Cell Type-specific Gene Expression Research Articles

Adult sex change leads to extensive forebrain reorganization in clownfish

BackgroundSexual differentiation of the brain occurs in all major vertebrate lineages but is not well understood at a molecular and cellular level. Unlike most vertebrates, sex-changing fishes have the remarkable ability to change reproductive sex during adulthood in response to social stimuli, offering a unique opportunity to understand mechanisms by which the nervous system can initiate and coordinate sexual differentiation.MethodsThis study explores sexual differentiation of the forebrain using single nucleus RNA-sequencing in the anemonefish Amphiprion ocellaris, producing the first cellular atlas of a sex-changing brain.ResultsWe uncover extensive sex differences in cell type-specific gene expression, relative proportions of cells, baseline neuronal excitation, and predicted inter-neuronal communication. Additionally, we identify the cholecystokinin, galanin, and estrogen systems as central molecular axes of sexual differentiation. Supported by these findings, we propose a model of sexual differentiation in the conserved vertebrate social decision-making network spanning multiple subtypes of neurons and glia, including neuronal subpopulations within the preoptic area that are positioned to regulate gonadal differentiation.ConclusionsThis work deepens our understanding of sexual differentiation in the vertebrate brain and defines a rich suite of molecular and cellular pathways that differentiate during adult sex change in anemonefish.

The myelin water imaging transcriptome: myelin water fraction regionally varies with oligodendrocyte-specific gene expression

Identifying sensitive and specific measures that can quantify myelin are instrumental in characterizing microstructural changes in neurological conditions. Neuroimaging transcriptomics is emerging as a valuable technique in this regard, offering insights into the molecular basis of promising candidates for myelin quantification, such as myelin water fraction (MWF). We aimed to demonstrate the utility of neuroimaging transcriptomics by validating MWF as a myelin measure. We utilized data from a normative MWF brain atlas, comprised of 50 healthy subjects (mean age = 25 years, range = 17–42 years) scanned at 3 Tesla. Magnetic resonance imaging data included myelin water imaging to extract MWF and T1 anatomical scans for image registration and segmentation. We investigated the inter-regional distributions of gene expression data from the Allen Human Brain Atlas in conjunction with inter-regional MWF distribution patterns. Pearson correlations were used to identify genes with expression profiles mirroring MWF. The Single Cell Type Atlas from the Human Protein Atlas was leveraged to classify genes into gene sets with high cell type specificity, and a control gene set with low cell type specificity. Then, we compared the Pearson correlation coefficients for each gene set to determine if cell type-specific gene expression signatures correlate with MWF. Pearson correlation coefficients between MWF and gene expression for oligodendrocytes and adipocytes were significantly higher than for the control gene set, whereas correlations between MWF and inhibitory/excitatory neurons were significantly lower. Our approach in integrating transcriptomics with neuroimaging measures supports an emerging technique for understanding and validating MRI-derived markers such as MWF.

Enhanced in situ spatial proteomics by effective combination of MALDI imaging and LC-MS/MS

Highly specialized cells are fundamental for proper functioning of complex organs. Variations in cell-type specific gene expression and protein composition have been linked to a variety of diseases. Investigation of the distinctive molecular makeup of these cells within tissues is therefore critical in biomedical research. Although several technologies have emerged as valuable tools to address this cellular heterogeneity, most workflows lack sufficient in situ resolution and are associated with high cost and extremely long analysis times. Here, we present a combination of experimental and computational approaches that allows a more comprehensive investigation of molecular heterogeneity within tissues than by either shotgun LC-MS/MS or MALDI imaging alone. We applied our pipeline on mouse brain, which contains a wide variety of cell types that not only perform unique functions but also exhibit varying sensitivities to insults. We explored the distinct neuronal populations within the hippocampus, a brain region crucial for learning and memory that is involved in various neurological disorders. As an example, we identified the groups of proteins distinguishing the neuronal populations of dentate gyrus (DG) and the cornu ammonis (CA) in the same brain section. Most of the annotated proteins matched the regional enrichment of their transcripts, thereby validating the method. As the method is highly reproducible, the identification of individual masses through the combination of MALDI-IMS and LC-MS/MS methods can be used for the much faster and more precise interpretation of MALDI-IMS measurements only. This greatly speeds up spatial proteomic analyses and allows the detection of local protein variations within the same population of cells. The method's general applicability has the potential to be used to investigate different biological conditions and tissues and a much higher throughput than other techniques making it a promising approach for clinical routine applications.

