Biology subjects

Ward, J. M.

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

14 recordsLinked to original sources

bioRxiv2026

Loss of NKX2-1 predisposes thyroid to neoplasm development through regulation of oxidative stress

Many factors including ionizing radiation and iodine deficiency are known to increase thyroid carcinogenesis risk. Our dataset analysis of The Cancer Genome Atlas (TCGA) showed that lower mRNA expression of NK2 homeobox 1 (NKX2-1) transcription factor, a master regulator of genesis, homeostasis, and function of thyroid, is linked to poor prognosis of papillary thyroid cancer patients. Here we provide the findings that thyroid-specific Nkx2-1 conditional knockout (Nkx2-1{Delta}T) mice develop thyroid adenoma and carcinoma in higher frequency with combined exposure to radiation and iodine deficiency than control Nkx2-1fl/fl mice. Iodine deficiency caused oxidative stress, which subsequently resulted in DNA damage, leading to transformation of thyroid follicular cells. RNA-seq gene set enrichment analysis indicated higher production of reactive oxygen species (ROS) in the thyroids of Nkx2-1{Delta}T as compared to Nkx2-1fl/fl mice with combined exposure to radiation and iodine deficiency. This was accompanied by a feedback induction of SOD3 (superoxide dismutase 3) and GPX2 (glutathione peroxidase 2). These antioxidants were naturally expressed at higher levels in the thyroids of Nkx2-1{Delta}T than Nkx2-1fl/fl mice without iodine deficiency or radiation. Nkx2-1{Delta}T thyroids exhibited abnormal follicle architecture and up-regulation of Acox2 (encoding acyl-CoA oxidase 2), which produces hydrogen peroxide. These results suggest that loss of NKX2-1 may contribute to excess ROS production, which elevates basal oxidative stress resulting in the promotion of ROS-induced carcinogenesis. We propose a role for NKX2-1 as a regulator of ROS production homeostasis in the thyroid. Its disturbance would dispose thyroid follicular cells more vulnerable to the ROS-producing carcinogens.

cancer biology↗

bioRxiv2025

Commensal skin bacteria interact with the innate immunesystem to promote tail regeneration in Xenopus laevis tadpoles.

Xenopus laevis tadpoles regenerate their tails following partial amputation, however, for a brief developmental window, some undertake wound healing rather than regeneration. Inspired by links between microbiomes and human inflammatory disease, we asked how tadpole skin microbiomes and innate immunity influence the ability to regenerate tails. Having previously shown that lipopolysaccharide-Tlr4 signalling promotes regeneration, here, we demonstrate that the peptidoglycan-Tlr2 pathway is also involved. While tadpoles acquire their commensal skin bacteria from their mothers, analysis of mitochondrial haplotypes did not explain familial regenerative bias. Levels of endogenous lipopolysaccharides on tail tips were also not predictive of regenerative success, and shotgun sequencing indicated no difference in bacterial loads. To see if microbiome composition correlated with regenerative success, we sequenced 16S rRNA amplicons from 503 tadpole tail tips and mapped these to regenerative outcomes. While no one taxon appeared critical for regenerative success, higher proportions of Gram-positives correlated with successful regeneration. Our results suggest a previously undocumented role for Tlr2, peptidoglycan and Gram-positive commensal skin bacteria in tipping the balance from wound repair to regenerative programmes in Xenopus laevis refractory stage tadpoles.

developmental biology↗

bioRxiv2024

SL/Kh Pre-B Lymphomas Originate in the Thymus and are a Model for Primary Mediastinal (Thymic) Large B-Cell Lymphoma

