Biology subjects

Swaminathan, G.

Publications and source records attributed to Swaminathan, G..

9 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↗

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

CES1 DEFICIENCY IS ASSOCIATED WITH METABOLIC REPROGRAMING AND ENDOTHELIAL DYSFUNCTION IN PULMONARY ARTERIAL HYPERTENSION

BackgroundPulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary microvascular loss and obliterative remodeling, driven by metabolic reprogramming, oxidative stress, and endothelial dysfunction. While BMPR2 mutations contribute to metabolic shifts in pulmonary microvascular endothelial cells (PMVECs), their low penetrance suggests additional genetic modifiers play a role. A genetic screen of PAH PMVECs identified carboxylesterase 1 (CES1)--an endoplasmic reticulum (ER) enzyme involved in lipid metabolism and detoxification--as a candidate regulator of endothelial metabolism and angiogenesis. We hypothesize that CES1 loss promotes endothelial dysfunction via metabolic reprogramming, lipotoxicity, and oxidative stress. MethodsPAH and healthy PMVECs and lung tissues were obtained from transplant donors and commercial sources. CES1 expression was modulated in PMVECs using siRNA knockdown and plasmid overexpression. Mitochondrial and ER function were assessed via confocal microscopy and proteomics. CES1 heterozygous knockout (HET KO) and endothelial-specific knockout (ECKO) mice were exposed to normoxia or hypoxia, with lung tissues analyzed by single-cell RNA sequencing (scRNA-seq) and histopathology. ResultsCES1 expression was significantly reduced in PAH PMVECs and vascular lesions. CES1-deficient PMVECs exhibited increased apoptosis, reactive oxygen species (ROS) production, mitochondrial fragmentation, ER stress, and impaired angiogenesis. Confocal imaging and metabolic studies revealed lipid droplet accumulation, reduced fatty acid oxidation, and a glycolytic shift-- phenotypes reversed by CES1 restoration. Mechanistically, CES1 transcription was induced by BMPR2 via NRF2 activation, a key regulator of redox and metabolic homeostasis. In vivo, CES1-deficient mice developed severe pulmonary hypertension (PH) under hypoxia, with extensive vascular remodeling, right ventricular dysfunction, and dysregulated angiogenesis and lipid metabolism pathways, as confirmed by lung scRNA-seq. ConclusionsCES1 is essential for pulmonary endothelial homeostasis and serves as a critical modifier of BMPR2 signaling. Given the limited efficacy of current PAH therapies in reversing endothelial dysfunction and small-vessel loss, restoring CES1 expression represents a promising therapeutic strategy.

biochemistry↗

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↗

bioRxiv2021

Pre-vaccination Frequency of Circulatory Tfh is associated with Robust Immune Response to TV003 Dengue Vaccine

It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents. Author summaryDengue virus (DENV) is a worldwide threat that causes significant health and economic burden. Currently, there are several challenges in the development of a DENV vaccine including the existence of four different serotypes all; capable of causing disease and antibody dependent enhancement (ADE). For complete protection, a vaccine must be able to generate neutralizing antibodies against all 4 serotypes to avoid ADE. Currently, there is one licensed DENV vaccine, CYD-TDV (DENGVAXIATM). However, this vaccine is only efficacious in protecting against severe disease in DENV seropositive individuals therefore serostatus effect must be further studied for optimal vaccine design. A subset of CD4+ T cells called T-follicular helper (Tfh) cells have been well known to play a major role in aiding high affinity antibody production. Therefore, we chose to look at subsets of Tfh and the cytokines they produce in human blood that can serve as biomarkers for effective vaccine design. We found that DENV sero-positive participants had increased pre-vaccination frequencies of Tfh cells and higher levels of the Tfh related chemokine CXCL13/BLC that plays a role in directing antigen-specific responses. This pre-vaccination Tfh profile and CXCL13/BLC are then correlated positively with the vaccinees ability to produce neutralizing antibody against all four sero-types (breadth of the Response) of DENV, an important goal for all DENV vaccine trials.

