Biology subjects

Johnson, T. A.

Publications and source records attributed to Johnson, T. A..

15 recordsLinked to original sources

bioRxiv2026

Coordinated Temporal Dynamics of Glucocorticoid Receptor Binding and Chromatin Landscape Drive Transcriptional Regulation

Glucocorticoid receptor (GR) signaling elicits diverse transcriptional responses through dynamic and context-dependent interactions with chromatin. Here, we define a temporally resolved and mechanistically integrated framework for GR-mediated gene regulation. Time-resolved analyses identify three conserved classes of GR chromatin binding (sustained, transient, and late), distinguished by differences in motif strength, chromatin accessibility, and cofactors engagement. Early GR binding preferentially occurs at high-affinity glucocorticoid response elements (GREs) within pre-accessible regulatory regions, whereas late binding is associated with weaker motifs and requires chromatin remodeling activity. Enhancer activation, marked by H3K27ac deposition, closely tracks GR occupancy, supporting a model in which GR recruits acetyltransferase activity to drive coordinated enhancer activation. Concurrently, GR-centered interaction networks are dynamically reconfigured, and motif enrichment analyses identify distinct transcription factor signatures across binding classes, including AP-1/JUNB at transient sites and CEBP family members at late-binding regions. Integration of chromatin binding, chromatin interaction, and transcriptomic datasets reveals that temporal and combinatorial GR occupancy is functionally linked to gene expression programs. Distinct GR binding clusters are nonrandomly associated with specific transcriptional trajectories, including sustained, transient, and late gene induction. Moreover, combinatorial occupancy across multiple regulatory elements correlates quantitatively with transcriptional output, indicating that GR functions not as a simple binary regulator, but as an integrator of multilayered regulatory inputs. These findings support a unified model in which temporal binding dynamics, chromatin state, and combinatorial enhancer activity collectively encode transcriptional specificity, providing a general framework for stimulus-responsive nuclear receptor signaling.

molecular biology↗

bioRxiv2026

Kinetic proofreading as a mechanism for transcriptional specificity in living human cells

How target genes selectively respond to their specific transcription factors (TF) amid the vast excess of non-specific TFs in the nucleus remains a fundamental question in gene regulation. Here, we develop an integrated single-molecule imaging framework to quantitatively link TF dynamics with nascent transcription kinetics at endogenous gene loci to address the transcription factor specificity. Using endogenously Halo-tagged glucocorticoid receptor (GR), we show that ligand activation rapidly increases GR chromatin binding and residence times without substantially altering search kinetics. Live-cell nascent RNA imaging reveals that GR is essential for Dex induction of ERRFI1 transcription via increased burst frequency. Gene locus-specific, dual-color tracking demonstrates that GR display longer residence times in general near its target gene ERRFI1 compared to the non-target MYH9 locus, consistent with a kinetic proofreading model. A high-throughput imaging-based CRISPR screen identifies ATP-dependent processes, including neddylation and chromatin remodeling, and similarly, acute inhibition of the TFIIH XPB selectively impairs ERRFI1 transcription while sparing MYH9, implicating ATPase activity in GR target discrimination. In all, these findings establish that promoters function as dwell-time rather than occupancy detectors, discriminating specific from non-specific TF interactions through energy-dependent mechanisms.

biophysics↗

bioRxiv2025

Form and function of actin impacts actin health and aging.

