Biology subjects

O'Donnell, E.

Publications and source records attributed to O'Donnell, E..

8 recordsLinked to original sources

bioRxiv2026

A mammalian-specific domain of MSH5 drives the transition from crossover licensing to designation during meiotic prophase I

SUMMARYMeiotic recombination initiates with DNA double-strand breaks (DSBs) repaired as either crossovers (COs) or non-crossovers. Across eukaryotes, MSH4/MSH5 (MutS{gamma}) licenses DSB repair intermediates, directing repair into the class I CO pathway via recruitment of MLH1/MLH3 (MutL{gamma}). In mammals, excess MutS{gamma} sites relative to final MutL{gamma} foci suggest additional MutS{gamma} functions, including directing repair through the minor class II CO pathway. We investigated the role of a mammalian-specific 38-amino acid C-terminal domain of MSH5 using mice lacking this domain (Msh5{Delta}C/{Delta}C). Spermatocytes and oocytes load MSH4 normally to achieve CO licensing in zygonema, but these numbers decline precipitously in pachynema, leading to dramatically reduced MutL{gamma} foci and associated pro-CO factors HEI10 and CNTD1. Despite this, licensing factors RNF212B and MutS{gamma}-associated kinase CDK4 remain persistently upregulated in pachynema. Strikingly, the switch from licensing-associated CDK4 to CO-site-associated CDK2 fails to occur in Msh5{Delta}C/{Delta}C mice, even at residual class I CO events. The result is rapid germ cell death prior to prophase I completion in both sexes. Thus, the loss of the MSH5 C-terminus functionally uncouples the regulatory proteins that define the stepwise patterning of class I COs. Our findings reveal novel early roles for the C-terminus of mammalian MSH5 in converting licensed DSB repair intermediates to designated class I COs.

genetics↗

bioRxiv2026

Functional interplay between H2B ubiquitylation and H2A.Z deposition

The Bre1 ubiquitin conjugating enzyme catalyzes the monoubiquitylation of histone H2B-K120/K123 at promoter proximal nucleosomes, contributing to transcriptional regulation. These same nucleosomes are targeted by the yeast SWR1C chromatin remodeling enzyme that deposits the histone variant H2A.Z. Yeast strains that lack both Bre1 and Swr1 are inviable, indicating that together they contribute to an essential cell function. Interestingly, H2B-K123ub and H2A.Z levels are anticorrelated, and recent biochemical studies suggest a model in which H2B-K123ub inhibits SWR1C activity by blocking access to the nucleosome acidic patch. Here, we exploit recombinant nucleosomes to show that the H2A.Z deposition activity of SWR1C is strongly inhibited by H2B-K123ub. Surprisingly, the loss of H2B-K123ub does not lead to significant changes in the genomic organization of H2A.Z in cells grown in normal media, but we find that H2B-K123ub is required for a re-localization of H2A.Z from promoters to coding regions during replication stress. Together, these data indicate a complex functional interplay between H2B ubiquitylation and H2A.Z deposition.