SL/Kh mice develop a high frequency of retrovirally-induced pre-B lymphomas at 3-6 months of age. They also exhibit an abnormal transient expansion of pre-B cells in the bone marrow, although the relevance of this expansion for lymphomagenesis has remained unclear. Here, we use a dual approach that combines pathology with flow cytometry to more fully characterize the nature and origin of SL/Kh lymphomas. Unexpectedly, our studies showed that SL/Kh lymphomas arise from a rare population of pro/pre-B cells in the thymus. We also identified a 10-fold reduction in Notch1 expression in SL/Kh thymic T cells that is associated with a block in early T cell development, a reduction in the number of thymic T cells with age, and an expansion of thymic pro/pre-B cells. This phenotype is consistent with previous studies showing that Notch1 signaling is essential for lymphoid progenitors to undergo T cell commitment and for suppressing B cell development in the thymus. We propose that this developmental defect provides a niche for early B cells to accumulate in the thymus, which, when combined with subsequent retroviral insertional mutagenesis, results in the induction of pre-B lymphomas that originate in the thymus. This is also consistent with our analysis of the genes insertionally mutated in SL/Kh lymphomas, which shows that many function in signaling pathways such as JAK/STAT and RAS/MAPK/ERK that are commonly deregulated in B-cell lymphomas. Primary human mediastinal large B-cell lymphoma (MLBCL) is another lymphoma that is derived from thymic B cells, although virtually nothing is known about the cause of this rare disease. Our studies provide new insights into an underappreciated class of B-cell lymphomas and a mouse model for the study of MLBCL.

pathology↗

bioRxiv2024

Fate (or state) of CA2 neurons in a mineralocorticoid receptor knockout.

Hippocampal area CA2 has emerged as a functionally and molecularly distinct part of the hippocampus and is necessary for several types of social behavior, including social aggression. As part of the unique molecular profile of both mouse and human CA2, the mineralocorticoid receptor (MR; Nr3c2) appears to play a critical role in controlling CA2 neuron cellular and synaptic properties. To better understand the fate (or state) of the neurons resulting from MR conditional knockout, we used a spatial transcriptomics approach. We found that without MRs, CA2 neurons acquire a CA1-like molecular phenotype. Additionally, we found that neurons in this area appear to have a cell size and density more like that in CA1. These finding support the idea that MRs control at least CA2s state during development, resulting in a CA1-like fate.

neuroscience↗

bioRxiv2024

Characterization of Collaborative Cross mouse founder strain CAST/EiJ as a novel model for lethal COVID-19

Mutations in SARS-CoV-2 variants of concern (VOCs) have expanded the viral host range beyond primates, and a limited range of other mammals, to mice, affording the opportunity to exploit genetically diverse mouse panels to model the broad range of responses to infection in patient populations. Here we surveyed responses to VOC infection in genetically diverse Collaborative Cross (CC) founder strains. Infection of wild-derived CC founder strains produced a broad range of viral burden, disease susceptibility and survival, whereas most other strains were resistant to disease despite measurable lung viral titers. In particular, CAST/EiJ, a wild-derived strain, developed high lung viral burdens, more severe lung pathology than seen in other CC strains, and a dysregulated cytokine profile resulting in morbidity and mortality. These inbred mouse strains may serve as a valuable platform to evaluate therapeutic countermeasures against severe COVID-19 and other coronavirus pandemics in the future.

immunology↗

bioRxiv2023

Proteasome inhibition reprograms chromatin landscape in breast cancer

The 26S proteasome is the major protein degradation machinery in cells. Cancer cells use the proteasome to modulate gene expression networks that promote tumor growth. Proteasome inhibitors have emerged as effective cancer therapeutics, but how they work mechanistically remains unclear. Here, using integrative genomic analysis, we discovered unexpected reprogramming of the chromatin landscape and RNAPII transcription initiation in breast cancer cells treated with the proteasome inhibitor MG132. The cells acquired dynamic changes in chromatin accessibility at specific genomic loci termed Differentially Open Chromatin Regions (DOCRs). DOCRs with decreased accessibility were promoter proximal and exhibited unique chromatin architecture associated with divergent RNAPII transcription. Conversely, DOCRs with increased accessibility were primarily distal to transcription start sites and enriched in oncogenic super enhancers predominantly accessible in non-basal breast tumor subtypes. These findings describe the mechanisms by which the proteasome modulates the expression of gene networks intrinsic to breast cancer biology. HighlightsO_LIProteasome inhibition uncovers de novo Differential Open Chromatin Regions (DOCRs) in breast cancer cells. C_LIO_LIProteasome inhibitor sensitive promoters exhibit a distinctive chromatin architecture with discrete transcription initiation patterns. C_LIO_LIProteasome inhibition reprograms accessibility of super enhancers. C_LIO_LIProteasome inhibitor sensitive super enhancers distinguish basal from non-basal breast cancer subtypes. C_LI

