Biology subjects

Smith, C. M.

Publications and source records attributed to Smith, C. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Polyketide synthase 12 is an in vivo essential source of novel mycolyl lipids in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a major pathogen worldwide that infects and transmits only among humans, yet nearly all in vivo virulence research occurs in non-human hosts. To overcome this central challenge in tuberculosis research, we leveraged a dataset of more than 50,000 sequenced isolates to identify Mtb genes that that are functionally preserved during natural infection, disease causation, and transmission between humans. This whole-genome ranking identified polyketide synthase 12 (pks12 ) as an exceptionally in vivo essential gene in human tuberculosis, which we validated in zebrafish and mouse models. Though Pks12 produces a mycoketide lipid in only trace amounts, pks12 deletion severely altered the host-facing surface and arabinoglycan architecture of Mtb. This amplified effect was explained through the discovery of mannosyl-{beta}-1-phosphomycoketide monomycolate (MPMMM), which is synthesized from the known Pks12 product at higher abundance by antigen 85 mycolyltransferases. Thus, we used a new host-facing genomic-metabolomic-phenotypic approach to discover the functions of an in vivo essential Mtb gene, which controls the physical structure of the Mtb-host interface.

microbiology↗

bioRxiv2026

The canine respiratory epithelium is a permissive ecosystem for influenza interspecies transmission and emergence

The outcome of virus spillover ranges from dead-end infections to pandemics and is underpinned by host-pathogen interactions as well as evolutionary and epidemiological processes. The emergence of novel influenza A viruses (IAVs) has been associated with reassortment events involving multiple species, highlighting the importance of reservoir and intermediate hosts in viral emergence. Highly pathogenic H5N1 IAVs of the 2.3.4.4b genotype have caused a panzootic affecting a broad range of mammals. The role of dogs--arguably the most popular companion animal and a natural host of IAVs--in the ecology of IAVs under this new zooepidemiological scenario is unknown. To address this, we characterised the glycome of the dog respiratory epithelium, infected canine tracheal explants with multiple IAVs (including canine H3N2 and H3N8, equine H3N8, avian H3N8 and H5N1, swine H1N1, human H1N1 and H3N2, and bovine H5N1 viruses), and determined their cellular tropism. We show that the respiratory tract of dogs presents abundant sialylated glycans known to act as IAV receptors. Further, most IAVs (including 2.3.4.4b viruses) infected and replicated in dog tracheas, targeting mainly ciliated cells. Serological testing showed evidence of influenza spillover infections in dogs from the UK. Overall, our results show that the canine respiratory tract can provide a suitable environment for the generation of new IAVs. Given the multi-host contact networks of dogs in nature, they could act as recipients, bridging hosts, and/or mixing vessels for multiple IAV lineages, playing a central role in the ecology of influenza emergence.

microbiology↗

bioRxiv2026

Bovine H5N1 influenza viruses have adapted to more efficiently use receptors abundant in cattle

Sustained mammal-to-mammal transmission of high pathogenicity H5N1 avian influenza viruses is reshaping the host range of these pathogens. One of the longest-running mammalian transmission chains involves the B3.13 genotype circulating in U.S. dairy cattle which was detected in early 2024. Genomic analyses revealed selection and rapid fixation of haemagglutinin mutations D104G and V147M. We demonstate, via glycomic profiling, that bovine tissues, including the mammary gland, are enriched in N- and O-linked glycans capped with N-glycolylneuraminic acid (NeuGc), a sialic acid absent in humans and birds, which instead express only N-acetylneuraminic acid (NeuAc). Early cattle H5 viruses poorly recognized NeuGc, but D104G and V147M enabled efficient engagement of both NeuAc- and NeuGc-containing receptors. These mutations enhanced replication in bovine mammary tissue without major attenuation of replication in human lung and primary nasal epithelial cells. NeuGc-driven receptor adaptation therefore promotes viral fitness in cattle while potentially limiting immediate zoonotic risk. Deep mutational scanning further identified alternative haemagglutinin substitutions that confer NeuGc usage and represent surveillance markers for emerging cattle H5 lineages.

