Biology subjects

Xu, X.

Publications and source records attributed to Xu, X..

At least 181 records · Page 10Linked to original sources

bioRxiv2025

A multi-step immune-competent genetic mouse model reveals phenotypic plasticity in uveal melanoma

Uveal melanoma (UM) is a highly aggressive intraocular malignancy with limited therapeutic options for metastatic disease. Existing transgenic UM mouse models inadequately recapitulate human disease progression, while transplant models lack immune competence for studying the tumor immune microenvironment and therapeutic interventions. To address these limitations, we developed a genetically engineered mouse model incorporating stepwise genetic alterations implicated in human UM progression. Spatiotemporally controlled expression of mutant GNAQQ209L from the endogenous locus induced choroidal nevi with limited penetrance. Concomitant BAP1 deletion enhanced nevus formation, while further MYC activation led to fully penetrant intraocular tumors with metastatic potential. Single-cell RNA sequencing revealed malignant cells segregated into Melanocytic and Neural Crest-like subpopulations characterized by distinct transcriptional and biosynthetic programs. Trajectory analyses inferred dedifferentiation from the Melanocytic toward the Neural Crest-like state during tumor progression. Comparison to human UM revealed commonalities with highly aggressive Class 2 UM, including gene expression signatures and copy number gains affecting genes that map to human chromosome 8q beyond the activated MYC allele, suggesting cooperative effects of multiple drivers in this chromosomal region. The tumor microenvironment featured immunosuppressive macrophage populations and exhausted T cells, closely resembling human UM. This physiologically relevant, immune-competent model provides a platform for investigating UM biology, functionally characterizing candidate driver genes, and developing immune-based therapeutic strategies. SIGNIFICANCE STATEMENTWe developed a mouse model that resembles the genetic progression and phenotypic plasticity of human UM. This spatially controlled model confirms the critical role of driver mutations in GNAQ and BAP1, proposes MYC as a promoter of malignant transformation in coordination with other chromosome 8q genes, and reveals UM progression through distinct cellular states. This model offers an urgently needed preclinical platform for understanding the immunogenomics of UM and for testing immune and targeted treatments for this lethal cancer.

cancer biology↗

bioRxiv2025

A Cell Type-Specific Role for Tubb6 in Ciliogenesis of Xenopus Epidermal Multiciliated Cells

Cilia are microtubule-based organelles found on the surface of most eukaryotic cells. These microtubules are composed of - and {beta}-tubulin heterodimers, and different tubulin isotypes can confer distinct properties to microtubules. Despite their importance, the contribution of individual tubulin isotype to cilia formation and function remains largely unexplored in vertebrates. Here, we identify a critical role for the {beta}-tubulin isotype Tubb6 in the formation of motile cilia in Xenopus epidermal multiciliated cells (MCCs). Tubb6 mRNA is selectively expressed in MCCs, and its protein product localizes to ciliary axonemes. Loss of Tubb6 leads to a marked reduction in cilia number and length, resulting in defective MCC function. In contrast, mono-motile cilia in the gastrocoel roof plate are unaffected by Tubb6 depletion, suggesting a selective requirement for ciliogenesis in MCCs. Together, our findings uncover a cell type-specific role for Tubb6 in motile cilia formation and highlight the functional specialization of tubulin isotypes in vertebrate cilia assembly.

developmental biology↗

bioRxiv2025

FEDRANN: effective overlap graph construction based on dimensionality reduction and approximate nearest neighbors

Overlap detection is a key step in de novo genome assembly pipelines based on the Overlap-Layout-Consensus (OLC) paradigm. However, existing methods for overlap detection either rely on heuristic seed-and-extension strategies or locality-sensitive hashing (LSH), both of which struggle to handle repetitive genomic regions and the computational burden of large-scale datasets. Here, we present FEDRANN, a novel strategy for overlap graph construction that integrates feature extraction, dimensionality reduction (DR), and approximate nearest neighbor (ANN) search. We find the pipeline combining inverse document frequency (IDF) transformation, sparse random projection (SRP), and NNDescent enables accurate detection of overlaps across diverse datasets. We developed an efficient open-source implementation of this pipeline named Fedrann (https://github.com/jzhang-dev/fedrann). Through systematic benchmarking on real long-read sequencing data, we demonstrate that Fedrann produces overlap graphs comparable to or better than those generated by existing state-of-the-art tools, including MECAT2, minimap2, and wtdbg2, while maintaining competitive runtime. Despite being implemented primarily in Python, Fedrann achieves performance on par with tools written in compiled languages, owing to matrix-based representations and C-accelerated numerical libraries. Our results suggest that DR and ANN techniques offer a promising new direction for scalable and accurate overlap detection in long-read assembly and broader sequence similarity search tasks.

