Biology subjects

Benoit, J. M.

Publications and source records attributed to Benoit, J. M..

6 recordsLinked to original sources

bioRxiv2025

Nucleosome positioning and sensitivity suggest novel functional organization of the purple sea urchin (Strongylocentrotus purpuratus) genome

The eukaryotic nucleosome is the fundamental subunit of chromatin and plays functional roles in DNA templated processes, including replication and transcription. In eukaryotic promoters, nucleosome organization is highly structured, with nucleosomes occupying canonical positions flanking the transcription start site (TSS), thereby regulating access of the transcriptional machinery to the underlying DNA. We determine whether this canonical distribution is present in the purple sea urchin, Strongylocentrotus purpuratus, a species of ecological importance and a model organism for developmental biology and climate science. We used titrations of micrococcal nuclease to produce high throughput maps of nucleosome distribution and sensitivity to digestion from female urchin gonad tissue. Unlike yeast, flies, zebrafish, maize, mice or humans, urchins have extended nucleosome repeat lengths and lack a nucleosome depleted region over TSSs. Urchin promoters are dominated by strongly positioned and highly occupied +1 and +2 nucleosomes which are most prominent in highly expressed genes. Additionally, urchin promoters exhibit distinct patterns of susceptibility to nuclease digestion, with heightened sensitivity upstream of the TSS and limited resistance to nuclease digestion. Discretely positioned sensitive nucleosomes were enriched in promoters of highly expressed genes, suggesting a relationship between nucleosome sensitivity and transcriptional regulation. Collectively, we present a comprehensive overview of the unique interplay between nucleosome positioning and chromatin sensitivity in sea urchins. Our study not only provides a better understanding of dynamics of gene expression in a key developmental organism, but also reveals potential heterogeneity in a key structural property of chromatin previously thought to be homogeneous in model eukaryotes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/653534v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@c724dforg.highwire.dtl.DTLVardef@45c163org.highwire.dtl.DTLVardef@19b6043org.highwire.dtl.DTLVardef@1ccf475_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

The Cistrome Response to Hypoxia in Human Umbilical Vein Endothelial Cells

Hypoxic stress triggers transcriptional signaling mainly through hypoxia-inducible transcription factors (HIFs), which bind hypoxia response elements (HREs) in gene regulatory regions. However, only a small proportion ([~]1%) of known HREs are occupied by HIFs during hypoxia, suggesting the involvement of additional hypoxia-responsive factors. To address this gap, we utilized MNase-defined cistrome Occupancy Analysis sequencing (MOA-seq), with the term cistrome referring to all genomic regions where transcription factors and other trans-acting regulators are bound to cis-acting elements across the genome for a particular cell type or treatment. This MNase-based assay enables genome-wide, high-resolution (<30 bp) identification of transcription factor (TF) occupancy footprints embedded within larger regions, most of which were previously annotated as open or accessible chromatin. Applying this in situ cistrome mapping to fixed nuclei from endothelial cells under normoxia or hypoxia (1, 3, or 24 hours) revealed thousands of hypoxia-responsive genomic sites with dynamic TF footprints. The affected genes were enriched in canonical hypoxia-induced pathways, such as angiogenesis. Motif analysis identified over 100 candidate TFs potentially mediating these multifaceted genomic responses. By grouping hypoxia-modified occupancy signals across the hypoxia exposure times, we clustered differentially occupied MOA sites into defined 10 distinct TF kinetic clusters, half of which were associated with HIF1A. HIF1A-proximal binding sites suggested co-activators, while non-HIF1A clusters pointed to additional TFs that may have HIF1A-independent roles. This analysis provides insight into how multiple TF networks coordinate hypoxia responses and highlights the power of cistrome profiling to deepen our understanding of the complex genomic response to low oxygen conditions. KEY POINTSO_LIMOA-seq mapped TF occupancy at 21,765 sites in normoxia, including 7,444 beyond the known ENCODE cCREs. C_LIO_LIHypoxia for 1, 3, and 24h changes the cistrome occupancy at thousands of genes. C_LIO_LIClustering analysis of hypoxia-responsive footprints consolidated cistrome kinetics into HIF1A-associated and HIF1A-independent TFs. C_LI

genomics↗

bioRxiv2025

SARS-CoV-2 nucleocapsid uniquely disrupts chromatin over pathophysiologically relevant gene promoters