microbiology↗

bioRxiv2026

Sensorimotor mapping of volitional facial movements in Tourette Syndrome

BackgroundTourette Syndrome (TS) is a neurodevelopmental movement disorder involving involuntary motor and vocal tics believed to be characterised by disordered neural inhibition. Cortical representations have previously been manipulated by disruptions in the inhibitory neurotransmitter {gamma}-aminobutyric acid (GABA). However, while facial tics are the most reported motor tic, it is unclear if facial sensorimotor representations differ in TS. MethodsSixteen individuals with Tourette Syndrome (TS) or chronic tic disorder and twenty typically developing (TD) control participants underwent 3-Tesla functional magnetic resonance imaging (fMRI). Blood-oxygenation level-dependent (BOLD) responses were measured during a block-design task comprising cued facial movements of common facial tics (blinking, grimacing and jaw clenching). Activations in bilateral pre- and post-central cortices and supplementary motor areas (SMA) were examined. Conjunction analyses identified voxels commonly and uniquely activated across movements within each group. ResultsBoth groups showed significant activations in the bilateral sensorimotor cortices and SMA in response to blink, grimace and jaw clench movements, with no significant between-group differences. Between-group similarities were lowest for unique blink maps. Common voxel maps also revealed low between-group similarity, with reduced sensorimotor activation and no shared SMA activation across movements in the TS group. ConclusionVoluntary facial sensorimotor representations do not differ between groups. However, low similarities between group unique blink maps may reflect greater prevalence of blinking tics in TS. Additionally, reduced overlap in sensorimotor activation and absent common SMA engagement across cued movements in the TS group may indicate altered motor integration or action initiation.

neuroscience↗

bioRxiv2026

Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

Despite the huge global health burden presented by respiratory viruses, effective broad-spectrum antiviral therapeutic options remain limited. Here we evaluated the antiviral activity of four RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir, ribavirin, favipiravir, and molnupiravir, against respiratory syncytial virus (RSV) and human parainfluenza (hPIV) using monotherapy or dual-drug combinations with epithelial cell lines and primary human airway culture models. Remdesivir showed the greatest potency across both viruses, while ribavirin and favipiravir also demonstrated inhibition. Molnupiravir was active against RSVA but not hPIV3. Several dual-drug combinations, including remdesivir-favipiravir, remdesivir-molnupiravir and favipiravir-molnupiravir, produced marked synergy against RSVA, and more limited synergy for hPIV3. Antiviral efficacy was validated in primary airway epithelial cultures, where effective concentrations preserved epithelial integrity and attenuated viral disruption of ciliary function. Across both viruses, increasing antiviral exposure was associated with dose-dependent signature mutagenesis. Importantly, antivirals induced significantly higher RSVA mutation burden in the primary airway model. These findings highlight the therapeutic potential of RdRp inhibitor combinations for RSVA and hPIV3, provide mechanistic insight through antiviral-related mutational signatures and demonstrate advantages of the primary human airway culture model for development of effective multi-drug regimens and broad-spectrum antiviral preparedness. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/699296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1eb00dforg.highwire.dtl.DTLVardef@186358forg.highwire.dtl.DTLVardef@265181org.highwire.dtl.DTLVardef@1b10d0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Disrupting action control with transcranial ultrasound neuromodulation: a step forward for Tourette syndrome

BackgroundWhile Tourette syndrome (TS) is characterised by the presence of tics, premonitory urges are involved in both triggering them and as a lever to control them. The right insular cortex is known to play a key role in these processes, yet its precise functional contribution remains unclear. Transcranial ultrasound stimulation (TUS) is currently one of the only non-invasive neuromodulation techniques capable of safely and precisely modulating the activity of deep brain structures such as the insula. ObjectiveThis study used TUS to causally investigate the respective roles of the anterior and posterior insula in urge and action control during a blink suppression task. MethodsTwenty healthy adults underwent three TUS sessions targeting the right anterior insula, posterior insula, and posterior ventricle (control site) in a within-subject, crossover design. Behavioural outcomes included blink counts and urge ratings. Additionally, voxel-based correlations were conducted to further estimate the role of each reached area with acoustic stimulation, allowing for a better mechanical understanding. ResultsTUS of the anterior insula induced a clear and acute increase in blink frequency without altering urge, reinforced by voxel-based analyses, confirming its role in voluntary action control. Posterior insula stimulation induced delayed increases in both blinks and urge, which are essentially explained by an involuntary stimulation of the right inferior frontal gyrus. Ventricular stimulation unexpectedly reduced blink frequency during the stimulation only, correlated with acoustic energy delivered within the posterior internal capsule. No significant side effects or temperature rises were observed. ConclusionsThese findings provide the first causal evidence that TUS can modulate the two main features of TS: premonitory urge and action control. Distinct effects across targets highlight the anterior insulas critical role in action control and suggest the internal capsule as a potential therapeutic target for TS.