Molecular basis of neurodegeneration in a mouse model of Polr3-related disease.

Pathogenic variants in subunits of RNA polymerase (Pol) III cause a spectrum of Polr3-related neurodegenerative diseases including 4H leukodystrophy. Disease onset occurs from infancy to early adulthood and is associated with a variable range and severity of neurological and non-neurological features. The molecular basis of Polr3-related disease pathogenesis is unknown. We developed a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes. Polr3a mutant mice exhibit behavioral deficits, cerebral pathology and exocrine pancreatic atrophy. Transcriptome and immunohistochemistry analyses of cerebra during disease progression show a reduction in most Pol III transcripts, induction of innate immune and integrated stress responses and cell type-specific gene expression changes reflecting neuron and oligodendrocyte loss and microglial activation. Earlier in the disease when integrated stress and innate immune responses are minimally induced, mature tRNA sequencing revealed a global reduction in tRNA levels and an altered tRNA profile but no changes in other Pol III transcripts. Thus, changes in the size and/or composition of the tRNA pool have a causal role in disease initiation. Our findings reveal different tissue- and brain region-specific sensitivities to a defect in Pol III transcription.

Deciphering cellular heterogeneity in Spodoptera frugiperda midgut cell line through single cell RNA sequencing

Using the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) platform, we discovered unexpected heterogeneity in an established cell line developed from the midgut of the Fall armyworm, Spodoptera frugiperda, a major global pest. We analyzed the sequences of 18,794 cells and identified ten unique cellular clusters, including stem cells, enteroblasts, goblet cells and enteroendocrine cells, characterized by the expression of specific marker genes. Additionally, these studies addressed an important knowledge gap by investigating the expression of genes coding for respiratory and midgut membrane insecticide targets classified by the Insecticide Resistance Action Committee. Dual-fluorescence tagging method, fluorescence microscopy and fluorescence-activated cell sorting confirmed the expression of midgut cell type-specific genes. Stem cells were isolated from the heterogeneous population of SfMG-0617 cells. Our results, validated by KEGG and Gene Ontology analyses and supported by Monocle 3.0, advance the fields of midgut cellular biology and establish standards for scRNA-seq studies in non-model organisms.

Dan forms condensates in neuroblasts and regulates nuclear architecture and progenitor competence in vivo

Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In Drosophila, a developmentally-timed genome reorganization in neural progenitors terminates competence to produce early-born neurons. These events require downregulation of Distal antenna (Dan), part of the conserved pipsqueak DNA-binding superfamily. Here we find that Dan forms liquid-like condensates with high protein mobility, and whose size and subnuclear distribution are balanced with its DNA-binding. Further, we identify a LARKS domain, a structural motif associated with condensate-forming proteins. Deleting just 13 amino acids from LARKS abrogates Dan’s ability to retain the early-born neural fate gene, hunchback, in the neuroblast nuclear interior and maintain competence in vivo. Conversely, domain-swapping with LARKS from known phase-separating proteins rescues Dan’s effects on competence. Together, we provide in vivo evidence for condensate formation and the regulation of progenitor nuclear architecture underlying neuronal diversification.

FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement.

