Biology subjects

O'Hara, R.

Publications and source records attributed to O'Hara, R..

11 recordsLinked to original sources

bioRxiv2025

Gene dosage effects of 22q11.2 copy number variants on in-vivo measures of white matter axonal density and dispersion

22q11.2 deletion (22qDel) and duplication (22qDup) carriers have an increased risk of neurodevelopmental disorders and exhibit altered brain structure, including white matter microstructure. However, the underlying cellular architecture and age-related changes contributing to these white matter alterations remain poorly understood. Neurite orientation dispersion and density imaging (NODDI) was used on mixed cross-sectional and longitudinal data to examine group differences and age-related trajectories in measures of axonal density (i.e., intracellular volume fraction; ICVF), axonal orientation (orientation dispersion index; ODI) and free water diffusion (isotropic volume fraction; ISO) in 50 22qDel (n scans = 69, mean age = 20.7, age range = 7.4-51.1, 64.0% female) and 24 22qDup (n scans = 34, mean age = 21.6, age range = 8.3-49.4, 54.2% female) carriers, and 890 controls (n scans = 901, mean age = 21.9, age range = 7.8-51.1, 54.5% female). The results showed widespread gene dosage effects, with higher ICVF in 22qDel and lower ICVF in 22qDup compared to controls, and region-specific effects of the 22qDel and 22qDup on ODI and ISO measures. However, 22qDel and 22qDup carriers did not exhibit altered age-related trajectories relative to controls. Observed differences in ICVF suggest higher white matter axonal density in 22qDel and lower axonal density in 22qDup compared to controls. Conversely, differences in ODI are highly localized, indicating region-specific effects on axonal dispersion in white matter. We do not find evidence for altered developmental trajectories of axonal density or dispersion among 22q11.2 CNV carriers, suggesting stable disruptions to neurodevelopmental events before childhood.

neuroscience↗

bioRxiv2025

Cellular mechanisms of early brain overgrowth in autistic children: elevated levels of GPX4 and resistance to ferroptosis

Autistic individuals with disproportionate megalencephaly (ASD-DM), characterized by enlarged brains relative to body height, have higher rates of intellectual disability and face more severe cognitive challenges than autistic children with average brain sizes. The cellular and molecular mechanisms underlying this neurophenotype remain poorly understood. To investigate these mechanisms, we generated human induced pluripotent stem cells from non-autistic typically developing children and autistic children with and without disproportionate megalencephaly. We assessed these children longitudinally from ages two to twelve years using magnetic resonance imaging and comprehensive cognitive and medical evaluations. We show that neural progenitor cells (NPCs) derived from ASD-DM children exhibit increased rates of cell survival and suppressed cell death, accompanied by heightened oxidative stress and ferrous iron accumulation. Despite these stressors, ASD-DM NPCs actively suppress apoptosis and ferroptosis by regulating proteins such as caspase-3 (CASP3), poly(ADP-ribose) polymerase 1 (PARP1), and glutathione peroxidase 4 (GPX4). Cellular ferroptotic signatures are further supported by elevated expression of selenocysteine genes, including GPX4, in the blood of ASD-DM children and their mothers, suggesting potential hereditary or environmental influences. Furthermore, we show that peripheral expression of GPX4 and other selenocysteine genes correlate with cognitive outcomes (IQ). These findings underscore the role of ferroptosis in autism, pointing to potential diagnostic biomarkers and targets for intervention.

neuroscience↗

bioRxiv2024

Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts

Pediatric malignancies frequently harbor chromosomal translocations that induce expression of fusion oncoproteins. The EWSR1-FLI1 fusion oncoprotein acts as a neomorphic transcription factor and is the dominant genetic driver of Ewings sarcoma. Interrogation of the mechanisms by which EWSR1-FLI1 drives tumorigenesis has been limited by a lack of model systems to precisely and selectively control its expression in patient-derived cell lines and xenografts. Here, we report the generation of a panel of patient-derived EWS cell lines in which inducible protein degrons were engineered into the endogenous EWSR1-FLI1 locus. These alleles enabled rapid and efficient depletion of EWSR1-FLI1. Complete suppression of EWSR1-FLI1 induced a reversible cell cycle arrest at the G1-S checkpoint, and we identified a core set of transcripts downstream of EWSR1-FLI1 across multiple cell lines and degron systems. Additionally, depletion of EWSR1-FLI1 potently suppressed tumor growth in xenograft models validating efforts to directly target EWSR1-FLI1 in Ewings sarcoma.

cancer biology↗

bioRxiv2024

Developmental convergence and divergence in human stem cell models of autism spectrum disorder

Two decades of genetic studies in autism spectrum disorder (ASD) have identified over a hundred genes harboring rare risk mutations. Despite this substantial heterogeneity, transcriptomic and epigenetic analyses have identified convergent patterns of dysregulation across ASD post-mortem brain tissue. To identify shared and distinct mutational mechanisms, we assembled the largest hiPS cell patient cohort to date, consisting of 70 hiPS cell lines after stringent quality control representing 8 ASD-associated mutations, idiopathic ASD, and 20 lines from non-affected controls. We used these hiPS lines to generate human cortical organoids (hCO), profiling by RNAseq at four distinct timepoints up to 100 days of in vitro differentiation. Early timepoints harbored the largest mutation-specific changes, but different genetic forms converged on shared transcriptional changes as development progressed. We identified a shared RNA and protein interaction network, which was enriched in ASD risk genes and predicted to drive the observed down-stream changes in gene expression. CRISPR-Cas9 screening of these candidate transcriptional regulators in induced human neural progenitors validated their downstream molecular convergent effects. These data illustrate how genetic risk can propagate via transcriptional regulation to impact convergently dysregulated pathways, providing new insight into the convergent impact of ASD genetic risk on human neurodevelopment.