neuroscience↗

bioRxiv2025

Design and development of a SARS and MERS Combination Vaccine

This work was conducted during the COVID-19 pandemic prior to the licensing of any vaccines against COVID-19. Although several COVID-19 vaccines are now commercially available, this research on the development of a combination Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS) is still relevant and shows how a combination vaccine can be designed, produced and rapidly tested in the laboratory. We present the development of a combination vaccine designed to provide immunity against Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS). The primary objective of this vaccine design is twofold: to mitigate the burden of coronavirus and to address the specific vulnerability of regions prone to recurrent MERS outbreaks. Our combination vaccine incorporates antigenic components from zoonotic sources, specifically SARS-CoV, SARS-CoV-2, and MERS. We assess the impact of combining different Spike proteins S1 subunit antigens, due to its recognised immunogenic potential, and adjuvants on serum antibody titres, virus neutralizing capabilities, and inter-antigen immune responses. We report a robust and broad antibody response against SARS-CoV-2 and related coronaviruses, which was amplified by different adjuvant formulations, including alum, MPLA, CpG, and Squalene-in-Water Emulsion.

immunology↗

bioRxiv2025

Neutrophil myeloperoxidase as a functional biomarker for RSV severity: implications for in vitro therapeutic screening.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet effective therapeutics are lacking. The aim of this study is to develop an in vitro model that recapitulates key clinical outcomes in infants with RSV bronchiolitis to help accelerate the discovery of effective therapeutics. Neutrophil activation and influx into the airways are hallmarks of severe RSV infection, but these responses are difficult to quantify in clinical trials. Here we profile peripheral blood-derived neutrophils from infants with RSV admitted to the Paediatric Intensive Care Unit (PICU) and identify myeloperoxidase (MPO) as a key indicator of disease severity when compared to age matched controls. To mechanistically model this response, we established a paediatric airway epithelial air-liquid interface (ALI) system incorporating an endothelial layer and primary neutrophils to recapitulate the tissue microenvironment at the blood-airway barrier. Following RSV infection, neutrophil migration and activation were assessed using flow cytometry. The inclusion of an endothelial layer enhanced physiological relevance and more accurately replicated in vivo MPO responses. We then evaluated two antiviral candidates (remdesivir (RDV) and RSV604) to assess their ability to modulate neutrophil activation. While both compounds reduced viral load at 24 hours post-infection, only RSV604 attenuated MPO expression. These findings establish MPO as both a biomarker of RSV disease severity and a functional readout of therapeutic efficacy and demonstrate that targeting neutrophildriven inflammatory pathways may be critical for reducing pathology in infant RSV infection. Take home messageAntiviral drug discovery should include neutrophil MPO reduction as a readout of therapeutic efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/682115v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@170058corg.highwire.dtl.DTLVardef@17c344corg.highwire.dtl.DTLVardef@14d79baorg.highwire.dtl.DTLVardef@172ac3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

CCR6 is essential for effective immunity against Mycobacterium tuberculosis infection in mice