bioinformatics↗

bioRxiv2025

Context-Dependent Variability Of HIF Heterodimers Influences Interactions With Macromolecular And Small Molecule Partners

Hypoxia inducible factors (HIFs) are transcription factors that coordinate cellular responses to low oxygen levels, functioning as an /{beta} heterodimer which binds a short hypoxia response element (HRE) DNA sequence. Prior studies suggest HIF/HRE complexes are augmented by the binding of additional factors nearby, but those interactions are not well understood. Here, we integrated structural and biochemical approaches to investigate several functionally relevant HIF assemblies with other protein, small molecule, and DNA partners. First, we used cryo-electron microscopy (cryo-EM) to establish HIF-1 and HIF-2 self-assemble to form novel "dimer-of-heterodimers" (DoHD) complexes on extended human EPO enhancer sequences, with one heterodimer bound at a canonical HRE site and the second binding in an inverted fashion to an HRE-adjacent sequence (HAS) 8 bp away. Consistent with ARNT PAS-B domains predominating interactions within a DoHD, we found HIF-1 and HIF-2 co-assemble mixed DoHD complexes on the same DNA. Second, we saw that despite the increased complexities of the larger complexes, ligands for the isolated ARNT or HIF-2 PAS-B domains are still capable of binding and disrupting both the heterodimer and DoHD complexes, albeit with variable potencies depending on the ligand. Finally, we combined cryo-EM and hydrogen- deuterium exchange by mass spectrometry (HDX-MS) to show how HIF-1 and HIF-2 heterodimers engage the transforming acidic coiled-coil containing protein 3 (TACC3) coactivator via both ARNT and HIF- subunits, though this was unseen in the larger DoHD. Our findings highlight the importance of both molecular context and dynamics in biomolecular complex formation, adding to the complexities of potential regulation. Significance StatementHypoxia inducible factors (HIFs) are transcription factors that regulate oxygen-dependent cellular processes with implications in certain types of cancers. Current molecular structures of HIFs bound to short DNA fragments provide insights into their function, but leave open questions about how they bind longer natural DNA fragments and interact with small molecules and protein coactivators. Integrating structural and biochemical techniques, we discovered a novel assembly in which two HIFs bind together on a single extended DNA fragment, forming a "dimer-of-heterodimers", which exhibits some differences in ligand and coactivator binding than heterodimers or isolated PAS domains. Our studies highlight how functional contexts can shift structural paradigms and provide greater insight into the mechanisms by which HIFs and similar bHLH-PAS transcription factors operate.

biochemistry↗

bioRxiv2025

In-cell Proteomics Enables High-Resolution Spatial and Temporal Mapping of Early Xenopus tropicalis Embryos

Early embryonic development requires tightly regulated molecular programs to coordinate cell division, fate specification, and spatial patterning. While transcriptomic profiling is widely performed, proteomic analyses of early vertebrate embryos remain limited due to technical challenges in embryonic sample preparation. Here, we propose an "in-cell proteomics" approach, which bypasses cell lysis and yolk depletion, processes individual embryos directly in functionalized filter devices, and generates liquid chromatography-mass spectrometry (LC-MS)-friendly samples in an extremely robust and streamlined manner. Combined with a single-shot data-independent acquisition (DIA) MS workflow, this approach enabled us to consistently quantify [~]6,200 proteins from a single Xenopus tropicalis embryo, representing the deepest proteomic coverage of early X. tropicalis development reported to date. Investigation of the temporal proteomes across five cleavage stages (1- to 16-cell) revealed a drastic proteomic shift between 2- and 4-cell stages, followed by more gradual transitions thereafter. Spatial analysis of dissected 8-cell blastomeres uncovered pronounced molecular asymmetry along the animal-vegetal axis, while dorsal-ventral differences were minimal. This study establishes a novel in-cell proteomics technology in conjunction with DIA-MS as a robust platform for high-resolution, low-input developmental proteomics analysis, and provides a comprehensive spatiotemporal protein atlas for early X. tropicalis embryos.

biochemistry↗

bioRxiv2025

TIP-seq: A Single-cell Multiomics Approach for Simultaneous Transcriptome and Intracellular Protein Profiling

