Biology subjects

Wingett, S. W.

Publications and source records attributed to Wingett, S. W..

8 recordsLinked to original sources

bioRxiv2025

Stalling of elongating Pol II triggers Ser7 phosphorylation in trans to drive transcription recovery

DNA is scattered with obstacles that stall RNA polymerase II (Pol II) and block the production of full-length transcripts. Two mechanisms are known to resolve stalled Pol II: transcription-coupled nucleotide excision repair (TC-NER) and the "last resort" Pol II ubiquitylation-degradation pathway. Here, we uncover a third, distinct mechanism that alerts incoming Pol II molecules to roadblocks ahead and primes them for efficient elongation. We show that transcription stalling triggers GSK3-mediated phosphorylation of Ser7 residues (Ser7P) on the Pol II C-terminal domain. Unexpectedly, this phosphorylation occurs in trans: obstacles in gene bodies induce Ser7P on Pol II complexes at gene beginnings. This modification enables processive transcription while restraining excessive Pol II degradation by the "last resort" pathway. Our findings reveal an adaptive system that preserves transcriptional homeostasis by coordinating polymerase behavior across the gene in response to elongation stress.

molecular biology↗

bioRxiv2024

Epigenetic restoration of differentiation competency via reversal of epiblast regionalisation

Although the epiblast in the embryo has the capacity to generate all tissues of the body, its in vitro counterparts often exhibit differentiation biases, posing significant challenges for both basic research and translational applications involving pluripotent stem cells (PSCs). The origins of these biases remain incompletely understood. In this study, we identify PSC differentiation biases as arising from fluctuations in repressive and activating histone posttranslational modifications, leading to the acquisition of a caudal epiblast-like phenotype. We present a novel approach to overcome this bias using a chemical chromatin restoration (CHR) treatment. This method restores transcriptional programs, chromatin accessibility, histone modification profiles, and differentiation potential, effectively recapitulating the competent anterior epiblast-like state. Furthermore, we propose that a high bivalency state is a defining feature of the anterior human epiblast. We suggest that fluctuations in histone modification marks drive epiblast regionalization, ultimately shaping cellular responses to differentiation cues.

developmental biology↗

bioRxiv2024

Identification of Cellular Signatures Associated with Chinese Hamster Ovary (CHO) Cell Adaptation for Secretion of Antibodies

The secretory capacity of Chinese hamster ovary (CHO) cells remains a fundamental bottleneck in the manufacturing of protein-based therapeutics. Unconventional biological drugs with complex structures and processing requirements are particularly problematic. Although engineered vector DNA elements can achieve rapid and high-level therapeutic protein production, a high metabolic and protein folding burden is imposed on the host cell. Cellular adaptations to these conditions include differential gene expression profiles that can in turn influence the productivity and quality control of recombinant proteins. In this study, we used quantitative transcriptomics and proteomics analyses to investigate how biological pathways change with antibody titre. Gene and protein expression profiles of CHO pools and clones producing a panel of different monoclonal and bispecific antibodies were analysed during fed-batch production. Antibody-expressing CHO pools were heterogeneous, resulting in few discernible genetic signatures. Clonal lines derived from these pools, selected for high and low production, yielded a small number of differentially expressed proteins that correlated with productivity and were shared across biotherapeutics. However, the dominant feature associated with higher protein production was transgene copy number and resulting mRNA expression level. Moreover, variability between clones suggested that the process of cellular adaptation is variable with diverse cellular changes associated with individual adaptation events.

bioengineering↗

bioRxiv2023

Multi-omic analysis of guided and unguided forebrain organoids reveal differences in cellular composition and metabolic profiles

Neural organoids are invaluable model systems for studying neurodevelopment and neurological diseases. For this purpose, reproducible differentiation protocols are needed that minimize inter-organoid variability whilst generating neural organoids that physiologically resemble the brain area of interest. Currently, two main approaches are used: guided, where the differentiation towards neuroectoderm and subsequently specific CNS regions is driven by applying extrinsic signalling molecules, and unguided, where the intrinsic capability of pluripotent stem cells to generate neuroectoderm without external signalling is promoted. Despite the importance for the field, the resulting differences between these models have not been directly investigated. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated using a guided and unguided approach focusing on proteomic, lipidomic and metabolomic differences. Furthermore, we characterised differences in phosphorylation and sialylation states of proteins, two key post-translational modifications (PTMs) in neurodevelopment, and performed single cell transcriptomics (scRNAseq). The multi-omic analysis revealed considerable differences in neuronal-, synaptic and glial content, indicating that guided forebrain organoids contain a larger proportion of neurons, including GABAergic interneurons, and synapses whereas unguided organoids contain significantly more GFAP+ cells and choroid plexus. Furthermore, substantial differences in mitochondrial- and metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid {beta}-oxidation in unguided forebrain organoids and a higher reliance on glycolysis in guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences arising when generating guided and unguided forebrain organoids and provide an important resource for the organoid field studying neurodevelopment and -disease.