neuroscience↗

bioRxiv2024

TASOR expression in naive embryonic stem cells safeguards their developmental potential

The seamless transition through stages of pluripotency relies on a delicate balance between transcription factor networks and epigenetic silencing mechanisms that ensure proper regulation of the developmental program, critical for normal development. Here, we uncover the pivotal role of the transgene activation suppressor (TASOR), a component of the human silencing hub (HUSH) complex, in sustaining cell viability during the transition from naive to primed pluripotency, despite its rapid downregulation during this transition. Loss of TASOR in naive cells triggers replication stress, disrupts H3K9me3 heterochromatin formation, and compromise the transcriptional and post-transcriptional silencing of LINE-1 (L1) transposable elements (TEs), with these effects become more pronounced in primed cells. Remarkably, the survival of Tasor-knockout cells during naive to primed transition can be restored through the inhibition of cysteine-aspartic acid protease (Caspase) or deletion of mitochondrial antiviral signaling protein (MAVS). This suggests that unscheduled L1 expression activates an innate immune response, leading to programmed cell death, specifically in cells exiting naive pluripotency. Additionally, we propose that HUSH-promoted H3K9me3 in naive PSCs sets the stage for ensuing DNA methylation in primed cells, establishing long-term silencing during differentiation. Our findings shed insights on the crucial impact of epigenetic programs established in early developmental stages on subsequent phases, underscoring their significance in the developmental process.

developmental biology↗

bioRxiv2022

PointedSDMs -- an R package to help facilitate the construction of integrated species distribution models

1O_LIIntegration of disparate datasets is a rapidly growing area in quantitative ecology, and is subsequently becoming a major asset in understanding the shifts and trends in species distributions. C_LIO_LIHowever, the tools and software available to construct statistical models to integrate these disparate datasets into a unified framework is lacking, stagnating the growth of data integration in applications. C_LIO_LIThis paper presents PointedSDMs: an easy to use R package used to construct integrated species distribution models using the integrated nested Laplace Approximation methodology. C_LIO_LIThe package is presented in this paper through an illustrative example for a selection of species found across Pennsylvania state. C_LI

ecology↗

bioRxiv2022

H3.3 contributes to chromatin accessibility and transcription factor binding at promoter-proximal regulatory elements

BackgroundThe histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood. ResultsWe performed genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and found evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 deposition at promoters reduces chromatin accessibility and transcription factor (TF) footprinting for nearly all TFs expressed in ESCs. H3.3 deletion leads to reduced promoter enrichment of the transcriptional coactivator and histone acetyltransferase, p300. Subsequently, histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the bromodomain-containing protein BRD4, an acetyl lysine reader. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TFs motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate. ConclusionsH3.3 facilitates DNA accessibility, TF binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it is required for TF binding at new promoters during differentiation.

genomics↗

bioRxiv2022

Loss of heterochromatin at endogenous retroviruses creates competition for transcription factor binding

The mammalian genome is partitioned into active and inactive regions, broadly termed euchromatin and heterochromatin, respectively. The majority of heterochromatin consists of repetitive elements, including endogenous retroviruses (ERVs). ERVs are enriched in regulatory elements containing transcription factor (TF) binding sites with individual families containing hundreds to thousands of distinct copies scattered throughout the genome. We hypothesized that epigenetic derepression of ERVs (such as that observed during early development) may alter the stoichiometry between TFs and their euchromatic target sites, with ERVs effectively competing for these factors. To test this, we modeled acute heterochromatin loss using inducible deletion of the co-repressor KAP1 in mouse embryonic stem cells (ESCs). Upon KAP1 deletion, we observe clear reductions in chromatin accessibility, histone acetylation, and TF binding at euchromatic regions. To directly test the concept of global binding site competition, we designed exogenous binding site arrays (EBSAs) to introduce upwards of 1500 copies of the OCT4 TF binding motif into ESCs. OCT4 EBSAs specifically reduce chromatin accessibility at POU family motifs and result in reduced transcription of the pluripotency machinery with subsequent differentiation. Overall, these data support a model in which heterochromatin at ERVs promotes euchromatic TF binding and transcriptional homoeostasis. We propose that regulated ERV derepression during pre-implantation may serve as a developmental siphon to weaken the robustness of ongoing transcription programs in favor of the plasticity required for cell fate specification.

genomics↗

bioRxiv2021

ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with the MCM replication complex and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

molecular biology↗

bioRxiv2020

Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19

Our understanding of protective vs. pathologic immune responses to SARS-CoV-2, the virus that causes Coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses reveal widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, with the most profound disturbances including a prominent neutrophil hyperactivation signature and monocytes with anti-inflammatory features. We further demonstrate that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention. One Sentence SummarySingle-cell profiling demonstrates multifarious dysregulation of innate immune phenotype associated with COVID-19 severity.

immunology↗

bioRxiv2019

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single cell resolution: a focus on aging and cancer

Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation and motility as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. Importantly, mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method (ddMDM; droplet digital mitochondrial DNA measurement) to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 hours, is optimized for 96-well plates and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. Importantly, we show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.

molecular biology↗