Single-cell multiomics technologies have significantly advanced our understanding of cellular heterogeneity; however, the concurrent profiling of mRNA transcripts and target proteins continues to pose a substantial challenge. To bridge the gap between single-cell sequencing and proteomic methods, we developed an innovative single-cell multiomics methodology, termed TIP-seq, which facilitates the simultaneous profiling of the transcriptome and intracellular proteins without prior cell manipulation. In TIP-seq, microfluidic technology is leveraged to integrate oligonucleotide-labeled antibodies with hydrogel beads, allowing for the capture of mRNAs and proteins from individual cells within droplets, thereby enabling high-throughput and precise dual-omics data acquisition. When applied to lung cell lines and non-small cell lung cancer (NSCLC) tissues, TIP-seq analysis revealed notable cellular heterogeneity and molecular dynamics, emphasizing the distinct immune cell interactions within NSCLC tissues. Key immune checkpoint interactions in NSCLC, such as SPP1-CD44, NECTIN2-TIGIT, and NECTIN2-CTLA4, along with functional alterations in tumor-associated dendritic cells (DCs) and T cells, were identified via TIP-seq, underscoring their pivotal roles in mediating immune suppression within the tumor microenvironment. Collectively, TIP-seq represents a powerful methodology for identifying novel therapeutic targets and biomarkers, thereby holding significant potential for the advancement of precision medicine in the treatment of lung cancer and other complex diseases.

biochemistry↗

bioRxiv2025

Adaptive Evolution of Gene Regulatory Networks in Mammalian Neocortical Neurons

Mammals have evolved a plethora of adaptations that have enabled them to thrive in diverse environments. Among the most significant is the emergence of a more complex brain, exemplified by the dramatic transformation of the dorsal cortex from a single layer of excitatory projection neurons (ExNs) in ancestors to a multilayered cerebral neocortex enriched with diverse intratelencephalic (IT) and extratelencephalic (ET) ExN subtypes. These ExNs established specialized projection systems, such as the corticospinal tract and corpus callosum, enhancing brain connectivity and functionality. However, the evolutionary mechanisms underlying these mammalian-specific adaptations remain elusive. By comparing the landscape of gene expression and cis-regulatory elements (CREs) in mouse ExN subtypes and by cross-species examination of mammalian and non-mammalian CREs, we identified mammalian-specific CREs and expression patterns. The mammalian-specific CREs include a subset bound by ZBTB18 that are associated with genes defining IT and ET subtypes and connectivity. Both ZBTB18 and these target genes have previously been implicated in intellectual disability and autism. Deletion of Zbtb18 in mouse ExNs dysregulated target gene expression, reduced molecular diversity, diminished corticospinal and callosal projections, and increased intrahemispheric cortico-cortical association projections to the prefrontal cortex, resembling features of non-mammalian dorsal pallium. Interestingly, ZBTB18 binding motifs are highly enriched in callosally projecting IT-biased CREs, where they show higher conservation specifically in mammals. This study uncovers critical components and mammalian-specific evolutionary adaptations within a regulatory node essential for neocortical ExN identity and connectivity, with implications for neurodevelopmental and neuropsychiatric disorders.

neuroscience↗

bioRxiv2025

Beyond the Axoneme: Whole-Cell Proteomics Identifies Novel Regulators of Ciliogenesis in Xenopus Epidermal Multiciliated Cells

Motile cilia generate directional fluid flow critical for development and tissue homeostasis, and their dysfunction underlies a range of human diseases. Prior proteomic studies have focused on isolated axonemes, leaving the regulatory components of ciliogenesis located in other cellular compartments of multiciliated cells (MCCs) largely unidentified. To bridge this gap, we performed whole-cell proteomic profiling of MCCs in Xenopus using an inducible system that enables selective MCC-enrichment. Leveraging a single-vessel in-cell proteomics approach with ultra-high sample preservation, we identified over 9,000 proteins, including 832 upregulated in MCCs. These encompassed both established ciliary components and novel candidates localized to distinct ciliary structures. Functional validation confirmed the involvement of previously uncharacterized proteins in ciliogenesis. This study presents a high-resolution, whole-cell proteome of vertebrate MCCs, providing critical insights into the molecular networks that govern ciliogenesis and offering a valuable resource for understanding the mechanisms of cilia-related disorders.

