Biology subjects

Johnston, E.

Publications and source records attributed to Johnston, E..

9 recordsLinked to original sources

bioRxiv2026

Environmental Regulation and Gene-by-Environment Interaction Influence RAP1 Activity and its Impact on Gene Expression

Gene-by-environment (GxE) interactions play a major role in shaping both phenotypic and molecular variation, with important implications for human health and disease. In this study, we used the Doxycycline (Dox) regulated, tetracycline-responsive (Tet-Off) promoter system to sequentially reduce or titrate gene expression levels of the essential yeast transcription factor Repressor Activator Protein 1 (RAP1) similar to a hypomorph allele series, across three distinct environments: Yeast Peptone Dextrose (YPD) media, YPD media with Heat Shock (HS), and Yeast Peptone Acetate (YPAC) media. We then performed RNA sequencing (RNA Seq) to assess global transcriptional responses to RAP1 reduction in these different growth environments. Our analysis first focused on the independent effects of varying RAP1 expression levels within and across environments. We then explored GxE interactions, revealing a subset of genes with significant consequences of reduced levels of RAP1 and environment-specific expression patterns. Notably, many genes exhibited opposite effects of RAP1 titration on gene expression when yeast were grown in YPAC media compared to YPD media and/or HS, suggesting environment-dependent regulatory architecture. This design reveals how cells integrate internal transcriptional and regulatory changes with external environmental cues, providing a deeper view of GxE architecture. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified co-regulated gene modules, and by combining this with transcription factor motif enrichment tests, our study identified candidate regulators driving their dynamics. Our findings demonstrate that gene regulatory networks can vary dramatically depending on the environmental context an organism experiences, which can then influence the specific phenotypes produced by a particular genetic perturbation. This illustrates the complexity of genotype-environment interactions and the importance of studying gene function in multiple environments to gain a truly comprehensive understanding of a genes sometimes numerous and diverse functions.

genomics↗

bioRxiv2026

A Genetic Method for Distinguishing Cryptic Pocillopora Species in French Polynesia without Sequencing

Pocillopora is a widespread, dominant reef-building coral genus in the Indo-Pacific that exhibits high morphological similarity and plasticity. Given this, genetic tools are needed to robustly identify Pocillopora individuals to the species level. Quick and accurate identification approaches for Pocillopora species are critical to estimating biodiversity patterns under current and future environmental challenges. In recent years, the mitochondrial open reading frame (mtORF) and a histone region (PocHistone) have been validated using genome-wide data to become the most widely used species-level markers for Pocillopora. However, Sanger sequencing of a large number of samples can be prohibitively expensive and sequencing facilities are not always readily available. Therefore, we present restriction fragment length polymorphism (RFLP) digests here that identify the six species of Pocillopora (P. acuta, P. cf. effusa, P. grandis, P. meandrina, P. tuahiniensis, and P. verrucosa) found in French Polynesia, without sequencing. In uninformed validation tests (in silico and in vitro), our protocol identified each Pocillopora species with 100% accuracy. Given their cost-effective, rapid nature, the tailoring of additional RFLP digest protocols to identify cryptic coral species in reef regions around the world will support foundational reef science, conservation and restoration initiatives.

molecular biology↗

bioRxiv2025

The PPARβ/δ-induced mesenchymal stromal cell secretome has cytoprotective effects via ANGPTL4 in a pre-clinical model of acute lung inflammation