The actin cytoskeleton is a fundamental and highly conserved structure that functions in diverse cellular processes, yet its direct contribution to organismal aging remains unclear. Here, we systematically interrogated how genetic and pharmacologic perturbations of actin structure and function influence lifespan and various hallmarks of aging in Caenorhabditis elegans. Whole-animal and tissue-specific knockdown of actin and key actin-binding proteins (ABPs) - arx-2 (Arp2/3), unc-60 (cofilin), and lev-11 (tropomyosin) - led to premature disruption of filament organization, reduced lifespan, and tissue-specific physiological defects. Bulk and single-nucleus RNA-sequencing revealed that ABP knockdowns elicited a strongly "aged" transcriptome. Actin dysfunction broadly exacerbated many age-associated phenotypes, including mitochondrial dysfunction, lipid dysregulation, loss of proteostasis, impaired autophagy, and intestinal barrier failure. Pharmacological destabilization with Latrunculin A mirrored genetic knockdowns, while mild stabilization with Jasplakinolide modestly extended lifespan, emphasizing that optimal and finely-tuned actin function is critical for healthy aging. Finally, analysis of human genome-wide association data revealed that common ACTB polymorphisms correlate with differences in age-related decline in gait speed, suggesting evolutionary conservation of actins role in healthy aging. Taken together, our results provide a comprehensive and publicly accessible resource that maps, for the first time, how actin integrity intersects with diverse aging pathways across tissues and scales. This descriptive framework is intended to enable future mechanistic discovery by offering a deep, unbiased dataset that can be integrated with emerging studies to define how actin dynamics contribute to aging.

cell biology↗

bioRxiv2025

Detection of five viruses commonly implicated with Bovine Respiratory Disease using loop-mediated isothermal amplification

Herein, we present novel quantitative loop-mediated isothermal amplification (qLAMP) and reverse-transcription qLAMP (RT-qLAMP) assays for the detection of five viruses commonly implicated with the onset and progression of bovine respiratory disease (BRD): Bovine Alphaherpesvirus Type 1 (BHV-1), Bovine Adenovirus Type 3 (BAV-3), Bovine Respiratory Syncytial Virus (BRSV), Bovine Viral Diarrhea Virus Type 1 (BVDV-1), and Bovine Parainfluenza Virus Type 3 (BPIV-3). Using contrived samples spiked with whole viruses, we found that our extraction-free assays have limits of detection between 30 and 1,057 copies per reaction (1.8% final sample concentration) with minimal sample processing. Using dual-tipped swabs and 1.4 mL resuspension volumes, these limits of detection are on the order of 2 x 105 copies per swab for BAV-3 and BHV-1 and between 6.31 x 106 to 8.22 x 106 copies per swab in the case of BPIV-3, BRSV, and BVDV-1. Analytical sensitivities ranged from 73 - 100% and analytical specificities ranged from 90 - 100%. Additionally, we introduced a streamlined pipeline to minimize the experimental workload to design, screen, select, and characterize LAMP performance for developing assays. The assays targeting these BRD viruses can be utilized to develop colorimetric LAMP assays that enable the sensitive and specific detection of these viruses chute side to aid in diagnosing and treating BRD. The associated development pipeline enables more rapid development of LAMP-based diagnostic tools targeting emerging pathogens.

bioengineering↗

bioRxiv2025

Development and Evaluation of Novel Quantitative PCR (qPCR) and Loop-Mediated Isothermal Amplification (LAMP) Assays for Bovine Adenovirus Type 7

Herein, we report the development of molecular assays for the specific detection of bovine adenovirus type 7 (BAV-7), a prevalent pathogen associated with bovine respiratory disease (BRD). To overcome the limitations of the current diagnostic methods, we developed and optimized a TaqMan quantitative polymerase chain reaction (qPCR) assay with a limit of detection (LOD) of 50 copies per reaction (10 copies/{micro}L) and 100% analytical specificity, showing no cross-reactivity with nine other viral pathogens commonly linked to BRD. Additionally, we designed a fluorescent quantitative loop-mediated isothermal amplification (qLAMP) assay demonstrating equivalent LOD and specificity. Both assays were evaluated using 24 bovine nasal swabs from BRD animals, revealing a 92% agreement between qPCR and qLAMP results. These assays provide powerful tools for advancing our understanding of BAV-7 epidemiology and improving disease management strategies. The qLAMP assay, with its rapid and isothermal amplification, presents potential for future development as a field-deployable diagnostic tool to enable timely detection of BAV-7 in cattle.

bioengineering↗

bioRxiv2025

Bile acids target an exposed cavity in the glucocorticoid receptor modulating receptor self-assembly, chromatin binding and transcriptional activity

