Biology subjects

Dewari, P. S.

Publications and source records attributed to Dewari, P. S..

5 recordsLinked to original sources

bioRxiv2026

A Single-Nucleus Transcriptomic Atlas Reveals Cell-Type-Specific Responses to OsHV-1 Infection in the Pacific Oyster

BackgroundThe Pacific oyster (Crassostrea/Magallana gigas) is increasingly recognised as a model marine invertebrate. Valued for both ecological and commercial importance, Pacific oysters are farmed widely, supporting global food security by providing a sustainable nutrient-rich source of protein. Despite the significant and recurring economic losses caused by Ostreid herpesvirus (OsHV-1) outbreaks, only a limited number of studies have examined host-pathogen interplay at single-cell resolution. The few available studies largely focus on circulating immune cells (haemocytes), thereby overlooking the complexity of host responses across different tissues and organs. ResultsWe present a detailed single-nucleus transcriptomic atlas of the whole Pacific oysters, including during OsHV-1 infection. A total of 18 distinct transcriptomic clusters were resolved, capturing major cell populations from the gill, mantle, hepatopancreas, adductor muscle, and haemocytes. Notably, three populations- gill ciliary cells, hepatopancreas cells, and an immune-enriched cluster 1- exhibited pronounced transcriptomic responses to OsHV-1 infection. Across the 6, 24, 72, and 96 hours post-infection (hpi) time course, viral transcripts were detected almost exclusively at 72 hpi, with enrichment primarily in adductor muscle cells and two immune cell populations- immature haemocytes, and hyalinocytes. ConclusionsOur findings suggest potential entry portals and tissue-specific replication sites for the OsHV-1 virus in Pacific oysters. This atlas resource provides a high-resolution cellular framework for understanding host-virus interactions and establishes a foundation for future investigations into herpesvirus pathogenesis in marine invertebrates.

genomics↗

bioRxiv2024

Multiomics uncovers the epigenomic and transcriptomic response to viral and bacterial stimulation in turbot

Uncovering the epigenomic regulation of immune responses is essential for a comprehensive understanding of host defence mechanisms, though remains poorly investigated in farmed fish. We report the first annotation of the innate immune regulatory response in the turbot genome (Scophthalmus maximus), integrating RNA-Seq with ATAC-Seq and ChIP-Seq (H3K4me3, H3K27ac and H3K27me3) data from head kidney (in vivo) and primary leukocyte cultures (in vitro) 24 hours post-stimulation with viral (poly I:C) and bacterial (inactive Vibrio anguillarum) mimics. Among the 8,797 differentially expressed genes (DEGs), we observed enrichment of transcriptional activation pathways in response to Vibrio and immune pathways - including interferon stimulated genes - for poly I:C. We identified notable differences in chromatin accessibility (20,617 in vitro, 59,892 in vivo) and H3K4me3-bound regions (11,454 in vitro, 10,275 in vivo) between stimulations and controls. Overlap of DEGs with promoters showing differential accessibility or histone mark binding revealed significant coupling of the transcriptome and chromatin state. DEGs with activation marks in their promoters were enriched for similar functions to the global DEG set, but not always, suggesting key regulatory genes being in poised state. Active promoters and putative enhancers were enriched in specific transcription factor binding motifs, many common to viral and bacterial responses. Finally, an in-depth analysis of immune response changes in chromatin state surrounding key DEGs encoding transcription factors was performed. This multi-omics investigation provides an improved understanding of the epigenomic basis for the turbot immune responses and provides novel functional genomic information, leverageable for disease resistance selective breeding.

genomics↗

bioRxiv2022

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional condensates, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harboring distinct ecDNA species (EGFR, MYC, PDGFR). We found no evidence that ecDNA cluster with one another or closely interact with transcriptional condensates. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy was similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with the cellular transcriptional machinery.

genetics↗

bioRxiv2021

The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation.

Several studies have shown a role for Stag proteins in cell identity. Our understanding of how Stag proteins contribute to cell identity have largely been focused on its roles in chromosome topology as part of the cohesin complex and the impact on protein-coding gene expression. Furthermore, several Stag paralogs exist in mammalian cells with non-reciprocal chromosome structure and cohesion functions. Why cells have so many Stag proteins and what specific functions each Stag protein performs to support a given cell state are poorly understood. Here we reveal that Stag1 is the dominant paralog in mouse embryonic stem cells (mESC) and is required for pluripotency. Through the discovery of diverse, naturally occurring Stag1 isoforms in mESCs, we shed new light not only on the unique ends of Stag1 but also the critical role that their levels play in stem cell identity. Furthermore, we revel a new role for Stag1, and specifically its unique N-terminal end, in regulating nucleolar integrity and safeguarding mESCs from totipotency. Stag1 is localised to repressive perinucleolar regions, bound at repeats and interacts with Nucleolin and TRIM28. Loss of the Stag1 N-terminus, leads to decreased LINE-1 and rRNA expression and disruption of nucleolar structure and function which consequently leads to activation of the two-cell-like (2C-LC)-specific transcription factor DUX and conversion of pluripotent mESCs to totipotent 2C-LCs. Our results move beyond protein-coding gene regulation via chromatin loops into a new role for Stag1 in repeat regulation and nucleolar structure, and offer fresh perspectives on how Stag proteins contribute to cell identity and disease.

cell biology↗

bioRxiv2018

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain tumour-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of sixty different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

cell biology↗