RationaleHuman bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are known to exert immunomodulatory and pro-reparative effects in vivo. This makes hBM-MSCs an enticing therapeutic candidate for inflammatory diseases, such as acute respiratory distress syndrome (ARDS). The ARDS microenvironment is complex and contains an abundance of free fatty acids (FFAs); which are known to differentially impact MSC functionality. PPAR{beta}/{delta} is a ubiquitously expressed nuclear receptor that is activated in response to FFA-binding. PPAR{beta}/{delta} has been shown to impact the therapeutic efficacy of mouse MSCs. ObjectiveThis study sought to investigate the impact of PPAR{beta}/{delta}-modulation on human MSC functionality in vitro and in vivo. MethodshBM-MSCs were exposed to a synthetic PPAR{beta}/{delta} agonist/antagonist in the presence or absence of ARDS patient serum and the immunomodulatory and pro-reparative capacity of the MSC secretome was investigated using in vitro assays and a pre-clinical model of LPS-induced acute lung inflammation (ALI). ResultsOur results highlighted enhanced pro-reparative capacity of PPAR{beta}/{delta}-agonised hBM-MSCs secretome in CALU-3 lung epithelial cells, mediated by MSC derived angiopoietin-like 4 (ANGPTL4). PPAR{beta}/{delta}-induced ANGPTL4-high MSC secretome facilitated enhanced endothelial barrier integrity in the lungs of ALI mice. Therapeutic effects of PPAR{beta}/{delta}-agonised hBM-MSCs secretome were further enhanced by licensing MSCs with human ARDS patient serum. ARDS-licensed PPAR{beta}/{delta}-induced ANGPTL4-high MSC secretome had reduced clinical score and weight loss. The role ANGPL4 in these protective effects was confirmed using an anti-ANGPTL4 antibody. ConclusionThese findings conclude that the MSC secretome therapeutic effects can be enhanced both in vitro and in vivo through licensing strategies that upregulate the angiogenic factor ANGPTL4. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/667170v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a03b3dorg.highwire.dtl.DTLVardef@1915253org.highwire.dtl.DTLVardef@1b3fd4aorg.highwire.dtl.DTLVardef@1ba27ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

SWI/SNF chromatin remodeling complexes regulate gene expression by modulating nucleosome positioning, yet their roles in metal homeostasis during skeletal muscle development remain unclear. Here, we uncover distinct functions of the BAF, PBAF, and ncBAF complexes in myoblast proliferation under metal stress. While knockdown (KD) of Baf250a (BAF-specific) or Brd9 (ncBAF-specific) reduces myoblast proliferation, Baf180 (PBAF-specific) KD does not impair cell proliferation under basal conditions. Interestingly, supplementation with copper (Cu) or zinc (Zn) rescues proliferation in Baf250a- and Brd9-deficient myoblasts but paradoxically inhibits growth in Baf180 KD cells. Mechanistically, Baf180 KD disrupts Cu and Zn homeostasis, leading to intracellular Cu accumulation without labile Cu pools and impaired expression of Atp7a, a key Cu exporter. Transcriptomic analyses reveal widespread gene dysregulation in metal-treated Baf180-deficient cells, while metal supplementation promotes pro-proliferative gene expression in Baf250a- and Brd9-KD myoblasts. CUT&RUN assays demonstrate that metal-responsive transcription factor Mtf1 exhibits increased chromatin binding upon Cu treatment, targeting genes involved in stress response and myogenesis. Notably, Mtf1 colocalizes with Baf180 in the nucleus and co-immunoprecipitates with both conserved SWI/SNF subunits and Baf180, suggesting a functional interplay between PBAF and Mtf1 in regulating metal-dependent gene expression. Our findings establish the PBAF complex as a crucial regulator of Cu/Zn homeostasis in myoblast proliferation via Mtf1, while metal supplementation compensates for BAF and ncBAF dysfunction but exacerbates defects in PBAF-deficient cells. This study reveals a novel link between chromatin remodeling, metal signaling, and muscle development, with implications for stress adaptation and metabolic regulation in myogenesis.

molecular biology↗

bioRxiv2024

Punctuated and continuous structural diversity of S-layers across the prokaryotic tree of life