Tuberculosis (TB) is a global epidemic that has threatened human health throughout recorded history. TB disease, caused by infection with Mycobacterium tuberculosis (M.tb), is heterogeneous between individuals, and clinical TB outcomes are impacted by genetic and environmental risk factors. Infected individuals must maintain a careful balance of cytokines and chemokines following M.tb infection to eliminate or contain the bacteria without causing excessive inflammation and lung damage. The CC chemokine receptor 6 (CCR6) is expressed by several immune cell populations that are classically involved in the pathogenesis of M.tb infection. However, the precise functions of CCR6 in the context of TB disease remain underexplored. Here, we show that mice lacking the CCR6 receptor (CCR6 KO) fail to restrict bacterial burden in the lungs, leading to increased disseminated infection at 4-weeks post-infection compared to wild-type C57BL/6J (B6) mice, with a more pronounced effect in females. CCR6 KO mice also developed necrotic pulmonary lesions, phenotypically distinct from B6 mice, and produced elevated levels of pro-inflammatory cytokines and chemokines at the onset of adaptive immunity, particularly IL-17. Long-term infection experiments revealed that the absence of CCR6 enhances risk for mortality following M.tb infection, particularly in females. This study provides insights into the role of CCR6 during TB pathogenesis and establishes its importance in maintaining protective immunity against M.tb within the context of a genetically tractable mouse model that forms necrotic pulmonary granulomas.

microbiology↗

bioRxiv2025

The mouse pangenome reveals the structural complexity of the murine protein coding landscape

We present the first mouse pangenome consisting of 17 high-quality inbred mouse strain genomes with complete annotation. This collection includes 12 widely used classical laboratory strains and 5 wild-derived strains. We have fully resolved previously incomplete genomic regions, including the major histocompatibility complex (MHC), the defensin cluster, T-cell receptor, and Ly49 complexes. Hundreds of non-reference genes identified in previous publications not found in GRCm39, like Defa1, Raet1a, and Klra20 (Ly49T), were localised in the new reference genomes. We conducted the first genome-wide scan of variable number tandem repeats (VNTRs) within the coding regions of mice, identifying over 400 genes with VNTR polymorphisms up to more than 600 repeat copies and repeat units reaching 990 nucleotides. Our strain-specific annotations enhance RNA-Seq analyses, as demonstrated in PWK/PhJ, where we observed a 5.1% improvement in read mapping and expression level differences in 2.1% of coding genes compared to using GRCm39.

genomics↗

bioRxiv2025

Cathepsin Z is a conserved susceptibility factor underlying tuberculosis severity

Tuberculosis (TB) outcomes vary widely, from asymptomatic infection to mortality, yet most animal models do not recapitulate human phenotypic and genotypic variation. The genetically diverse Collaborative Cross mouse panel models distinct facets of TB disease that occur in humans and allows identification of genomic loci underlying clinical outcomes. We previously mapped a TB susceptibility locus on mouse chromosome 2. Here, we identify cathepsin Z (Ctsz) as a lead candidate underlying this TB susceptibility and show that Ctsz ablation leads to increased bacterial burden, CXCL1 overproduction, and decreased survival in mice. Ctsz disturbance within murine macrophages enhances production of CXCL1, a known biomarker of TB severity. From a Ugandan household contact study, we identify significant associations between CTSZ variants and TB disease severity. Finally, we examine patient-derived TB granulomas and report CTSZ localization within granuloma-associated macrophages, placing human CTSZ at the host-pathogen interface. These findings implicate a conserved CTSZ-CXCL1 axis in humans and genetically diverse mice that mediates TB disease severity.

genetics↗

bioRxiv2025

Donor-derived airway ALI model for high-throughput screening of antiviral combinations with concurrent analysis of antiviral efficacy and epithelial toxicity using ciliR

Respiratory RNA viral infections significantly impact quality of life and productivity, both in terms of pandemics (e.g., SARS-CoV-2, influenza A viruses) and seasonal infections such as respiratory syncytial virus (RSV). Globally, RSV causes an estimated 33.8 million cases annually in children under five, leading to 2.8-4.3 million hospital admissions and up to 199,000 deaths. Human-relevant drug screening models are key to addressing the urgent need for effective antiviral treatments for RSV, particularly in vulnerable patient groups. Here, we present a donor-derived differentiated primary human nasal epithelial cell, 96-HTS Transwell air-liquid interface (ALI) model designed to investigate the effects of combination antiviral therapies on RSV infection in primary human ciliated airway epithelium. Additionally, we describe novel analytical tools using R (ciliR) to screen drug combinations by concurrently measuring efficacy and ciliary beat frequency as a sensitive marker of cell toxicity. Our results demonstrate that the smaller 96-HTS ALI cultures retain comparable epithelial composition and ciliary function to conventional ALI culture formats. These cultures are permissible to infection with an RSV-GFP reporter virus, enabling quantitative comparison and combination treatment across multiple epithelial cultures from the same donor. We anticipate that our disease-relevant system will serve as a foundation for larger-scale experiments aimed at optimizing combination therapy for RSV and other respiratory viruses.