The glucocorticoid receptor (GR) is an essential transcription factor that controls metabolism and homeostasis. Glucocorticoids (GCs) activate the GR upon occupying the internal ligand-binding pocket (LBP) of its ligand-binding domain (GR-LBD), which has been the focus of most previous structure-function studies. Synthetic GCs such as dexamethasone are widely used to treat inflammatory diseases, but their chronic use results in major side effects, whose molecular underpinnings remain unresolved. Here we present a thorough analysis of the topography of GR-LBD and its ability to bind small-molecule compounds, especially cholesterol derivatives. We show that one important class of steroids, bile acids, bind to previously unidentified and highly conserved, surface-exposed cavities on GR-LBD. We show that bile acids affect GR turnover and self-assembly in living cells, modulating receptor transcriptional activity. These findings reveal a previously unrecognized mechanism of GR regulation, with implications for the design of GCs with novel mechanisms of action. TeaserBile acids modulate the activity of the glucocorticoid receptor upon binding to an exposed allosteric pocket thereby influencing transcriptional regulation and receptor self-assembly in living cells.

molecular biology↗

bioRxiv2025

In-feed bacitracin methylene disalicylate alters microbiota function and increases antibiotic resistance in a dose-dependent manner

Antibiotics are commonly used in turkey production to prevent and treat infection which can improve animal growth and efficiency, but the mechanism by which antibiotics improve animal performance, and the resistance risks associated with the antibiotic inclusion levels remain unclear, particularly in turkey production. Therefore, we investigated the longitudinal effect of subtherapeutic and therapeutic doses of bacitracin methylene disalicylate (BMD) on antibiotic resistance genes, mobile genetic elements, and metabolism genes by analyzing the turkey cecal metagenome. The therapeutic dose of BMD increased a vast array of antibiotic resistant genes (ARGs), conjugation-related genes of type IV secretion system and transduction-related genes for the length of the experiment (78 days), while a smaller, transient effect was observed due to the subtherapeutic dose. Estimated bacterial growth rate, estimated by metagenome assembled genome sequence coverage, decreased after 7 days of in-feed BMD, but increased in the therapeutic group over time. Tryptophan synthesis from chorismate increased in a dose-dependent manner between days 7 - 35. Overall, the effects of subtherapeutic BMD on the turkey cecal microbiota was temporary while those of a therapeutic dose were longer lasting. This study shows that antimicrobial resistance genes belonging to multiple antibiotic classes, and mobile genetic elements (MGEs) were increased after BMD administration. The enrichment these genes by BMD shows the risk associated with antimicrobial feed additives. BMDs effect on tryptophan synthesis provides a potential metabolic target for developing non-antibiotic microbiome modulatory growth promoters for turkey production. ImportanceAntibiotic use in agricultural animals remains a hotly debated and important topic to human, animal, and environmental health. The dose-dependent responses to BMD, an antibiotic fed additive allowed at both therapeutic and subtherapeutic doses, are not well understood. This study highlights that therapeutic use of BMD is a stronger selective pressure than the subtherapeutic dose for antibiotic resistance genes and genes related to horizontal gene transfer. This indicates that use of BMD could select for antibiotic resistant bacteria that may pose a risk to animal, human and environmental health. Additionally, this study highlighted that BMD decreased the activity of beneficially bacteria, and therefore may be associated with a decreased concentration of bacterial metabolites (especially tryptophan related) in the cecum. This may indicate that growth promoting antibiotics suppress bacterial activity generally, rather than allowing beneficial bacteria to generate beneficial metabolites.

microbiology↗

bioRxiv2024

The multimerization pathway of the glucocorticoid receptor

The glucocorticoid receptor (GR) is a leading drug target due to its anti-inflammatory and immunosuppressive roles. The functional oligomeric conformation of full-length GR (FL-GR), which is key for its biological activity, remains disputed. Here we present a new crystal structure of agonist-bound GR ligand-binding domain (GR-LBD) comprising eight copies of a non-canonical dimer. The biological relevance of this dimer for receptor multimerization in living cells has been verified by studying single-and double-point mutants of FL-GR in fluorescence microscopy (Number & Brightness) and transcriptomic analysis. Self-association of this GR-LBD basic dimer in two mutually exclusive assemblies reveals clues for FL-GR multimerization and activity in cells. We propose a model for the structure of multidomain GR based on our new data and suggest a detailed oligomerization pathway. This model reconciles all currently available structural and functional information and provides a more comprehensive understanding of the rare glucocorticoid resistance disorder (Chrousos syndrome).