Surface layers (S-layers) are two-dimensional (2D) crystalline lattices that frequently coat prokaryotic cells, playing a crucial role in protection, maintaining cellular integrity, and mediating environmental interactions. However, the molecular landscape of these abundant proteins has remained underexplored due to a lack of structural data. By employing AlphaFold2multimer together with planar symmetry constraints in a workflow validated by electron cryomicroscopy structure determination, we have elucidated the lattice structures of over 150 S-layers from diverse archaea and bacteria. Our findings unveil a multifaceted evolutionary landscape for S-layer proteins, highlighting key differences in the evolution of bacterial and archaeal S-layers. Our study allows us to discover underlying patterns in S-layer structure, organisa-tion, and cell anchoring mechanisms across the prokaryotic tree of life, deepening our understanding of the intricately complex microbial cell surfaces, which appear to have evolved proteinaceous S-layers independently on multiple occasions. This work will open avenues for rational manipulation of prokaryotic cellular interactions in multicellular microbiomes, as well as for innovative 2D biomaterial design.

molecular biology↗

bioRxiv2023

Imaging proteins sensitive to direct fusions using transient peptide-peptide interactions

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of protein subcellular location and function. Some proteins, however, show altered localization and/or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to [~]200 nm, which is two orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs. FOR TABLE OF CONTENTS ONLY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/547312v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@ce1d9borg.highwire.dtl.DTLVardef@1f9b5c8org.highwire.dtl.DTLVardef@10d92ccorg.highwire.dtl.DTLVardef@20ad5d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

An experimental workflow identifies nitrogenase proteins ready for expression in plant mitochondria

Industrial nitrogen fertilizer is intrinsic to modern agriculture yet expensive and environmentally harmful. We aim to reconstitute bacterial nitrogenase function within plant mitochondria to reduce nitrogen fertilizer usage. Many nitrogen fixation (Nif) proteins are required for biosynthesis and function of the mature nitrogenase enzyme, and these will need to be correctly processed and soluble within mitochondria as a pre-requisite for function. Here we present our workflow that assessed processing, solubility and relative abundance of 16 Klebsiella oxytoca Nif proteins targeted to the plant mitochondrial matrix using an Arabidopsis mitochondrial targeting peptide (MTP). The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using bacterial nitrogenase assays. We found that despite the use of the same constitutive promoter and MTP, MTP::Nif processing and relative abundance in plant leaf varied considerably. Assessment of solubility for all MTP::Nif proteins found NifF, M, N, S, U, W, X, Y and Z were soluble, while NifB, E, H, J, K, Q and V were mostly insoluble. Although most Nif proteins tolerated the N-terminal extension as a consequence of mitochondrial processing, this extension in NifM reduced nitrogenase activity to 10% of controls. Using proteomics, we detected a [~]50-fold increase in the abundance of NifM when it contained the N-terminal MTP extension, which may account for this reduction seen in nitrogenase activity. Based on plant mitochondrial processing and solubility, and retention of function in a bacterial assay, our workflow has identified that NifF, N, S, U, W, Y and Z satisfied all these criteria. Future work can now focus on improving these parameters for the remaining Nif components to assemble a complete set of plant-ready Nif proteins for reconstituting nitrogen fixation in plant mitochondria.

synthetic biology↗

bioRxiv2019

Engineering a functional NifDK polyprotein resistant to mitochondrial degradation

To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

synthetic biology↗

bioRxiv2018

Tax4Fun2: a R-based tool for the rapid prediction of habitat-specific functional profiles and functional redundancy based on 16S rRNA gene marker gene sequences

Main text Main text Methods Statistical data analysis Data availability Author Contributions Competing interests References Assessing the functional capability and redundancy of a microbial community is a major challenge in environmental microbiology. To address this challenge, we developed Tax4Fun2, a R-based tool for the rapid prediction of functional profiles and functional redundancy of prokaryotic communities from 16S rRNA gene sequences. By incorporating user-defined, habitat-specific genomic information, the accuracy and robustness of predicted functional profiles can be substantially enhanced.\n\nMicroorganisms play a key role in ecosystem functioning1. High-throughput sequencing of 16S rRNA genes is a powerful and widely used approach to study the composi ...

bioinformatics↗