Biology subjects

Cusanovich, D.

Publications and source records attributed to Cusanovich, D..

7 recordsLinked to original sources

bioRxiv2026

Profiling Peripheral Blood with an Optimized, Multiplexed, Single-cell Multiome Approach Supports an Insulin-driven Asthma Subtype

RNA sequencing (RNA-seq) and the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) have become standard techniques for studying gene regulation in human populations. Single-cell (sc) "multiomic" genomic methodologies now enable researchers to dissect cellular heterogeneity while simultaneously measuring gene expression and chromatin accessibility within individual cells. However, single-cell approaches remain experimentally complex and cost-prohibitive, limiting their application in population studies, and motivating the development of new strategies for population-scale single-cell investigations. To this end, we have adapted and optimized a previous multiomic protocol, "Transcriptome, Epitope, and ATAC sequencing" (TEA-seq) through experimentation and simulation to incorporate sample multiplexing, thus resulting in our "multiplexed TEA-seq" (mTEA-seq) protocol. Using mTEA-seq, we sought to determine whether asthma that develops in conjunction with early-life elevated insulin levels might have an identifiable molecular signature. We studied samples from adult individuals (54 subjects, 272,003 cells) from the Tucson Childrens Respiratory Study (TCRS), a birth cohort phenotypically characterized over four decades, to identify unique molecular characteristics of blood cells from asthmatics who had high serum insulin levels at age 6. Using a Bayesian approach, we found striking sex-specific effects. Male asthmatic subjects with high insulin at age 6 displayed widespread immune transcriptional and epigenetic alterations into adulthood compared to male non-asthmatic subjects without elevated insulin at age 6. We also found that male non-asthmatics with early-life high insulin showed epigenetic perturbations in adulthood, but not transcriptional changes. The consistency of epigenetic signals between these two groups that had high insulin at age 6 was highly cell-type-specific. For example, CD14+ monocytes displayed broadly common insulin-associated chromatin remodeling regardless of asthma status, while NK cells exhibited unique patterns of insulin-associated epigenetic reprogramming depending on asthma status. Finally, genotyping performed directly from our single-cell data enabled cell type-specific cis-QTL mapping that suggested HLA-DQB1 and AHI as genes for future study in insulin-associated asthma. Our investigation of childhood insulin-associated asthma demonstrates a metabolically-driven alterations on immune cells persisting into adulthood, thus providing a molecular signature of this asthma subtype, and offering novel insights for disease prevention and therapeutic intervention.

genomics↗

bioRxiv2025

Early, adjuvant-responsive epigenetic programs in B cells imprint subsequent plasma cell survival and the duration of humoral immunity

The duration of antibody production varies across different infections and vaccines. To define molecular programs that promote durable humoral immunity, we used mice deficient in ZBTB20, a transcription factor that is highly expressed by plasma cells and required to maintain antibody production in vivo. However, genetic deletion of Zbtb20 in long-lived plasma cells had no impact on the duration of antibody production. Instead, deletion of Zbtb20 in B cells only within the first week after immunization caused a subsequent failure to maintain plasma cells. Through single cell ATAC-sequencing, we observed elevated IRF8- and Ets-dependent epigenetic programs in ZBTB20-deficient B cells at 7 days post-immunization, whereas the corresponding transcriptional changes manifested ~1 week later. Switching from alum to an oil-in-water adjuvant suppressed Ets-dependent epigenetic programs and rescued ZBTB20-deficient antibody responses. Deletion of Irf8 also rescued ZBTB20-deficient antibody responses. Thus, B cell-intrinsic epigenetic programs imprint durable antibody production at a very early stage, prior to major transcriptional consequences and weeks before most long-lived plasma cells are formed.

immunology↗

bioRxiv2023

Lactoylglutathione promotes inflammatory signaling in macrophages

Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH, while demonstrating a potentiated inflammatory response when exposed to lipopolysaccharides, corresponding with a rise in histone lactoylation. Interestingly, our data demonstrate that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state, however, upon stimulation, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is a primary contributing factor facilitating the inflammatory response.

biochemistry↗

bioRxiv2023

Multiomics of human aortic endothelial cells reveals cell subtypes with heterogeneous responses to canonical endothelial-to-mesenchymal perturbations