microbiology↗

bioRxiv2025

Wildfire ash impacts to photosynthesis and growth of marine phytoplankton cultures

Climate change includes increasing surface temperatures as well as extreme events--heatwaves, storms, floods, and droughts. In 2020, these factors produced a record 10,000 wildfires that burned 18,000 km2 in California USA. Air pollution, including airborne ash, from these fires was a widespread human health hazard. While the ecological effects of wildfires have been extensively documented in terrestrial and freshwater systems, impacts on ocean and coastal ecosystems are largely unexplored. Here, we describe the physical and chemical properties of ash from the CZU Lightning Complex fire and experimentally test its effects on the photosynthesis and growth of four unicellular marine phytoplankton. Sieved air-fall ash was primarily composed of particles 250-500 m and contained [~]1 {per thousand} of Fe, Mn, and Ba. Diagnostic indices of polycyclic aromatic hydrocarbons indicated the air-fall ash originated from combusted wood, and the total concentration of the EPA 16 high-priority PAHs exceeded 2.7 ppm. Pulse Amplitude Modulation fluorometry documented various declines in the photosynthetic efficiency of Isochrysis and Dunaliella cultures dosed with ash, and the bulk cellular growth of these cultures was inhibited. While our study demonstrated the impacts of wildfire ash on marine producers, the precise mechanisms are unclear. We provide recommendations for how future studies may further resolve the impacts of ash on phytoplankton productivity, community diversity, and trophic transfer of toxins and describe the long-term impacts of wildfires on coastal marine ecosystems.

ecology↗

bioRxiv2025

Polymerase mutations underlie adaptation of H5N1 influenza virus to dairy cattle and other mammals.

In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA1. As of mid-2025 the epidemic is ongoing, resulting in spillbacks into poultry, wild birds and other mammals including humans2. Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells and tissues, as well as cells of other mammalian species including humans and pigs. We find evidence of several mammalian adaptations gained early in the evolution of these viruses in cattle including PB2 M631L, which is found in all cattle sequences, and PA K497R, which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication. We show that this mutation adapts the polymerase to better interact with bovine ANP32 proteins, particularly ANP32A, and thereby enhances virus replication in bovine mammary systems and primary human airway cultures. Importantly, we show that ongoing evolution during 2024 and 2025 in the PB2 gene, including E627K and a convergently arising D740N substitution, further increase polymerase activity and virus replication in a range of mammalian cells. Thus, the continued circulation of H5N1 in dairy cattle not only allows virus adaption improving replicative ability in cattle, but also increases the risk of zoonotic spillover.

microbiology↗

bioRxiv2024

Cellular heterogeneity in hypertrophic burn scars in response to carbon dioxide laser therapy

Fractional carbon dioxide (AFCO2) laser therapy is used for treating pathological scarring, but the clinical outcomes are variable and the mechanisms of scar reduction poorly understood. We investigated the mechanisms underpinning efficacy of AFCO2 laser therapy, performing single-cell RNA sequencing in skin biopsies from patients with hypertrophic scars after AFCO2 laser therapy. Patients with younger scars (Good Responder, GR, <6 years from healing) had better scar reduction than patients with older scars (Poor Responder, PR, >6 years from healing) by various measures of scarring. scRNAseq analysis revealed that genes enriched in GR were associated with extracellular matrix and structure organisation (COL14A1, POSTN, SPARC); whereas genes enriched in PR were related to enhanced immune responses (IL-12, MSTN, HLA-DQA). The groups had distinct intercellular communication networks and differentiation trajectories after AFCO2, with regenerative Mesenchymal fibroblasts associated with a good response and inflammatory Secretory Papillary and Inflammatory Fibroblasts with a poor response.