immunology↗

bioRxiv2021

Effect of Substrate Stiffness on Human Intestinal Enteroids Infectivity by Enteroaggregative Escherichia coli

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft ([~]2 kPa), medium ([~]10 kPa), and stiff ([~]100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 {micro}m whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 {micro}m. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of >100 and even >1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/448533v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f9c7b3org.highwire.dtl.DTLVardef@a5d5feorg.highwire.dtl.DTLVardef@184224forg.highwire.dtl.DTLVardef@1e8a28d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗

bioRxiv2021

Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids

Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures. ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

molecular biology↗

bioRxiv2021

Vaccine Hyporesponse Induced By Individual Antibiotic Treatment In Mice And Non-Human Primates Is Diminished Upon Recovery Of The Gut Microbiome

Emerging evidence demonstrates a connection between microbiome composition and suboptimal response to vaccines (vaccine hyporesponse). Harnessing the interaction between microbes and the immune system could provide novel therapeutic strategies for improving vaccine response. Currently we do not fully understand the mechanisms and dynamics by which the microbiome influences vaccine response. Using both mouse and non-human primate models, we report that short-term oral treatment with a single antibiotic (vancomycin) results in disruption of the gut microbiome and this correlates with a decrease in systemic levels of antigen-specific IgG upon subsequent parenteral vaccination. We further show that recovery of microbial diversity before vaccination prevents antibiotic-induced vaccine hyporesponse, and that the antigen specific IgG response correlates with the recovery of microbiome diversity. RNA-sequencing analysis of small intestine, spleen, whole blood, and secondary lymphoid organs from antibiotic treated mice revealed a dramatic impact on the immune system, and a muted inflammatory signature is correlated with loss of bacteria from Lachnospiraceae, Ruminococcaceae, and Clostridiaceae. These results suggest that microbially modulated immune pathways may be leveraged to promote vaccine response and will inform future vaccine design and development strategies. ImportanceAntibiotic-induced gut microbiome disruption has been linked to reduced vaccine efficacy. Despite these observations, there remains a knowledge gap in the specific mechanisms by which antibiotics and the gut microbiome influence vaccine efficacy. We aim to contribute to the fields growing mechanistic understanding by presenting a detailed analysis of antibiotic treatment and recovery as it relates to vaccine response and the microbiome. Using animal models, we show that short-term antibiotic treatment prior to vaccination results in diminished vaccine-specific immune responses, and that these are correlated with specific microbiome signatures. We also demonstrate the converse, in which gut microbiome recovery can result in improved vaccine response. We further reveal that antibiotics can significantly alter multiple relevant immune pathways and this alteration in immune tone may contribute to the vaccine hyporesponse. We expect our findings will enable the continued prosecution of the role of the microbiome in modulating the host immune system.

immunology↗

bioRxiv2021

GPR15 in colon cancer development and anti-tumor immune responses.

G protein-coupled receptor 15 (GPR15) is a chemoattractant receptor which in response to its ligand, C10orf99/GPR15L, promotes colon homing of T cells in health and colitis. The functional role of GPR15 in colon cancer is largely unexplored, motivating our current studies using murine colon cancer models and human colorectal cancer (CRC) tissues. Our initial analysis of human CRC specimen revealed significant reduction in GPR15 expression and frequency of GPR15+ immune cells in tumors compared to tumor-free surgical margins. In the AOM/DSS murine model of colitis associated colon cancer (CAC), we observed increased colonic polyps/tumor burden and lower survival rate in Gpr15-deficient (KO) compared to Gpr15-sufficient (Het) mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. GPR15 deficiency thus alters the immune environment in colonic polyps to mitigate T cell-mediated anti-tumor responses resulting in severe disease. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38 CRC mouse model increased CD45+ cell infiltration, enhanced TNF expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden. Our findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.

immunology↗