genomics↗

bioRxiv2023

BRG1 establishes the neuroectodermal chromatin landscape to restrict dorsal cell fates

Cell fate decisions are achieved with gene expression changes driven by lineage-specific transcription factors (TFs). These TFs depend on chromatin remodelers including the BAF complex to activate target genes. BAF complex subunits are essential for development and frequently mutated in cancer. Thus, interrogating how BAF complexes contribute to cell fate decisions is critical for human health. We examined the requirement for the catalytic BAF subunit BRG1 in neural progenitor cell (NPC) specification from human embryonic stem cells. During the earliest stages of differentiation, BRG1 was required to establish chromatin accessibility at neuroectoderm-specific enhancers. BRG1 depletion resulted in abnormal NPC populations that differentially expressed neuroectodermal TFs, were more prone to neuronal differentiation, and precociously formed neural crest lineages. These findings demonstrate that BRG1 mediates NPC specification by ensuring proper expression of lineage-specific TFs and appropriate activation of their transcriptional programs.

molecular biology↗

bioRxiv2023

The Loss of the H1.4 Linker Histone Impacts Nascent Transcription and Chromatin Accessibility

The Chromatosome superstructure, comprised of core histone containing nucleosomes and linker histones, act in concert as physical barriers to genetic material in the mammalian nucleus to trans-acting factors. Appropriate arrangement, composition, and post-translational modification of the chromatosome is highly regulated and necessary for appropriate gene expression. These proteins act to radically condense the genetic material and linker H1 histone is essential for the further condensation of the chromatin fiber. However, the regulatory role of H1 in gene expression and chromatin organization is complicated by cell type specific expression and compensation of multiple H1 variants. Leveraging the UL3 osteosarcoma cell line which displays biased expression of H1 variants, and CRISPR/Cas9, we generated H1.4-deficient clones. Loss of H1.4 results in consistent changes to chromatin accessibility concomitant with changes to histone tail modifications, as well as a set of differentially expressed genes shared among {Delta}H1.4 genetic clones. We identified immune and inflammation immediate early genes as enriched in differentially expressed genes, skewed towards AP-1 regulated targets. Our data show that H1.4 is critical for the regulation of stress response pathways. Key Points for NAR(3 bullet points summarizing the manuscripts contribution to the field) O_LIH1.4 is essential for appropriate expression of over 6,000 nascent transcripts in UL3 cells. C_LIO_LILoss of H1.4 results in widespread changes in chromatin accessibility at enhancers and transcribed regions as well as heterochromatin and quiescent chromatin. C_LIO_LIImmediate early genes, and especially AP-1 family members, are highly sensitive to H1.4 loss and their binding sites coincide with losses in chromatin accessibility C_LI

genomics↗

bioRxiv2022

Recombinant expression and characterisation of a lipase from the Antarctic zooplankton Salpa thompsoni