Long-range glutamatergic inputs originating from the cortex and thalamus are indispensable for striatal development, providing the foundation for motor and cognitive functions. Despite their significance, transcriptional regulation governing these inputs remains largely unknown. We investigated the role of a transcription factor encoded by a high-risk autism-associated gene, FOXP1, in sculpting glutamatergic inputs onto spiny projection neurons (SPNs) within the striatum. We find a neuron subtype-specific role of FOXP1 in strengthening and maturing glutamatergic inputs onto dopamine receptor 2-expressing SPNs (D2 SPNs). We also find that FOXP1 promotes synaptically driven excitability in these neurons. Using single-nuclei RNA sequencing, we identify candidate genes that mediate these cell-autonomous processes through postnatal FOXP1 function at the post-synapse. Last, we demonstrate that postnatal FOXP1 reinstatement rescues electrophysiological deficits, cell type-specific gene expression changes, and behavioral phenotypes. Together, this study enhances our understanding of striatal circuit development and provides proof of concept for a therapeutic approach for FOXP1 syndrome and other neurodevelopmental disorders.

Linking Altered Nephrogenesis with Hypertension using Single Nuclei RNA Sequencing of Fetal Kidney at E15.5, E16.5 and E17.5 in a Novel Nephron Deficient Rat Model

This study sought to elucidate molecular mechanisms linking altered nephrogenesis in the HSRA rat, a novel model of nephron deficiency, to the development of hypertension and chronic kidney disease (CKD) later in life. The HSRA model gives rise to offspring born with a single kidney (-S) 50-75% of the time. The single kidneys from HSRA-S animals exhibit reduced nephron endowment compared to either kidney from two-kidney offspring (-C). We hypothesize that early molecular factors during development contribute to the observed reduction in nephron number in -S animals. Timed breeding and embryonic kidney isolation from -S and -C animals were performed at gestational days (GD) 15.5, 16.5, or 17.5. Single nuclei RNA sequencing (snRNA-seq) was conducted using n=3 -S kidneys and n=3 -C kidneys for each timepoint. The library was prepared using a 10x Chromium Single Cell 3’ kit, followed by sequencing on an Illumina NextSeq2000. The average number of cells captured per sample was 16,997, yielding an average of 25,335 reads/cell, with a median of 1,512 genes/cell identified. Bioinformatic analysis using Cell Ranger and Seurat (v4) revealed various renal progenitor cell types including metanephric mesenchymal (MM) and ureteric bud cells, alongside tubular, stromal, and glomerular endothelial progenitors. Podocytes emerged by GD17.5, originating from the MM cell population, as evidenced by their extension from this cluster. Global differential expression analysis revealed minimal transcriptional differences between -S and -C fetal kidneys at GD 16.5 and 17.5. However, a down-regulation of ribosomal genes was observed on GD15.5 in the -S group, suggesting premature cellular differentiation contributing to the diminished nephron formation. Markedly, cell type-specific gene expression differences were observed at GD 16.5 and 17.5, highlighting the superior resolution that snRNAseq provides compared to bulk RNA sequencing methods. We are currently performing trajectory analysis using Monocle3 to investigate cellular fate pathways and uncover genes involved during nephron progenitor cell differentiation. These preliminary findings shed light on the cellular and molecular landscape during critical developmental stages in the kidney of the HSRA rat that could link developmental factors affecting nephron number to hypertension and CKD pathologies later in life. This research was generously supported by the Graduate School of Health Sciences at UMMC. MRG is supported by NIH R01HL137673. The UMMC Molecular and Genomics Facility is supported, in part, by funds from the NIGMS, including Molecular Center of Health and Disease-COBRE (P20GM144041-Garrett), Mississippi INBRE (P20GM103476-Flynt), and Cardiorenal and Metabolic Diseases-COBRE (P20GM104357-Hall). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DISSECT: deep semi-supervised consistency regularization for accurate cell type fraction and gene expression estimation.