cell biology↗

bioRxiv2024

Rapid learning of temporal dependencies at multiple timescales

Our environment contains temporal information unfolding simultaneously at multiple timescales. How do we learn and represent these dynamic and overlapping information streams? We investigated these processes in a statistical learning paradigm with simultaneous short and long timescale contingencies. Human participants (N=96) played a game where they learned to quickly click on a target image when it appeared in one of 9 locations, in 8 different contexts. Across contexts, we manipulated the order of target locations: at a short timescale, the order of pairs of sequential locations in which the target appeared; at a longer timescale, the set of locations that appeared in the first vs. second half of the game. Participants periodically predicted the upcoming target location, and later performed similarity judgements comparing the games based on their order properties. Participants showed context dependent sensitivity to order information at both short and long timescales, with evidence of stronger learning for short timescales. We modeled the learning paradigm using a gated recurrent network trained to make immediate predictions, which demonstrated multilevel learning timecourses and patterns of sensitivity to the similarity structure of the games that mirrored human participants. The model grouped games with matching rule structure and dissociated games based on low-level order information more so than high-level order information. The work shows how humans and models can rapidly and concurrently acquire order information at different timescales.

neuroscience↗

bioRxiv2023

ciliR: a new R package for determining ciliary beat frequency using fast-Fourier transformation.

Cilia are motile hair-like structures that play a vital role in our body. Accurate assessment of ciliary beat frequency is pivotal for investigating ciliary dynamics and diagnosing ciliopathies. This study aims to develop software for accurately measuring the beat frequency of cilia captured using high-speed video microscopy. To achieve this, we developed the ciliR package in R, which was validated against manual counting and three other automated methods of counting cilia beat frequency. The results showed that ciliR produced results that were comparable to manual counting. The accuracy of ciliR was defined by its ability to reduce noise, including only counting data in a biologically significant range (0-60 Hz). Our software is a valuable tool for researchers in the field of ciliobiology as it offers a reliable method for detailed ciliary function analysis, thereby contributing to the broader understanding of mechanisms underlying ciliary-related disease. We encourage researchers to try this package and feed-back their findings to the authors. Instructions for use and processes for providing feedback are provided in supplementary material. SummaryciliR is a novel R package designed for analysing ciliary beat frequency (CBF) via ImageJ and RStudio. The advantage of the ciliR system, lies in its integration with the R environment, increasing processing speed and access to data visualization tools and analysis pipelines available in other R packages. The open-source platform invites community feedback to refine functionality, aiming to advance ciliopathy research with an accessible, comprehensive toolkit.

cell biology↗

bioRxiv2023

Multiple repeat regions within mouse DUX recruit chromatin regulators to facilitate an embryonic gene expression program

The embryonic transcription factor DUX regulates chromatin opening and gene expression in totipotent cleavage-stage mouse embryos, and its expression in embryonic stem cells promotes their conversion to 2-cell embryo-like cells (2CLCs) with extraembryonic potential. However, little is known regarding which domains within mouse DUX interact with particular chromatin and transcription regulators. Here, we reveal that the C-terminus of mouse DUX contains five uncharacterized [~]100 amino acid (aa) repeats followed by an acidic 14 amino acid tail. Unexpectedly, structure-function approaches classify two repeats as active and three as inactive in cleavage/2CLC transcription program enhancement, with differences narrowed to a key 6 amino acid section. Our proximity dependent biotin ligation (BioID) approach identified factors selectively associated with active DUX repeat derivatives (including the 14aa tail), including transcription and chromatin factors such as SWI/SNF (BAF) complex, as well as nucleolar factors that have been previously implicated in regulating the Dux locus. Finally, our mechanistic studies reveal cooperativity between DUX active repeats and the acidic tail in cofactor recruitment, DUX target opening, and transcription. Taken together, we provide several new insights into DUX structure-function, and mechanisms of chromatin and gene regulation.

developmental biology↗