Cold marine environments are abundant on earth and represent a rich resource for low temperature enzymes. Here we apply in silico bioprospecting methods followed by in vitro expression and biochemical analyses to characterise a novel low temperature lipase from the Antarctic tunicate Salpa thompsoni. A 586 amino acid pancreatic lipase-like gene was identified from S. thompsoni transcriptomic data, expressed as a hexahistadine fusion protein in Escherichia coli at 10{degrees}C and purified by affinity chromatography. Hydrolysis of the synthetic substrate {rho}-nitrophenyl butyrate (PNPB) showed that this recombinant protein has optimal activity at 20 {degrees}C and pH 7, and a specific activity of 3.16 U/mg under this condition. Over 60% of enzyme activity was maintained between 15 to 25 {degrees}C, with a sharp decrease outside this range. These results are indicative of cold active psychrophilic enzyme activity. A meta-analysis of lipase activities towards PNPB showed that the novel S. thompsoni lipase displays a higher activity at lower temperatures relative to previously characterised enzymes. The work demonstrates a methodology for conversion of transcriptomic to in vitro expression data for the discovery of new cold-active biocatalysts from marine organisms.

synthetic biology↗

bioRxiv2022

Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis.

BackgroundLimb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organising signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid) act early on pattern this axis. ResultsTranscriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal-distal axis, showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER-specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt and retinoic acid (RA) signalling as well as differential cell adhesion and proliferation. ConclusionsWe identify new candidate genes for early proximodistal limb patterning. Our analysis of RA-regulated genes supports a role for transient RA gradients in early limb bud in proximal-to-distal patterning in this anamniote model organism.

developmental biology↗

bioRxiv2020

Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

A central challenge in cancer genomics is the systematic identification of single and cooperating tumor suppressor genes driving cellular transformation and tumor progression in the absence of oncogenic driver mutation(s). Multiple in vitro and in vivo gene inactivation screens have enhanced our understanding of the tumor suppressor gene landscape in various cancers. However, these studies are limited to single or combination gene effects, specific organs, or require sensitizing mutations. In this study, we developed and utilized a Sleeping Beauty transposon mutagenesis system that functions only as a gene trap to exclusively inactivate tumor suppressor genes. Using whole body transposon mobilization in wild type mice, we observed that cumulative gene inactivation can drive tumorigenesis of solid cancers. We provide a quantitative landscape of the tumor suppressor genes inactivated in these cancers, and show that despite the absence of oncogenic drivers, these genes converge on key biological pathways and processes associated with cancer hallmarks.

cancer biology↗

bioRxiv2019

Transposon mutagenesis identifies cooperating genetic drivers during keratinocyte transformation and cutaneous squamous cell carcinoma progression

The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.

cancer biology↗

bioRxiv2019

Simultaneous optimization of donor and acceptor substrate specificity for transketolase by a small but smart library

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimization of both substrate specificities, is also required for the rapid expansion of accepted substrates. Transketolase catalyses the reversible transfer of a C2-ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for widely industrial applications. Herein, transketolase from E. coli was engineered to simultaneously accept both pyruvate as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid. Twenty single-mutant variants were firstly designed and characterized experimentally. Beneficial mutations were then recombined to construct a small but smart library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to WT. Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% ee for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-formylbenzoic acid. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor acceptances, that would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

bioengineering↗

bioRxiv2017

Distinct neuronal activity patterns induce different gene expression programs

Brief and sustained neuronal activity patterns can have opposite effects on synaptic strength that both require activity-regulated gene (ARG) expression. However, whether distinct patterns of activity induce different sets of ARGs is unknown. In genome-scale experiments, we reveal that a neurons activity-pattern history can be predicted from the ARGs it expresses. Surprisingly, brief activity selectively induces a small subset of the ARG program that that corresponds precisely to the first of three temporal waves of genes induced by sustained activity. These first-wave genes are distinguished by an open chromatin state, proximity to rapidly activated enhancers, and a requirement for MAPK/ERK signaling for their induction. MAPK/ERK mediates rapid RNA polymerase recruitment to promoters, as well as enhancer RNA induction but not histone acetylation at enhancers. Thus, the same mechanisms that establish the multi-wave temporal structure of ARG induction also enable different sets of genes to be induced by distinct activity patterns.

neuroscience↗