Cell deconvolution is the estimation of cell type fractions and cell type-specific gene expression from mixed data. An unmet challenge in cell deconvolution is the scarcity of realistic training data and the domain shift often observed in synthetic training data. Here, we show that two novel deep neural networks with simultaneous consistency regularization of the target and training domains significantly improve deconvolution performance. Our algorithm, DISSECT, outperforms competing algorithms in cell fraction and gene expression estimation by up to 14 percentage points. DISSECT can be easily adapted to other biomedical data types, as exemplified by our proteomic deconvolution experiments.

Identifying tumor type and cell type-specific gene expression alterations in pediatric central nervous system tumors.

Central nervous system (CNS) tumors are the leading cause of pediatric cancer death, and these patients have an increased risk for developing secondary neoplasms. Due to the low prevalence of pediatric CNS tumors, major advances in targeted therapies have been lagging compared to other adult tumors. We collect single nuclei RNA-seq data from 84,700 nuclei of 35 pediatric CNS tumors and three non-tumoral pediatric brain tissues and characterize tumor heterogeneity and transcriptomic alterations. We distinguish cell subpopulations associated with specific tumor types including radial glial cells in ependymomas and oligodendrocyte precursor cells in astrocytomas. In tumors, we observe pathways important in neural stem cell-like populations, a cell type previously associated with therapy resistance. Lastly, we identify transcriptomic alterations among pediatric CNS tumor types compared to non-tumor tissues, while accounting for cell type effects on gene expression. Our results suggest potential tumor type and cell type-specific targets for pediatric CNS tumor treatment. Here we address current gaps in understanding single nuclei gene expression profiles of previously under-investigated tumor types and enhance current knowledge of gene expression profiles of single cells of various pediatric CNS tumors.

Mitochondrial complex I deficiency stratifies idiopathic Parkinson’s disease

Idiopathic Parkinson’s disease (iPD) is believed to have a heterogeneous pathophysiology, but molecular disease subtypes have not been identified. Here, we show that iPD can be stratified according to the severity of neuronal respiratory complex I (CI) deficiency, and identify two emerging disease subtypes with distinct molecular and clinical profiles. The CI deficient (CI-PD) subtype accounts for approximately a fourth of all cases, and is characterized by anatomically widespread neuronal CI deficiency, a distinct cell type-specific gene expression profile, increased load of neuronal mtDNA deletions, and a predilection for non-tremor dominant motor phenotypes. In contrast, the non-CI deficient (nCI-PD) subtype exhibits no evidence of mitochondrial impairment outside the dopaminergic substantia nigra and has a predilection for a tremor dominant phenotype. These findings constitute a step towards resolving the biological heterogeneity of iPD with implications for both mechanistic understanding and treatment strategies.

Single-cell transcriptomics unveiled that early life BDE-99 exposure reprogrammed the gut-liver axis to promote a proinflammatory metabolic signature in male mice at late adulthood.

Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants that bioaccumulate in the environment. The gut microbiome is an important regulator of liver functions including xenobiotic biotransformation and immune regulation. We recently showed that neonatal exposure to polybrominated diphenyl ether-99 (BDE-99), a human breast milk-enriched PBDE congener, up-regulated proinflammation-related and down-regulated drug metabolism-related genes predominantly in males in young adulthood. However, the persistence of this dysregulation into late adulthood, differential impact among hepatic cell types, and the involvement of the gut microbiome from neonatal BDE-99 exposure remain unknown. To address these knowledge gaps, male C57BL/6 mouse pups were orally exposed to corn oil (10 ml/kg) or BDE-99 (57 mg/kg) once daily from postnatal days 2-4. At 15 months of age, neonatal BDE-99 exposure down-regulated xenobiotic and lipid-metabolizing enzymes and up-regulated genes involved in microbial influx in hepatocytes. Neonatal BDE-99 exposure also increased the hepatic proportion of neutrophils and led to a predicted increase of macrophage migration inhibitory factor signaling. This was associated with decreased intestinal tight junction protein (Tjp) transcripts, altered gut environment, and dysregulation of inflammation-related metabolites. ScRNA-seq using germ-free (GF) mice demonstrated the necessity of a normal gut microbiome in maintaining hepatic immune tolerance. Microbiota transplant to GF mice using large intestinal microbiome from adults neonatally exposed to BDE-99 down-regulated Tjp transcripts and up-regulated several cytokines in large intestine. In conclusion, neonatal BDE-99 exposure reprogrammed cell type-specific gene expression and cell-cell communication in liver towards proinflammation, and this may be partly due to the dysregulated gut environment.