developmental biology↗

bioRxiv2025

A domestication change at PvMYB26 in common bean sheds light on the origins of Middle American agriculture

Domestication imposed radically different selection pressures on plants, eventually transforming them into the cultivated forms that support global populations today. Here, we investigate the loss of seed dispersal via pod shattering during common bean (Phaseolus vulgaris L.) domestication. We identified an eight-kb deletion eliminating the transcription start site and promoter of the candidate gene PvMYB26. Mutants express PvMYB26 at < 1% of the level of wild types and produce 44% less pod lignin. Whole-genome sequencing revealed that the mutation is nearly diagnostic for domestication status among Middle American common bean, indicating its importance in domestication. We also identified a high-frequency PvMYB26 frameshift/premature stop mutation unique to Andean domesticates. Wild haplotypes most like Middle American domesticates are found in eastern Jalisco, Mexico. Our results suggest that West-Central Mexico was the site of common bean domestication and suggest that this region may have been important in the rise of Middle American agriculture.

evolutionary biology↗

bioRxiv2025

NETSseq Reveals Inflammatory and Aging Mechanisms in Distinct Cell Types Driving Cerebellar Decline in Ataxia Telangiectasia

The cellular and molecular changes driving the neurological abnormalities associated with ataxia - telangiectasia (A-T) are not well understood. Here, we applied our proprietary Nuclear Enriched Transcript Sort sequencing (NETSseq) platform to investigate changes in cell type composition and gene expression in human cerebellar post-mortem tissue from A-T and control donors. Compared to single-nuclei technologies, NETSseq provided a more robust detection of genes with low abundance, a higher cell type specific expression pattern, and significantly lower levels of cross-contamination. We found dysregulation in neurotransmitter signaling in granule neurons, potentially underlying the impaired motor coordination in A-T. Astrocytes and microglia have evidence of accelerated aging, with astrocytes being characterized by neurotoxic signatures, while microglia showed activation of DNA damage response pathways. These findings highlight the importance of NETSseq as a resource for investigating mechanisms and biological processes associated with disease, providing high-sensitivity, cell-specific insights to advance targeted therapies for neurodegenerative diseases.

neuroscience↗

bioRxiv2025

HADDOCK3: A modular and versatile platform for integrative modelling of biomolecular complexes

HADDOCK is a widely used resource for integrative modelling of a variety of biomolecular complexes that is able to incorporate experimental knowledge into physics-based calculations during complex prediction, refinement, scoring and analysis. Here we introduce HADDOCK3, the new modular version of the program, in which the original, parameterisable albeit rigid pipeline has been first broken down in a catalogue of independent modules and then enriched with powerful analysis tools and third-party integrations. Thanks to this increased flexibility, HADDOCK3 can now handle multiple integrative modelling scenarios, providing a valuable, physics-based tool to enrich and complement the predictions made by machine learning algorithms in the post-AlphaFold era. We present examples of successful applications of HADDOCK3 that were not feasible with the previous versions of HADDOCK, highlighting its expanded capabilities. The HADDOCK3 software source code is freely available from the GitHub repository (https://github.com/haddocking/haddock3) and comes with an online user guide (www.bonvinlab.org/haddock3-user-manual). All example data described in this manuscript are available at https://github.com/haddocking/haddock3-paper-data.

bioinformatics↗

bioRxiv2025

Locus Coeruleus to Medial Prefrontal Cortex Noradrenergic Neural Circuit Modulates States of Consciousness during Sevoflurane Anesthesia in Mice

How exactly does general anesthesia achieve its effects? It is so widely used in surgeries and medical treatments to enhance comfort for nearly one hundred years, yet its precise mechanisms underlying the loss and recovery of consciousness still remain unclear. Utilizing a variety of research methods-pharmacological, optogenetic, chemogenetic, fiber photometry, and gene knockdown approaches-our study has shed light on the significant modulatory function of locus coeruleus noradrenergic neurons during the transition from sevoflurane anesthesia to awakening. Furthermore, the activation of the LC-mPFC circuit has been found to have a substantial arousal effect, with 1-AR playing a crucial role in this process. Additionally, GABAA-R has been identified as the key binding site for sevoflurane within the locus coeruleus. These findings collectively offer novel insights into the neural network mechanisms underlying general anesthesia, advancing our understanding of this complex medical phenomenon.

neuroscience↗

bioRxiv2025

EpiMII: Integrating Structure and Graph Neural Networks for MHC-II Epitope and Neoantigen Design