biochemistry↗

bioRxiv2024

Transcription factors form a ternary complex with NIPBL/MAU2 to localize cohesin at enhancers

While the cohesin complex is a key player in genome architecture, how it localizes to specific chromatin sites is not understood. Recently, we and others have proposed that direct interactions with transcription factors lead to the localization of the cohesin-loader complex (NIPBL/MAU2) within enhancers. Here, we identify two clusters of LxxLL motifs within the NIPBL sequence that regulate NIPBL dynamics, interactome, and NIPBL-dependent transcriptional programs. One of these clusters interacts with MAU2 and is necessary for the maintenance of the NIPBL-MAU2 heterodimer. The second cluster binds specifically to the ligand-binding domains of steroid receptors. For the glucocorticoid receptor (GR), we examine in detail its interaction surfaces with NIPBL and MAU2. Using AlphaFold2 and molecular docking algorithms, we uncover a GR-NIPBL-MAU2 ternary complex and describe its importance in GR-dependent gene regulation. Finally, we show that multiple transcription factors interact with NIPBL-MAU2, likely using interfaces other than those characterized for GR.

molecular biology↗

bioRxiv2024

Birth weight and in-feed antibiotics alter the assembly and succession of the swine gut microbiota

Understanding the principles of gut microbiota assembly and succession during host development is critical for effective gut microbiome manipulation to optimize host health and growth. The objective of this study was to conduct a high-frequency sampling of the swine gut microbiota from controlled groups of pigs to increase understanding of the dynamics of microbial community development. Here, a total of 924 fecal samples from 44 piglets (22 low-birth-weight, LBW; 22 normal-birth-weight, NBW) over 21 time points (1-41 days of age) collected every two days. Community composition, assembly, and succession was determined using 16S rRNA gene amplicon sequencing. Alpha diversity continuously increased during the suckling stage, yet no significant increase was observed during the days post-weaning. Post-weaning in-feed antibiotics consistently decreased microbial diversity and changed the community structure in both LBW and normal birth weight (NBW) piglets. Delayed post-weaning gut microbial community maturation was observed in LBW piglets compared with NBW. Heterogeneity of the gut microbial community between piglets linearly decreased over time, as revealed by the within-time Bray-Curtis dissimilarities. Individuality analysis on the relatively stable stage revealed that the gut microbiota composition of some individuals over time, and the abundance of most genera between individuals, were highly variable. Dirichlet multinomial mixtures analysis supported an age-dependent microbiota developmental pattern and identified the age-discriminatory taxa. The importance of stochastic processes in microbial community assembly increased over time within primary and secondary successions, despite the fact that the most dominant factors influencing community assembly were homogeneous selection and dispersal limitation, which are deterministic. IMPORTANCEOur understanding of the assembly and succession of the swine gut microbial community is limited, and scientific advancement in this interdisciplinary topic is hampered by individuality and transient dynamics. The solution to the above foundational questions is not only ecologically relevant but also useful for practical swine production. Our study addresses ecological processes shaping the swine gut microbiota between piglets with contrasting birth weights and receiving post-weaning antibiotics. Persistent gut microbiota immaturity in LBW piglets suggests that efforts to accelerate microbial community succession might improve LBW piglet growth performance and disease resistance. Intra-individual variance both in community structure and genus abundance during the post-weaning period indicates the importance of repeated measurements for reliable observations. Additionally, neutral (stochastic) processes increased as a factor of community assembly within each stage of pig growth, indicating that early intervention and multiple follow ups may be critical in manipulating the gut microbiota development.