neuroscience↗

bioRxiv2023

Genome-wide analysis of promoter contacts identifies novel regulators of late-stage adipogenesis

Adipogenesis is a multi-stage process essential for healthy fat storage and metabolic regulation. While early regulatory mechanisms are well characterized, the control of late-stage adipocyte differentiation remains poorly understood. Integrating CAGE-seq, promoter capture Hi-C, and a high-throughput siRNA screen of druggable genes, we report here that chromatin architecture rewiring promotes gene regulation changes essential for terminal adipogenesis. We identified nine clusters of dynamic promoter-anchored chromosomal interactions, many involving distal enhancers. Functional screening of genes engaged in these interactions revealed 19 novel regulators of late adipogenesis, including proteins with peptidase and ubiquitin ligase activity. Human genetic variant-to-gene mapping, coupled with cross-species chromatin interaction and synteny analyses, highlighted new gene-trait associations relevant to lipid traits (FXYD5, LAP3, SGPP1) and type 2 diabetes (FBXO17, FN3KRP, ZFAND6, TTC3). Our findings define the 3D gene regulatory landscape of late adipogenesis. The molecular links uncovered here provide mechanistic insight into metabolic disease risk and potential interventions.

genetics↗

bioRxiv2020

The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle

Circadian gene expression is essential for organisms to adjust cellular responses and anticipate daily changes in the environment. In addition to its physiological importance, the clock circuit represents an ideal, temporally resolved, system to study transcription regulation. Here, we analysed changes in spatial mouse liver chromatin conformation using genome-wide and promoter-capture Hi-C alongside daily oscillations in gene transcription in mouse liver. We found circadian topologically associated domains switched assignments to the transcriptionally active, open chromatin compartment and the inactive compartment at different hours of the day while their boundaries stably maintain their structure over time. Individual circadian gene promoters displayed maximal chromatin contacts at times of peak transcriptional output and the expression of circadian genes and contacted transcribed regulatory elements, or other circadian genes, was phase-coherent. Anchor sites of promoter chromatin loops were enriched in binding sites for liver nuclear receptors and transcription factors, some exclusively present in either rhythmic or stable contacts. The circadian 3D chromatin maps provided here identify the scales of chromatin conformation that parallel oscillatory gene expression and protein factors specifically associated with circadian or stable chromatin configurations.

genomics↗

bioRxiv2017

Spatial RNA proximities reveal a bipartite nuclear transcriptome and territories of differential density and transcription elongation rates

Spatial transcriptomics aims to understand how the ensemble of RNA molecules in tissues and cells is organized in 3D space. Here we introduce Proximity RNA-seq, which enriches for nascent transcripts, and identifies contact preferences for individual RNAs in cell nuclei. Proximity RNA-seq is based on massive-throughput RNA-barcoding of sub-nuclear particles in water-in-oil emulsion droplets, followed by sequencing. We show a bipartite organization of the nuclear transcriptome in which compartments of different RNA density correlate with transcript families, tissue specificity and extent of alternative splicing. Integration of proximity measurements at the DNA and NA level identify transcriptionally active genomic regions with increased nucleic acid density and faster RNA polymerase II elongation located close to compact chromatin.

molecular biology↗

bioRxiv2017

Chromosome contacts in activated T cells identify autoimmune disease-candidate genes

BackgroundAutoimmune disease-associated variants are preferentially found in regulatory regions in immune cells, particularly CD4+ T cells. Linking such regulatory regions to gene promoters in disease-relevant cell contexts facilitates identification of candidate disease genes.\n\nResultsWithin four hours, activation of CD4+ T cells invokes changes in histone modifications and enhancer RNA transcription that correspond to altered expression of the interacting genes identified by promoter capture Hi-C. By integrating promoter capture Hi-C data with genetic associations for five autoimmune diseases we prioritised 245 candidate genes with a median distance from peak signal to prioritised gene of 153 kb. Just under half (108/245) prioritised genes related to activation-sensitive interactions. This included IL2RA, where allele-specific expression analyses were consistent with its interaction-mediated regulation, illustrating the utility of the approach.\n\nConclusionsOur systematic experimental framework offers an alternative approach to candidate causal gene identification for variants with cell state-specific functional effects, with achievable sample sizes.

genomics↗