MHC-II neoantigens play a critical role in immunotherapy, either as direct effectors or through their influence on CD8+ T cells. However, only a small fraction of tumor DNA mutations qualify as functional neoantigens, and current prediction tools often lack accuracy, leading to the low immunogenicity of predicted neoantigens in vivo. Here, we present EpiMII, a Graph Neural Network model for MHC-II epitope design, which learns from the structural features of epitopes to predict their sequences. To train EpiMII, we constructed a reliable, large dataset containing 142,934 MHC-II epitope structures. This approach achieves a 4.2x improvement over ProteinMPNN, with a sequence recovery rate of 78.0% for known MHC-II epitopes in the Protein Data Bank. As a case study, we designed a neoantigen from hepatocellular carcinoma. All five designed epitopes significantly activated CD4+ T cells in vitro and induced secretion of IFN-{gamma} and TNF-. Notably, one epitope treatment significantly reduced tumor volume in mice in vivo. EpiMII offers a novel and efficient approach for identifying MHC-II epitopes/neoantigens, potentially contributing to vaccine development.

immunology↗

bioRxiv2025

iSHARC: Integrating scMultiome data for heterogeneity and regulatory analysis in cancer

SummaryThe 10x Genomics single cell Multiome (scMultiome) assay enables the simultaneous profiling of chromatin accessibility and gene expression from the same nucleus, and has increasingly been utilized in revealing cellular heterogeneity and gene regulation in cancers. However, a dedicated bioinformatics pipeline specifically designed for this type of data is still lacking. Here we present iSHARC, a streamlined pipeline for quality control, modality integration, clustering, cell type annotation, and regulatory mechanism analysis of individual scMultiome data, as well as for integrating multiple samples. The main advantages of iSHARC are: 1) easy implementation, execution and extension through a modular Snakemake workflow management system; 2) flexible analysis and parameters customization via a single configuration file; and 3) comprehensive accessibility by providing different access points to results and detailed summary reports from a single run. Availability and implementationThis pipeline is an open-source software under the MIT license and it is freely available at https://github.com/yzeng-lol/iSHARC. Contactyong.zeng@uhn.ca or hansen.he@uhn.ca or mathieu.lupien@uhn.ca Supplementary informationSupplementary data are appended.

bioinformatics↗

bioRxiv2025

DNA repair under heat: DNA Polymerase {lambda} modulates heat stress-induced mutagenesis in plants

Mutation rates can increase substantially under environmental stress, known as stress-induced mutagenesis. Specifically, heat stress has been shown to elevate mutation rates, thereby enhancing genetic variability and facilitating adaptation. However, the underlying mechanisms remain elusive in eukaryotes. Here, we investigated how heat stress affects DNA repair and induces mutations both locally and globally through CRISPR-Cas9 targeted DNA breaks and whole genome sequencing analyses in Arabidopsis thaliana. Heat stress was found to enhance CRISPR editing efficiency across all chromatin contexts, with particularly significant increases, up to 29.9-fold, in heterochromatic regions. Moreover, heat stress consistently shifts mutation outcomes toward 1-bp insertions regardless of chromatin states. We identified a heat-inducible, error-prone DNA polymerase, Pol{lambda}, as the key mediator of mutation profile changes. When extending our investigation from targeted mutations to genome-wide effects, we found that increases in global mutation rates under heat stress are also dependent on Pol{lambda}. Single-cell transcriptomic analysis further demonstrated that Pol{lambda} expression is tightly regulated and cell-type specific, with the highest expression levels in central zone meristematic cells. Together, these findings provide practical applications for improving editing efficiency in heterochromatic regions and fundamental insights into heat-induced mutagenesis, establishing Pol{lambda} as a crucial mediator of stress-induced genetic variation in plants.

plant biology↗

bioRxiv2025

IL-18 metabolically reprograms CAR-expressing natural killer T cells and enhances their antitumor activity against neuroblastoma