biochemistry↗

bioRxiv2026

Hunting for microsatellite instability in long-read data with Owl

Microsatellite instability (MSI) is a key biomarker of mismatch repair deficiency and response to immunotherapy, yet most existing genomic detection methods are optimized for short-read sequencing and rely on small panels of homopolymer markers, limiting the ability to characterize genome-wide and motif-specific patterns of instability. Here we present Owl, a bioinformatic tool for quantifying MSI from long-read (PacBio) genomic data. Owl leverages a genome-wide marker set of more than 140,000 microsatellite repeats ranging from 1-6 bp in length to measure MSI across a phased genome. Using a wrap-around alignment algorithm, Owl constructs repeat-length distributions at each marker site and flags somatic instability using the coefficient of variation. We applied Owl to screen for markers with stable coverage, phasing, and baseline variation across 131 diverse genomes from the Human Pangenome Reference Consortium, where Owl scores ranged from 1.4% to 5.4% of markers exceeding the instability threshold. When applied to 19 cancer cell lines and one diffuse astrocytoma tumor-normal pair, Owl identified five MSI-high genomes with 15-18% unstable markers and showed close concordance with an Illumina DRAGEN MSI assay for the astrocytoma sample. Motif-level analyses revealed shared enrichment of short homopolymer and dinucleotide (A- and AT-rich) repeats across MSI-high cancers, and additionally uncovered a distinct pattern of elevated GGAA microsatellite instability in Ewing sarcoma cell lines, consistent with the known role of the EWS::FLI1 fusion protein at GGAA-rich regulatory elements. Owl is implemented in Rust and integrated into the PacBio HiFi Somatic workflow, providing a scalable framework for MSI analysis from long-read sequencing focused on repeat instability specifically in tumor samples.

bioinformatics↗

bioRxiv2025

Single-nuclear transcriptomics of lymphedema-associated adipose reveals a pro-lymphangiogenic stromal cell population

Chronic lymphedema is a progressive, disfiguring disease that results from dysfunction of the lymphatic vasculature, causing distal accumulation of interstitial fluid, localized development of tissue edema, and expansion of subcutaneous adipose tissue (SAT). As the molecular mechanisms governing SAT remodeling in this disease are unclear, we performed single-nucleus RNA sequencing on paired control and affected SAT biopsies from patients with unilateral lymphedema. Lymphedema samples were characterized by expansion of SAA+ adipocytes, pro-adipogenic stem cells, and proliferation of lymphatic capillaries. A GRIA1+ lymphedema-enriched stromal cell population expressing VEGFC, ADAMTS3, and CCBE1 was identified, suggesting an enhanced axis of communication between adipose stem and progenitor cells (ASPCs) and lymphatic endothelial cells. Furthermore, lymphedema ASPC-conditioned media promoted lymphatic endothelial tube elongation and proliferation in vitro. These findings indicate a critical role for ASPCs in regulating adipocyte differentiation and lymphatic vascular remodeling in lymphedema, and provide a valuable resource for better understanding this disease.

cell biology↗

bioRxiv2024

Genetic and Epigenetic Characterization of Sarcoma Stem Cells Across Subtypes Identifies EZH2 as a Therapeutic Target

High-grade complex karyotype soft tissue sarcomas (STS) are a heterogeneous and aggressive set of cancers that share a common treatment strategy. Disease progression and failure to respond to anthracycline based chemotherapy, standard first-line treatment, is associated with poor patient outcomes. To address this, we investigated the contribution of STS cancer stem cells (STS-CSCs) to doxorubicin resistance. We identified a positive correlation between CSC abundance and doxorubicin IC50 in resistant cell lines. We investigated if a common genetic signature across STS-CSCs could be targeted. Utilizing patient derived samples from five sarcoma subtypes we identified Enhancer of Zeste homolog 2 (EZH2), a member of the polycomb repressive complex 2 (PRC2) responsible for H3K27 methylation as being enriched in the CSC population. EZH2 activity and a shared epigenetic profile was observed across subtypes. Targeting of EZH2 using Tazemetostat, an FDA approved inhibitor specifically ablated the STS-CSC population. Treatment of doxorubicin resistant cell lines with tazemetostat resulted in a decrease in the STS-CSC population. Further, co-treatment was not only synergistic in the parent cell lines, but restored chemosensitivity in doxorubicin resistant lines. These data confirm the presence of shared genetic programs across distinct subtypes of CSC-STS that can be therapeutically targeted.

cancer biology↗

bioRxiv2024

Peripheral mutations underlie promiscuous transport of quaternary ammonium antiseptics by Small Multidrug Resistance transporters