microbiology↗

bioRxiv2023

Investigating impacts of marine sponge derived mycothiazole and its acetylated derivative on mitochondrial function and aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), a widely used ETC complex I inhibitor, these two molecules showed cytotoxicity to cancer cells but strikingly demonstrate a lack of toxicity to non-cancer cells, a highly beneficial feature in the development of anti-cancer therapeutics. Furthermore, in vivo experiments with these small molecules utilizing C.elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. Interestingly, we also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilize the transcription factors ATFS-1 and HSF-1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF-1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

cell biology↗

bioRxiv2023

The mineralocorticoid receptor forms higher order oligomers upon DNA binding

The prevailing model of steroid hormone nuclear receptor function assumes ligand-induced homodimer formation followed by binding to DNA hormone response elements (HREs). This model has been challenged by evidence showing that the glucocorticoid receptor (GR) forms tetramers upon ligand and DNA binding, which then drive receptor-mediated gene transactivation and transrepression. GR and the closely-related mineralocorticoid receptors (MR) interact to transduce corticosteroid hormone signaling, but whether they share the same quaternary arrangement is unknown. Here, we used a fluorescence imaging technique, Number & Brightness, to study oligomerization in a cell system allowing real-time analysis of receptor-DNA interactions. Agonist-bound MR forms tetramers in the nucleoplasm and higher order oligomers upon binding to HREs. Antagonists form intermediate quaternary arrangements, suggesting that large oligomers are essential for function. Divergence between MR and GR quaternary structure is driven by different functionality of known and new multimerization interfaces, which does not preclude formation of heteromers. Thus, influencing oligomerization may be important to selectively modulate corticosteroid signaling.

biophysics↗

bioRxiv2023

The Glucocorticoid Receptor is Required for Efficient Aldosterone-Induced Transcription by the Mineralocorticoid Receptor

The glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) have distinct, yet overlapping physiological and pathophysiological functions. There are indications that both receptors interact functionally and physically, but the precise role of this interdependence is poorly understood. Here, we analyzed the impact of GR co-expression on MR genome-wide chromatin binding and transcriptional responses to aldosterone and glucocorticoids, both physiological ligands of this receptor. Our data show that GR co-expression alters MR genome-wide binding to consensus DNA sequences in a locus-and ligand-specific way. MR binding to consensus DNA sequences is affected by GR. Transcriptional responses of MR in the absence of GR are weak and show poor correlation with chromatin binding. In contrast, co-expression of GR potentiates MR-mediated transcription, particularly in response to aldosterone. Finally, single-molecule tracking of MR suggests that the presence of GR contributes to productive binding of MR/aldosterone complexes to chromatin. Together, our data indicate that co-expression of GR potentiates aldosterone-mediated MR transcriptional activity, even in the absence of glucocorticoids.

molecular biology↗

bioRxiv2021

The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution and quantitative fluorescence microscopy in living cells. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work unveils likely pathophysiologically relevant quaternary assemblies of the nuclear receptor with important implications for glucocorticoid action and drug design.

biochemistry↗

bioRxiv2020

Swine Growth Promotion with Antibiotics or Alternatives Increases Antibiotic Resistance Gene Mobility Potential in the Fecal Microbiome

Even though the use of antibiotics for food-producing animals may contribute to the emergence of antimicrobial resistance, antibiotics are still used as growth promoters. Due to consumer and regulatory pressures, the use of alternatives to antibiotics as growth promoters is increasing, thus more information is needed on their capability to disseminate antimicrobial resistance compared to antibiotics. We investigated the impacts of carbadox (antibiotic), copper sulfate and zinc oxide (metals) and mushroom powder (natural product) on the pig fecal resistome and microbiome. Antibiotic resistance gene (ARG) and mobile genetic element (MGE) abundances were measured using a high-throughput qPCR array with 382 primer pairs. Bacterial community composition was determined by 16S rRNA gene sequencing. More ARGs co-occurred with MGEs in the growth promoter group samples than in the control group samples. Community composition could not be linked to resistome in the growth promoter group samples, indicating a potential decoupling of ARGs and phylogeny. Additionally, machine-learning methods aided in defining the community and resistome differences in response to treatments. Since increased ARG mobility potential was the primary response to the dietary additives used in this study, we suggest that ARG mobility should be considered when designing antimicrobial use policies and antimicrobial resistance surveillances.

microbiology↗