Identifying gene expression programs in single-cell RNA-seq data using linear correlation explanation

ObjectiveGene expression analysis through single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of gene regulation in diverse cell types, tissues, and organisms. While existing methods primarily focus on identifying cell type-specific gene expression programs (GEPs), the characterization of GEPs associated with biological processes and stimuli responses remains limited. In this study, we aim to infer biologically meaningful GEPs that are associated with both cellular phenotypes and activity programs directly from scRNA-seq data. MethodsWe applied linear CorEx, a machine-learning-based approach, to infer GEPs by grouping genes based on total correlation optimization function in simulated and real-world scRNA-seq datasets. Additionally, we utilized a transfer learning approach to project CorEx-inferred GEPs to other scRNA-seq datasets. ResultsBy leveraging total correlation optimization, linear CorEx groups genes and demonstrates superior performance in identifying cell types and activity programs compared to similar methods using simulated data. Furthermore, we apply this same approach to real-world scRNA-seq data from the mouse dentate gyrus and embryonic colon development, uncovering biologically relevant GEPs related to cell types, developmental ages, and cell cycle programs. We also demonstrate the potential for transfer learning by evaluating similar datasets, showcasing the cross-species sensitivity of linear CorEx. ConclusionOur findings validate linear CorEx as a valuable tool for comprehensively analyzing complex signals in scRNA-seq data, leading to deeper insights into gene expression dynamics, cellular heterogeneity, and regulatory mechanisms.

Metacell-based differential expression analysis identifies cell type specific temporal gene response programs in COVID-19 patient PBMCs

By profiling gene expression in individual cells, single-cell RNA-sequencing (scRNA-seq) can resolve cellular heterogeneity and cell-type gene expression dynamics. Its application to time-series samples can identify temporal gene programs active in different cell types, for example, immune cells’ responses to viral infection. However, current scRNA-seq analysis has limitations. One is the low number of genes detected per cell. The second is insufficient replicates (often 1-2) due to high experimental cost. The third lies in the data analysis—treating individual cells as independent measurements leads to inflated statistics. To address these, we explore a new computational framework, specifically whether “metacells” constructed to maintain cellular heterogeneity within individual cell types (or clusters) can be used as “replicates” for increasing statistical rigor. Toward this, we applied SEACells to a time-series scRNA-seq dataset from peripheral blood mononuclear cells (PBMCs) after SARS-CoV-2 infection to construct metacells, and used them in maSigPro for quadratic regression to find significantly differentially expressed genes (DEGs) over time, followed by clustering expression velocity trends. We showed that such metacells retained greater expression variances and produced more biologically meaningful DEGs compared to either metacells generated randomly or from simple pseudobulk methods. More specifically, this approach correctly identified the known ISG15 interferon response program in almost all PBMC cell types and many DEGs enriched in the previously defined SARS-CoV-2 infection response pathway. It also uncovered additional and more cell type-specific temporal gene expression programs. Overall, our results demonstrate that the metacell-pseudoreplicate strategy could potentially overcome the limitation of 1-2 replicates.