SARS-CoV-2, the causative agent of COVID-19, is a positive-sense, single-stranded RNA virus that causes a spectrum of disease severity, from asymptomatic infection to severe illness to long-term sequelae. Similar to other human coronaviruses, SARS-CoV-2 proteins modulate host genomic responses through epigenomic modifications, facilitating viral replication and immune evasion. While the nucleocapsid protein is well known for its role in RNA stability and immune modulation, its impact on host chromatin organization remains unclear. To investigate this, we generated stable human alveolar cell lines expressing nucleocapsid proteins from endemic and pandemic human coronaviruses. Our analysis revealed that nucleocapsid proteins from all tested coronaviruses induced changes in nucleosome positioning and occupancy at specific gene promoters involved in coagulation pathways, hormone signaling, and innate immune responses. Additionally, SARS-CoV-2-specific alterations were identified in genes dysregulated in severe infections, suggesting a direct role for epigenomic modifications in disease pathophysiology. We also observed extensive changes in nucleosome susceptibility to nuclease digestion in SARS-CoV and SARS-CoV-2 samples that were not observed in common cold cell lines. Promoters with altered sensitivity and resistance to nuclease were linked to innate immune, metabolic, olfactory, and signaling pathways known to be dysregulated in severe COVID-19 and post-acute sequelae (PASC). These findings demonstrate that nucleocapsid protein expression alters chromatin structure at specific loci, implicating viral proteins in host genome dysregulation. Furthermore, we identified both shared and unique chromatin targets of SARS-CoV-2 and common cold coronaviruses, highlighting pathways for further investigation and potential therapeutic intervention. ImportanceHost chromatin is known to be modulated by coronaviruses during infections. However, the role of the nucleocapsid protein in these alterations are unknown. Here, we show that nucleocapsids from seven human coronaviruses alter nucleosome distribution and susceptibility to enzymatic digestion over specific gene promoters in a human lung cell line. Nucleocapsids from SARS-CoV and SARS-CoV-2 have the most prominent effects which are seen over genes involved in immune responses, metabolism, hormone signaling, and other pathways that are known to be dysregulated in severe COVID-19 and post-acute sequelae of COVID-19. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/639619v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@d97a79org.highwire.dtl.DTLVardef@1f7d5c3org.highwire.dtl.DTLVardef@1f312f0org.highwire.dtl.DTLVardef@1606a56_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Transient alterations in nucleosome distribution and sensitivity to nuclease define the THP-1 monocyte to macrophage transition

The monocyte to macrophage transition is marked by alterations to both the structure and function of the genome, including changes in histone post-translational modifications, DNA methylation, 3D nuclear architecture, and expression of lineage specific genes. The nucleosome is the fundamental organizational unit of the eukaryotic genome and underpins both genome structure and function. However, nucleosome dynamics at promoters, which are essential for transcriptional regulation, are understudied in cellular differentiation. We conducted high-resolution chromatin structure profiling at promoters in the THP-1 cell line at eight different time points spanning PMA-induced monocyte to macrophage differentiation. We found that fewer than 10% of nucleosomes within promoters were redistributed during differentiation and only a subset of these were associated with immediate transcriptional alterations. Nucleosomes within the promoters of PMA-responsive genes were strongly positioned prior to differentiation and experienced minimal alterations during differentiation thus implying the existence of a pre-differentiation primed chromatin state. Additionally, we observed pronounced alterations in nucleosome sensitivity to MNase digestion within one hour of PMA-induced differentiation and the emergence of a highly resistant phenotype in fully differentiated cells. We found that resistance is correlated with active chromatin marks, transcription factor binding, gene expression, and higher order chromatin structure demonstrating that it is a useful measure of both genome structure and function. Together this suggests that, unlike more stable nucleosome distribution, transient sensitivity alterations may underpin new genomic functions in differentiating cells. Our results offer a framework for understanding how chromatin structural alterations potentiate cellular differentiation in a monocyte model and use methodology that is widely applicable to other systems. Summary sentenceNucleosome distribution is largely static during PMA induced monocyte differentiation while nucleosome sensitivity is highly dynamic and is associated with gene expression, active chromatin marks, transcription factor binding, and higher order chromatin structure.