ObjectiveEndothelial cells (ECs), macrophages, and vascular smooth muscle cells (VSMCs) are major cell types in atherosclerosis progression, and heterogeneity in EC sub-phenotypes are becoming increasingly appreciated. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Approach and ResultsTo create an in vitro dataset to study human EC heterogeneity, multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs). To model pro-inflammatory and activating environments characteristic of the atherosclerotic microenvironment in vitro, HAECs from at least three donors were exposed to three distinct perturbations with their respective controls: transforming growth factor beta-2 (TGFB2), interleukin-1 beta (IL1B), and siRNA-mediated knock-down of the endothelial transcription factor ERG (siERG). To form a comprehensive in vivo/ex vivo dataset of human atherosclerotic cell types, meta-analysis of single cell transcriptomes across 17 human arterial specimens was performed. Two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and in vivo cell profiles. HAEC cultures were reproducibly populated by 4 major clusters with distinct pathway enrichment profiles: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Exposure to siERG, IL1B or TGFB2 elicited mostly distinct transcriptional and accessible chromatin responses. EC1 and EC2, the most canonically healthy EC populations, were affected predominantly by siERG; the activated cluster EC3 was most responsive to IL1B; and the mesenchymal population EC4 was most affected by TGFB2. Quantitative comparisons between in vitro and in vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated SNPs from GWAS, suggesting these cell phenotypes harbor CAD-modulating mechanisms. ConclusionPrimary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here, which have been reported by others to be involved in the pathogenesis of atherosclerosis as well as induce endothelial-to-mesenchymal transition (EndMT), only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and in vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

genomics↗

bioRxiv2020

Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes

Recently developed single cell technologies allow researchers to characterize cell states at ever greater resolution and scale. C. elegans is a particularly tractable system for studying development, and recent single cell RNA-seq studies characterized the gene expression patterns for nearly every cell type in the embryo and at the second larval stage (L2). Gene expression patterns are useful for learning about gene function and give insight into the biochemical state of different cell types; however, in order to understand these cell types, we must also determine how these gene expression levels are regulated. We present the first single cell ATAC-seq study in C. elegans. We collected data in L2 larvae to match the available single cell RNA-seq data set, and we identify tissue-specific chromatin accessibility patterns that align well with existing data, including the L2 single cell RNA-seq results. Using a novel implementation of the latent Dirichlet allocation algorithm, we leverage the single-cell resolution of the sci-ATAC-seq data to identify accessible loci at the level of individual cell types, providing new maps of putative cell type-specific gene regulatory sites, with promise for better understanding of cellular differentiation and gene regulation in the worm.

genomics↗

bioRxiv2019

Breast tumor stiffness instructs bone metastasis via mechanical memory

The mechanical microenvironment of primary breast tumors plays a substantial role in promoting tumor progression. While the transitory response of cancer cells to pathological stiffness in their native microenvironment has been well described, it is still unclear how mechanical stimuli in the primary tumor influence distant, late-stage metastatic phenotypes across time and space in absentia. Here, we show that primary tumor stiffness promotes stable, non-genetically heritable phenotypes in breast cancer cells. This mechanical memory instructs cancer cells to adopt and maintain increased cytoskeletal dynamics, traction force, and 3D invasion in vitro, in addition to promoting osteolytic bone metastasis in vivo. Furthermore, we established a mechanical conditioning (MeCo) score comprised of mechanically-regulated genes as a global gene expression measurement of tumor stiffness response. Clinically, we show that a high MeCo score is strongly associated with bone metastasis in patients. Using a discovery approach, we mechanistically traced mechanical memory in part to ERK-mediated mechanotransductive activation of RUNX2, an osteogenic gene bookmarker and bone metastasis driver. The combination of these RUNX2 traits permits the stable transactivation of osteolytic target genes that remain upregulated after cancer cells disseminate from their activating microenvironment in order to modify a distant microenvironment. Using genetic, epigenetic, and functional approaches, we were able to simulate, repress, select and extend RUNX2-mediated mechanical memory and alter cancer cell behavior accordingly. In concert with previous studies detailing the influence of biochemical properties of the primary tumor stroma on distinct metastatic phenotypes, our findings detailing the influence of biomechanical properties support a generalized model of cancer progression in which the integrated properties of the primary tumor microenvironment govern the secondary tumor microenvironment, i.e., soil instructs soil.

cancer biology↗

bioRxiv2017

Chromatin accessibility dynamics of myogenesis at single cell resolution

Over a million DNA regulatory elements have been cataloged in the human genome, but linking these elements to the genes that they regulate remains challenging. We introduce Cicero, a statistical method that connects regulatory elements to target genes using single cell chromatin accessibility data. We apply Cicero to investigate how thousands of dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of co-accessible regulatory elements linked by Cicero meet criteria of \"chromatin hubs\", in that they are physically proximal, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of gene expression. Pseudotemporal analysis revealed a subset of elements bound by MYOD in myoblasts that exhibit early opening, potentially serving as the initial sites of recruitment of chromatin remodeling and histone-modifying enzymes. The methodological framework described here constitutes a powerful new approach for elucidating the architecture, grammar and mechanisms of cis-regulation on a genome-wide basis.

genomics↗