272 Integrative Genomics Implicates Primary Impact on Cortical Development in Spina Bifida

INTRODUCTION: Spina bifida is one of the most common malformation of the central nervous system that is associated with poor neurodevelopmental outcomes. Altered neurodevelopment in spina bifida has been attributed to venting of CSF through the neural tube defect. Despite correction of the spinal defect, some patients still develop hydrocephalus with poor neurodevelopmental outcomes, suggesting incomplete understanding of disease pathogenesis. Recent human genetic efforts have identified candidate genes that confer spina bifida risk, however, the mechanisms linking gene mutations to altered neurodevelopment remain poorly understood. METHODS: We curated a list of 439 genes that confer risk to spina bifida from a whole-genome sequencing study of 310 individuals. We then mapped the spatiotemporal and cell-type specific expression of these risk genes onto bulk and single-cell RNA sequencing data of normally developing human brains covering the entire lifespan of human development. RESULTS: We found that spina bifida risk genes are enriched at post-conception weeks 5-6 and 25-38 in the developing human brain. After birth, spina bifida risk genes are enriched at postnatal months 6-10 and then after 60 years of age. At the cell type level, spina bifida risk genes are enriched in proliferative neuroprogenitor cells and microglia in the prenatal human cortex. CONCLUSIONS: Our analyses show that spina bifida risk genes are expressed in the developing human brain, suggesting a primary impact on brain development that is independent from the open caudal end of the neural tube defect. The finding that spina bifida risk genes continue to be expressed postnatally suggests a window for therapeutic intervention to optimize neurodevelopmental outcomes after birth in at least a subset of patients.

Abstract 1243: Shifted mSWI/SNF complex assembly and function underlie therapeutically targetable dependencies in endometrial carcinoma

Abstract The mammalian SWI/SNF (mSWI/SNF) family of chromatin remodelers govern cell type-specific chromatin accessibility and gene expression and assemble as three distinct complexes: canonical BAF (cBAF), Polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF). ARID1A and ARID1B are paralog subunits that specifically nucleate the assembly of cBAF complexes and are the most frequently mutated mSWI/SNF components in human cancer, including in endometrial cancer. However, the biochemical and functional impacts of ARID1A/B loss, and hence cBAF complex disruption, on remaining PBAF and ncBAF complex activities, chromatin architecture, and gene expression remain poorly understood. Here, we define the molecular consequences of complete cBAF loss in models of aggressive de-differentiated endometrial carcinoma (DEC) harboring dual ARID1A/ARID1B mutations. Notably, re-expression of ARID1A in DEC cellular model systems biochemically restores cBAF complex assembly while reducing that of PBAF and ncBAF complexes, implicating functional contributions of differential mSWI/SNF family stoichiometry. We observe genome-wide increases in DNA accessibility at distal enhancer regions following ARID1A rescue, coupled with transcriptional changes at nearest genes including those associated with epithelial lineage differentiation, estrogen receptor signaling, and PI3K/AKT pathway signaling. Importantly, differential gene regulatory changes aligned with simultaneous cBAF restoration and reduction in ncBAF, and PBAF complex function mirror signatures derived from well-differentiated (WEC) and dedifferentiated (DEC) compartments of a collection of n= 15 primary human DEC cases profiled. Reduced ncBAF- and PBAF-mediated chromatin regulation resulting from either ARID1A rescue or ncBAF/PBAF genetic disruption attenuated oncogenic gene signature features such as MYC target genes, cell cycle checkpoint and DNA repair pathways and significantly reduced proliferative capacity. Finally, treatment of ARID1A/1B-mutant and ARID1A-mutant endometrial cell lines in culture and PDX models in vivo with a clinical-grade SMARCA4/2 ATPase inhibitor, FHD-286, markedly attenuated both cell proliferation and tumor growth. Further, FHD-286 synergized with carboplatin, leading to significant reduction in tumor burden in ARID1A- and ARID1A/B-mutant PDX models. Taken together, these findings reveal the oncogenic contributions of shifted of mSWI/SNF family complex abundance and chromatin-level gene regulatory function and suggest therapeutic utility of mSWI/SNF complex small molecule inhibitors in endometrial carcinoma and other cBAF-disrupted cancer types. Citation Format: Jessica Diane St. Laurent, Grace Xu, Alexander Ying, Bengul Gokbayrak, Ajinkya Patil, Akshay Sankar, Chae Young Shin, Daniel Same Guerra, David Kolin, David Huntsman, Yemin Wang, Cigall Kadoch. Shifted mSWI/SNF complex assembly and function underlie therapeutically targetable dependencies in endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1243.