genomics↗

bioRxiv2025

Dynamic nucleosome redistribution and increases innucleosome sensitivity underpin THP-1 macrophage response to LPS

Macrophages detect lipopolysaccharide (LPS) through toll-like receptor 4 (TLR-4) on the cell surface which initiates a signaling cascade resulting in the recruitment of regulatory factors to chromatin and subsequent expression of chemokine and cytokine genes. Primary response genes, marked by poised promoters and enhancers, are rapidly expressed after LPS stimulation, and their gene products activate secondary response genes via paracrine and autocrine signaling pathways. While the signaling cascades following macrophage activation are well understood, the dynamics of nucleosome architecture and regulatory factor binding in promoter regions during early and late LPS responses remain unclear. Here, we stimulated THP-1 derived macrophages with LPS and assessed nucleosome distribution and MNase sensitivity across promoters at eight time points spanning primary and secondary responses. We found that while nucleosome distribution was static over most promoters, LPS stimulation resulted in transient remodeling of a subset of immune and DNA repair gene promoters. We also observed distinct MNase sensitivity alterations in two phases which aligned with early and late gene expression patterns. Notably, while most Pol II promoters showed altered chromatin sensitivity, only a subset exhibited transcriptional changes, suggesting that widespread alterations in nucleosome distribution and sensitivity occur at promoters with or without alterations in gene expression. These findings provide new temporal insights into the transient and long-term effects of immune stimulation on promoter architecture and offer a methodological framework for additional time-resolved studies of chromatin remodeling in other systems. Summary sentenceFollowing LPS stimulation, a subset of nucleosomes in macrophage immune promoters undergo transient redistribution, whereas the majority of nucleosomes show changes in MNase sensitivity that are largely uncoupled from gene expression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/637695v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@198f47org.highwire.dtl.DTLVardef@dab80org.highwire.dtl.DTLVardef@1769d0corg.highwire.dtl.DTLVardef@40be27_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO LPS stimulation of THP-1 derived macrophages leads to altered nucleosome occupancy and positioning within a subset of promoters which are enriched for LPS response genes. Altered distribution patterns permit regulatory factor binding and gene expression. The majority of promoters have altered nucleosome sensitivity with a trend towards increased sensitivity after LPS stimulation. Altered sensitivity results in regulatory factor binding and expression of LPS response genes. C_FIG

genomics↗

bioRxiv2023

Targeting the MR1-MAIT Cell Axis Improves Vaccine Efficacy and Affords Protection against Viral Pathogens

Mucosa-associated invariant T (MAIT) cells are MR1-restricted, innate-like T lymphocytes with tremendous antibacterial and immunomodulatory functions. MAIT cells also sense and respond to viral infections in an MR1-independent fashion. However, whether they can be directly targeted in immunization strategies against viral pathogens is unknown. We addressed this question in multiple wild-type and genetically altered but clinically relevant mouse strains using several vaccine platforms against influenza viruses, poxviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), a riboflavin-based MR1 ligand of bacterial origin, can synergize with viral vaccines to expand MAIT cells in multiple tissues, reprogram them towards a pro-inflammatory MAIT1 phenotype, license them to bolster mainstream virus-specific CD8+ T cell responses, and potentiate heterosubtypic antiviral protection. Repeated 5-OP-RU administration did not render MAIT cells anergic, thus allowing for its inclusion in prime-boost immunization protocols. Mechanistically, tissue MAIT cell accumulation was due to their robust proliferation, as opposed to altered migratory behavior, and required viral vaccine replication competency, Toll-like receptor 3 (TLR3) and cell-autonomous type I interferon receptor signaling. Furthermore, the observed phenomenon was manifest in young and old female and male mice, and could also be recapitulated in a human cell culture system in which peripheral blood mononuclear cells were exposed to replicating virions and 5-OP-RU. In conclusion, although viruses and virus-based vaccines are devoid of the riboflavin biosynthesis machinery that supplies MR1 ligands, targeting MR1 enhances the efficacy of vaccine-elicited antiviral immunity. We propose 5-OP-RU as a non-classic but potent and versatile vaccine adjuvant against respiratory viruses.

immunology↗