The mechanistic basis of transport promiscuity in multidrug exporters is not well understood. We examine this question using the Small Multidrug Resistance (SMR) transporters. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this new activity. Using x-ray crystallography, electrophysiology, and a novel proteoliposome-based antiseptic transport assay, we tease apart the mechanistic roles that these residues play in transport polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate, but through mutations peripheral to the binding pocket. Our new molecular insights into substrate promiscuity among the SMRs can be applied to understand multidrug export and the evolution of novel transport functions more generally.

biochemistry↗

bioRxiv2022

Transformation of Human Mesenchymal Stem Cells into High-Grade Sarcomas by YAP1 and K-RAS Reflects the Undifferentiated Pleomorphic Sarcoma-Myxofibrosarcoma Disease Spectrum

High-grade complex karyotype sarcomas are a heterogeneous group of more than seventy tumors that vary in histology, clinical course, and patient demographics. Despite these clear differences, these high-grade sarcomas are treated similarly with a uniformly high metastatic rate. Pre-clinical models that allow for rigorous comparisons of distinct human sarcoma subtypes would advance insights into the relationships between sarcomas and inform therapeutic decisions. We describe the robust transformation of human mesenchymal stem cells into multiple subtypes of high-grade sarcoma. Using a pooled genetic screening approach, we identified key drivers and potential modifiers of transformation. YAP1 and KRAS were validated as drivers of two distinct sarcoma subtypes, undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma (MFS), respectively. In addition, the pathology of tumors driven by CDK4 and PIK3CA reflected leiomyosarcoma (LMS) and osteosarcoma (OS) indicating that further iterations of this model could result in additional sarcoma subtypes. Histologically and phenotypically these tumors reflect human sarcomas including the pathognomonic complex karyotype. In addition, CDK4 and PIK3CA driven tumors demonstrated endogenous YAP1 amplification which is seen across a subset of human tumors. While all tumors overlapped transcriptionally with the TCGA sarcoma data, further analysis confirmed that YAP1 and KRAS tumors recapitulate the UPS and MFS subtypes. Co-analysis of TCGA and model tumors support that these sarcoma subtypes lie along a spectrum of disease and adds guidance for further transcriptome-based refinement of sarcoma subtyping. Within complex karyotype sarcomas, there are multiple genetic changes but identifying those that are clinically relevant has been challenging. Comparing differentially expressed genes in YAP1 and KRAS tumors to human UPS and MFS identified the enrichment of oxidative phosphorylation pathways in both YAP1 tumors and UPS. Treatment of a panel of sarcoma cell lines with the combination of an oxidative phosphorylation inhibitor and Hippo pathway inhibitor led to a significant impairment in growth identifying new therapeutic targets. A subset of human UPS tumors showed an even greater enrichment in these pathways indicating this model can be used to identify clinically relevant subtypes. This model can be used to begin to understand pathways and mechanisms driving human sarcoma development, the relationship between sarcoma subtypes and to identify and test new therapeutic vulnerabilities for this aggressive and heterogeneous disease. Statement of SignificanceWe have created the first model to study the development, growth, and metastasis of multiple human sarcoma subtypes. This system can be used as a platform to investigate sarcoma biology and identify new therapeutic targets across a heterogeneous disease.

cancer biology↗

bioRxiv2018

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy

The success of immunotherapy has led to a myriad of new clinical trials. Connected to these trials are efforts to discover biomarkers providing mechanistic insight and predictive signatures for personalization. Still, the plethora of immune monitoring technologies can face investigator bias, missing unanticipated cellular responses in limited clinical material. We here present a mass cytometry workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate human immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. The resulting assay enumerated [≥] 98% of peripheral immune cells with [≥] 4 positively identifying antigens. Robustness and reproducibility were demonstrated on multiple samples types, across research centers and by orthogonal measurements. Using automated analysis, we monitored complex immune dynamics, identifying signatures in bone-marrow transplantation associated graft-versus-host disease. This validated and available workflow ensures comprehensive immunophenotypic analysis, data comparability and will accelerate biomarker discovery in immunomodulatory therapeutics.

immunology↗