Invariant natural killer T cells (NKTs) have intrinsic anti-tumor properties that make them promising candidates for chimeric antigen receptor (CAR)-based immunotherapies. Transgenic cytokine expression has been shown to enhance the potency of cellular immunotherapies, and we hypothesized that co-expressing IL-18 alone or with IL-15 would boost CAR-NKT therapeutic potential. To test this hypothesis, we generated retroviral constructs expressing IL-15 and/or IL-18 with the inducible caspase 9 (iC9) safety switch and co-transduced them with a GD2-specific CAR into human NKTs. Co-expression of IL-18 or IL-15/IL-18 increased GD2.CAR-NKT in vitro cytotoxicity, proliferation, and cytokine secretion compared to IL-15 alone. In a metastatic neuroblastoma model, GD2.CAR-NKTs expressing constructs with IL-18 controlled tumor growth better than cells expressing IL-15 only, but mice in the IL-15/IL-18 group developed severe toxicities not observed in the IL-18-only group. Mechanistically, we found that IL-18 drives a distinct transcriptional profile from IL-15 in CAR-NKTs marked by lower expression of exhaustion gene signatures and enrichment of metabolism-related processes. Finally, targeted metabolomics revealed that IL-18 induces broad metabolic reprogramming in CAR-NKTs including enhancement of oxidative phosphorylation, glycolysis, glutaminolysis and purine metabolism. These results support the use of IL-18 in developing the next generation of cytokine-armed CAR-NKT cancer immunotherapy.

immunology↗

bioRxiv2025

Saccades orchestrate intraocular glucose to shape visual responses in birds

Birds exhibit remarkable vision despite lacking the typical network of blood vessels in their eyes. The characteristic poses a long-standing question about how avian retinas fulfill high energy demands necessary for sight. Here we show that natural, rhythmic eye movements, known as oscillatory saccades, orchestrate intraocular metabolic dynamics and attention-guided visual processing in pigeons. In a series of integrated experiments, we monitored eye movements along with glucose levels in the eye and neuronal activity in key brain regions receiving direct input from the retina. We found that these saccades generate fluctuations in intraocular glucose concentrations, closely linked to changes in neuronal visual responses over timescales of seconds to minutes. Moreover, pharmacological manipulations that altered glucose availability and eliminated these oscillatory saccades resulted in corresponding shifts in neuronal responses, demonstrating a causal linkage between oscillatory saccades, metabolic regulation, and visual processing. These findings reveal a mechanism by which birds actively evoked saccades during attention-driven information gathering, propelling retinal metabolism and facilitating their vision in the absence of retinal vasculature. This study underscores the interplay among eye movements, metabolic regulation, and high-level visual performance, suggesting broader implications for how eye movements contribute to retinal health, attention, and visual function across species.

neuroscience↗

bioRxiv2025

Molecular Basis of EWS Interdomain Self-Association and Its Role in Condensate Formation

Ewing sarcoma, the second most common pediatric bone and soft tissue cancer, is caused by aberrant fusion of the EWS low-complexity domain (EWSLCD) to the DNA-binding domain of the transcription factor FLI1. The resulting fusion, EWS::FLI1, directly interacts with and engages in a dynamic interplay with EWS that drives tumorigenesis and regulates the function of both proteins. While EWSLCD is known to promote self-association, the role of the RNA-binding domains (RBDs) of EWS, which include RGG repeat regions and a structured RNA-recognition motif (RRM), remains less well understood. Here, we investigate the interplay between EWSLCD and RBDs using biomolecular condensation assays, microscopy, NMR spectroscopy, and molecular simulations. Our studies reveal that RBDs differentially influence EWSLCD condensate formation and suggest that electrostatics and polypeptide-chain length likely contribute to this interaction. NMR spectroscopy and molecular dynamics simulations further demonstrate that EWSLCD and the central RNA-binding region, comprising the RRM and RGG2 domains, engage in transient, non-specific interactions that are broadly distributed across both regions and involve diverse residue types. Specifically, tyrosine, polar residues, and proline within EWSLCD preferentially interact with arginine, glycine, and proline residues in the RBD. Atomistic simulations of EWS confirm that the full-length protein exhibits a similar interaction profile with conserved chemical specificity, supporting a model in which a network of weak, distributed interdomain contacts underlies EWS self-association. Together, these findings provide molecular insight into the mechanisms of EWS condensate formation and lay the groundwork for understanding how interdomain interactions regulate EWS and EWS::FLI1 function. SIGNIFICANCEThis study shows that the RNA-binding regions of the RNA-binding protein EWS subtly tune biomolecular condensation through numerous transient contacts with the low complexity domain that is common to both EWS and the oncogenic fusion EWS::FLI1. These results provide insight into condensate formation of EWS, which may be useful for understanding the oncogenic mechanisms of EWS::FLI1 and broadly, into pathogenic biomolecular condensate formation.

biophysics↗