Beyond Quiescent and Active: Intermediate Microglial Transcriptomic States in a Mouse Model of Down Syndrome.

Research on microglia in Down syndrome (DS) has shown that microglial activation, increased inflammatory gene expression, and oxidative stress occur at different ages in DS brains. However, most studies resulted in simplistic definitions of microglia as quiescent or active, ignoring potential intermediate states. Indeed, recent work on microglial cells in young DS brains indicated that those evolve through different intermediate activation phenotypes before reaching a fully activated state. Here we used single nucleus RNA sequencing, to examine how trisomy affects microglial states in the Ts65Dn mouse model of DS. Despite no substantial changes in the proportion of glial populations, differential expression analysis revealed cell type-specific gene expression changes, most notably in astroglia, microglia, and oligodendroglia. Focusing on microglia, we identified differential expression of genes associated with different microglial states, including disease-associated microglia (DAMs), activated response microglia (ARMs), and human Alzheimer's disease microglia (HAMs), in trisomic microglia. Furthermore, pseudotime analysis reveals a unique reactivity profile in Ts65Dn microglia, with fewer in a homeostatic state and more in an intermediate aberrantly reactive state than in euploid microglia. This comprehensive understanding of microglial transcriptional dynamics sheds light on potential pathogenetic mechanisms but also possible avenues for therapy for neurodevelopmental disorders.

Abstract B039: Identification of sex-specific molecular features in glioma transcriptome

Abstract Glioma is a lethal disease with limited treatment options available. Sex differences in glioma are well-documented in clinical settings, with males having twice the risk of developing the disease and increased mortality compared to female counterparts. To investigate the molecular basis of sex differences in glioma, we analyzed transcriptomic sex differences in a collection of three independent glioma datasets and validated them in additional independent studies. The impact of differentially expressed genes were assessed through Gene Set Enrichment Analysis (GSEA) on approximately 5000 gene sets to interpret their biological relevance. Given the well-recognized heterogeneity of glioma, we also integrated three single-cell RNA (scRNA) sequencing datasets to decipher the sex differences in cellular composition and cell-type specific differential gene expression between male and female tumors. The effects of sex on gene expression are modest and primarily driven by the X chromosome (14 out of 16 differentially expressed genes were on the X chromosome). These genes are associated with escape from X-chromosome inactivation (e.g., KDM5C, KDM6A) and are also sex-differentially expressed (DE) in the healthy human brain according to Genotype-Tissue Expression (GTEx) data. GSEA results suggest increased activity in cell proliferation and neuronal signaling in male GBM samples, whereas females show enrichment in glycoprotein/lipid metabolism. By deconvoluting the cell-type composition, we identified a higher percentage of pericytes in the male glioma tumor microenvironment compared to those in females, which is consistent with recent spatial-omic discoveries in glioma. By integrating scRNA sequencing data, we found that both differentially expressed genes and pathways show cell-type-specific enrichment. For example, the female-biased gene GBGT1 is exclusively expressed in the myeloid lineage, while male-biased neuronal pathways are enriched in cancer stem cells. Integrating with the inference of cell trajectories, we observed that male tumors harbor more unconnected, invasive malignant cells than females, which might explain the poor prognosis in male glioma patients. In summary, our results provide a comprehensive multi-level characterization of sex differences in glioma tumors, and these findings will provide novel insights into glioma disease progression and development of new therapy. Citation Format: Yingbo Huang, Yuting Shan, Weijie Zhang, Christina Printzis, Lorenzo Pesce, Barbara Elaine Stranger, R. Stephanie Huang. Identification of sex-specific molecular features in glioma transcriptome [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B039.

