Biology subjects

Hollingsworth, E. W.

Publications and source records attributed to Hollingsworth, E. W..

4 recordsLinked to original sources

bioRxiv2023

Rapid and Quantitative Functional Interrogation of Human Enhancer Variant Activity in Live Mice

Functional analysis of non-coding variants associated with human congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice of any genetic background. We use this new technology to examine and measure the gain- and loss-of-function effects of enhancer variants linked to limb polydactyly, autism, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterize variant enhancer alleles at cellular resolution, thereby implicating candidate molecular pathways in pathogenic enhancer misregulation. We further show that independent, polydactyly-linked enhancer variants lead to ectopic expression in the same cell populations, indicating shared genetic mechanisms underlying non-coding variant pathogenesis. Finally, we streamline dual-enSERT for analysis in F0 animals by placing both reporters on the same transgene separated by a synthetic insulator. Dual-enSERT allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks.

genomics↗

bioRxiv2022

Dysregulation of The Chromatin Environment Leads to Differential Alternative Splicing as A Mechanism Of Disease In a Human Model of Autism Spectrum Disorders

Autism spectrum disorder (ASD) affects 1 in 44 children. Chromatin regulatory proteins are overrepresented among genes that contain high risk variants in ASD. Disruption of the chromatin environment leads to widespread dysregulation of gene expression, which is traditionally thought as a mechanism of disease pathogenesis associated with ASD. Alternatively, alterations in chromatin dynamics could also lead to dysregulation of alternative splicing, which is understudied as a mechanism of ASD pathogenesis. The anticonvulsant valproic acid (VPA) is a well-known environmental risk factor for ASD that acts as a class I histone deacetylase (HDAC) inhibitor. However, the precise molecular mechanisms underlying defects in human neuronal development associated with exposure to VPA are understudied. To dissect how VPA exposure and subsequent chromatin hyperacetylation influence molecular signatures involved in ASD pathogenesis, we conducted RNA sequencing (RNA-seq) in human cortical neurons that were treated with VPA. We observed that differentially expressed genes (DEGs) were enriched for mRNA splicing, mRNA processing, histone modification, and metabolism related gene sets. Furthermore, we observed widespread increase in the number and the type of alternative splicing events. Analysis of differential transcript usage (DTU) showed that exposure to VPA induces extensive alterations in transcript isoform usage across neurodevelopmentally important genes. Finally, we find that DEGs and genes that display DTU overlap with known ASD-risk genes. Together, these findings suggest that, in addition to differential gene expression, changes in alternative splicing correlated with alterations in the chromatin environment could act as an additional mechanism of disease in ASD.

neuroscience↗

bioRxiv2022

Modeling of Aryl Hydrocarbon Receptor Pathway Intrinsic Immunometabolic Role using Glioblastoma Stem Cells and Patient-Derived Organoids

The intrinsic genetic program of glioblastoma (GBM) stem cells is critical for tumor evolution and recurrence. We recently identified intrinsic phenotypes and immune-like genetic programs of GBM organoids (GBMO)1 from patient derived glioblastoma stem cells (GSCs), replicating genomic, metabolic, and cellular aspects of GBM in vivo. Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is a key regulator of infiltrating immune cells in gliomas2, 3 and associated with poor prognosis, but its role in GSC biology is unknown2. Here, we show that AHR is a patient-specific regulator of the glioma intrinsic gene program in GSCs and GSC-derived GBMO that are enriched for AHR. We find that AHR is required for GSC self-renewal, GBMO expansion, radial glia-like cell proliferation, and expression of immune mediators seen in the mesenchymal subtype. CRISPR-Cas9 genetic ablation and pharmacological inhibition revealed that AHR regulates genes linked to intrinsic immunity, proliferation, and migration in GBMO. Genomic analysis of GBMO treated with AHR inhibitors identified expression signatures and candidate markers associated with survival of gliomas. Our work defines the glioma intrinsic function of AHR in a model of early GBM formation, offering a rationale for clinical exploration of a potential two-hit target of both GBM cells and infiltrating immune cells in patients with GBM expressing high levels of AHR.

cancer biology↗

bioRxiv2021

Patient-Derived Organoids Recapitulate Intrinsic Immune Landscapes and Progenitor Populations of Glioblastoma.

Glioblastoma stem cells (GSCs) are highly self-renewing, resistant to therapy, and are able to form lethal tumors1, 2. Tumor organoids have been developed to study tumor evolution1-4, and while GSCs can form organoids for glioblastoma multiforme, our understanding of their intrinsic immune, metabolic, genetic, and molecular programs is limited. To address this, we deeply characterized GSC-derived GBM organoids using a modified protocol (GBMOsm) from several patient-derived GSCs and found they develop into complex 3D tissues with unique self-organization, cancerous metabolic states, and burdensome genetic landscapes. We discovered that GBMOsc recapitulate the presence of two important cell populations thought to drive GBM progression, SATB2+ and HOPX+ progenitors. Despite being devoid of immune cells, transcriptomic analysis across GBMOsc revealed an immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We determined that SATB2+ and HOPX+ populations contribute to this immune and interferon landscape in GBM in vivo and GBMOsm. Our work deepens our understanding of the intrinsic molecular and cellular architecture of GSC-derived GBMO and defines a novel GBMOsc intrinsic immune-like program.

neuroscience↗