicted to increase antibiotic sensitivity to fluoroquinolone antibiotics. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%) and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S rRNA external transcribed spacer that was transferred to P. aeruginosa by EVs was sufficient to reduce planktonic growth of P. aeruginosa in the presence of ciprofloxacin, to reduce the minimum inhibitory concentration (MIC) of P. aeruginosa for ciprofloxacin by over 50%, and to significantly reduce protein levels of MexH and OpmD. In conclusion, an rRNA fragment secreted by AEC in EVs increases the ciprofloxacin sensitivity of P. aeruginosa by targeting and down-regulating the fluoroquinolone efflux pump MexHI-OpmD. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections. Author SummaryAccording to the World Health Organization and the U.S. Centers for Disease Control and Prevention the development of antibiotic resistant strains of bacteria, including Pseudomonas aeruginosa, are a significant global threat to human health. Thus, development of new approaches to eliminate antibiotic resistant infections is required. In this study, we report that lung epithelial cells secrete extracellular vesicles (EVs) that fuse with and deliver small rRNAs to P. aeruginosa, and that the rRNAs increase the sensitivity of P. aeruginosa to the antibiotic ciprofloxacin by reducing protein levels of the drug efflux pump MexHI-OpmD. We identified one rRNA fragment that by itself significantly reduced the protein levels of MexH and OpmD and increased the ability of ciprofloxacin to kill P. aeruginosa. We propose that developing synthetic vesicles containing a combination of the rRNA that inhibits antibiotic efflux pumps and ciprofloxacin would benefit patients with antibiotic resistant P. aeruginosa infections.

microbiology↗

bioRxiv2021

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation

Robust and effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain energetically costly processes like growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By employing complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fuels biosynthetic processes while controlling the ratio of -ketoglutarate/succinate to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells causes proliferation and survival defects when the TCA cycle is truncated, blocking carbon flux to support nucleosides biosynthesis. Accordingly, replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. Conversely, SDH deficiency induces a pro-inflammatory gene signature in T cells and promotes T helper 1 (TH1) and T helper 17 (TH17) lineage differentiation. Mechanistically, the hypoxia-inducible factor 1 (HIF-1) is not required for succinate-induced inflammation in T cells. A reduced -ketoglutarate/succinate ratio in SDH deficient T cells promotes inflammation through changing the pattern of the transcriptional and chromatin-accessibility signatures and consequentially increasing the expression of the transcription factor, B lymphocyte-induced maturation protein-1 (Blimp-1). Collectively, our studies revealed a critical role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

immunology↗

bioRxiv2021

Epigenetic Regulation of Nuclear Lamina-Associated Heterochromatin by HAT1 and the Acetylation of Newly Synthesized Histones

During S phase, eukaryotic cells must faithfully duplicate both the sequence of the genome and the regulatory information found in the epigenome. A central component of the epigenome is the pattern of histone post-translational modifications that play a critical role in the formation of specific chromatin states. During DNA replication, parental nucleosomes are disrupted and re-deposited on the nascent DNA near their original location to preserve the spatial memory of the epigenetic modifications. Newly synthesized histones must also be incorporated into the nascent chromatin to maintain nucleosome density. Transfer of modification patterns from parental histones to new histones is a fundamental step in epigenetic inheritance. Whether new histones play an active or passive role in epigenetic inheritance is unknown. Here we report that HAT1, which acetylates lysines 5 and 12 of newly synthesized histone H4 during replication-coupled chromatin assembly, regulates the epigenetic inheritance of chromatin states. HAT1 regulates the accessibility of large domains of heterochromatin termed HAT1-dependent Accessibility Domains (HADs). HADs are mega base-scale domains that comprise ~10% of the mouse genome. HAT1 functions as a global negative regulator of H3 K9me2/3 and HADs correspond to the regions of the genome that display HAT1-dependent increases in H3 K9me3 peak density. HADs display a high degree of overlap with a subset of Lamin-Associated Domains (LADs). HAT1 is required to maintain nuclear structure and integrity. These results indicate that HAT1 and the acetylation of newly synthesized histones are critical regulators of the epigenetic inheritance of heterochromatin and suggest a new mechanism for the epigenetic regulation of nuclear lamina-heterochromatin interactions.

genomics↗