Abstract PR006: ARID1A/B mutations retarget mSWI/SNF chromatin remodeler activity and define a spectrum of dedifferentiation in endometrial carcinoma

Abstract Dedifferentiated/undifferentiated endometrial carcinoma (DEC/UEC) is a highly aggressive histologic subtype of uterine cancer with a higher incidence among non-Hispanic Black women. Recent exome-wide sequencing studies have revealed frequent and often concomitant mutations in the ARID1A and ARID1B genes in over 50% of DEC/UEC cases and 30% of all endometrial carcinomas. The mSWI/SNF family of chromatin remodelers are critical regulators of cell type-specific chromatin accessibility and gene expression, existing in three distinct forms: canonical BAF (cBAF), Poly bromodomain-associated BAF (PBAF), and non-canonical BAF (ncBAF). The paralog subunits, ARID1A and ARID1B, specifically nucleate the assembly of cBAF complexes. However, the precise role ARID1A/ARID1B mutations (cBAF loss) play in the development of dedifferentiated endometrial carcinoma remains unclear. In this study, we define an expression signature associated with endometrial “dedifferentiation” by profiling the well-differentiated and dedifferentiated components of cBAF loss DEC cases through laser capture microdissection followed by RNA sequencing. Introduction of ARID1A or ARID1B in cBAF loss DEC cell lines demonstrated the restoration of cBAF assembly, confirmed by glycerol gradient and IP mass spectrometry. Upon the expression of ARID1A or ARID1B, we observed widespread chromatin occupancy and DNA accessibility at distinct paralog-specific sites, along with common sites associated with epithelial lineage differentiation, estrogen receptor, and PI3K/AKT pathway signaling. These findings align with the differentiation signature, defined in DEC cases demonstrating downregulation of genes associated with Myc targets, cell cycle checkpoint pathways, and DNA repair, suggesting these pathways represent potential therapeutic avenues for targeting DEC/UEC. Collectively, these data establish that well-differentiated endometrial carcinoma and DEC/UEC with mSWI/SNF mutations represent a spectrum of cBAF loss, defining the unique roles of ARID1A and ARID1B in endometrial carcinoma development. Furthermore, these findings imply that cBAF loss drives the widespread changes in DEC morphology, gene expression, and clinical behavior, enhancing our understanding of the etiology and therapeutic options for these cancers. Citation Format: Jessica D. St. Laurent, Grace Xu, Kasey Cervantas, Ajinkya Patil, Akshay Sankar, Shary Chen, David Kolin, Yemin Wang, David G. Huntsman, Cigall Kadoch. ARID1A/B mutations retarget mSWI/SNF chromatin remodeler activity and define a spectrum of dedifferentiation in endometrial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR006.

Single-cell chromatin profiling reveals genetic programs activating proregenerative states in nonmyocyte cells.

Cardiac regeneration requires coordinated participation of multiple cell types whereby their communications result in transient activation of proregenerative cell states. Although the molecular characteristics and lineage origins of these activated cell states and their contribution to cardiac regeneration have been studied, the extracellular signaling and the intrinsic genetic program underlying the activation of the transient functional cell states remain largely unexplored. In this study, we delineated the chromatin landscapes of the noncardiomyocytes (nonCMs) of the regenerating heart at the single-cell level and inferred the cis-regulatory architectures and trans-acting factors that control cell type-specific gene expression programs. Moreover, further motif analysis and cell-specific genetic manipulations suggest that the macrophage-derived inflammatory signal tumor necrosis factor-α, acting via its downstream transcription factor complex activator protein-1, functions cooperatively with discrete transcription regulators to activate respective nonCM cell types critical for cardiac regeneration. Thus, our study defines the regulatory architectures and intercellular communication principles in zebrafish heart regeneration.