Darren A Cusanovich

Interests

Teaching

Genomics, Bioinformatics, Complex disease biology ,Molecular biology

Research

Asthma, Genomics ,Single cell genomics ,Genomic technology development, Transcriptional regulation, Complex disease biology

Courses

Scholarly Contributions

Journals/Publications

• Cabel, C. R., Guzman, B. A., Alizadeh, E., Li, S., Holberg, C., Wichaidit, C., Cusanovich, D. A., Paek, A. L., Thatcher, G. R., Van Doorslaer, K., Nargi, R. S., Sutton, R. E., Suryadevara, N., Crowe, J. E., Carnahan, R. H., Campos, S. K., & Thorne, C. A. (2025). Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors. Frontiers in Cell and Developmental Biology, 13(Issue). doi:10.3389/fcell.2025.1538934
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  The sudden rise of the SARS-CoV-2 virus and the delay in development of effective therapeutics for mitigation made evident a need for ways to screen compounds that can block infection and prevent further pathogenesis and spread. However, identifying effective drugs that are efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, but as the SARS-CoV-2 mutated, these antibodies became ineffective. Small-molecule antivirals were identified using pseudovirus constructs to recapitulate infection in nonhuman cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce viral infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e., PIK3i, mTORi, and multiple RTKi) that could be effective against the SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.
• Kang, H. J., Fitch, J. C., Varghese, R. P., Thorne, C. A., & Cusanovich, D. A. (2025). Optimization of a Cas12a-Driven Synthetic Gene Regulatory Network System. ACS Synthetic Biology. doi:10.1021/acssynbio.5c00084
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  Gene regulatory networks, which control gene expression patterns in development and in response to stimuli, use regulatory logic modules to coordinate inputs and outputs. One example of a regulatory logic module is the gene regulatory cascade (GRC), where a series of transcription factor genes turn on in order. Synthetic biologists have derived artificial systems that encode regulatory rules, including GRCs. Furthermore, the development of single-cell approaches has enabled the discovery of gene regulatory modules in a variety of experimental settings. However, the tools available for validating these observations remain limited. Based on a synthetic GRC using DNA cutting-defective Cas9 (dCas9), we designed and implemented an alternative synthetic GRC utilizing DNA cutting-defective Cas12a (dCas12a). Comparing the ability of these two systems to express a fluorescent reporter, the dCas9 system was initially more active, while the dCas12a system was more streamlined. Investigating the influence of individual components of the systems identified nuclear localization as a major driver of differences in activity. Improving nuclear localization for the dCas12a system resulted in 1.5-fold more reporter-positive cells and a 15-fold increase in reporter intensity relative to the dCas9 system. We call this optimized system the “Synthetic Gene Regulatory Network” (SGRN, pronounced “sojourn”).
• Adelus, M. L., Ding, J., Tran, B. T., Conklin, A. C., Golebiewski, A. K., Stolze, L. K., Whalen, M. B., Cusanovich, D. A., & Romanoski, C. E. (2024). Author response: Single-cell ‘omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations. eLife. doi:10.7554/elife.91729.3.sa2
• Adelus, M. L., Ding, J., Tran, B. T., Conklin, A. C., Golebiewski, A. K., Stolze, L. K., Whalen, M. B., Cusanovich, D. A., & Romanoski, C. E. (2024). Single cell ‘omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveals heterogeneity of endothelial subtype and response to activating perturbations. eLife, 12. doi:10.7554/elife.91729.2
• Trujillo, M. N., Jennings, E. Q., Hoffman, E. A., Zhang, H., Phoebe, A. M., Mastin, G. E., Kitamura, N., Reisz, J. A., Megill, E., Kantner, D., Marcinkiewicz, M. M., Twardy, S. M., Lebario, F., Chapman, E., McCullough, R. L., D'Alessandro, A., Snyder, N. W., Cusanovich, D. A., & Galligan, J. J. (2024). Lactoylglutathione promotes inflammatory signaling in macrophages through histone lactoylation. Molecular Metabolism, 81. doi:10.1016/j.molmet.2024.101888
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  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 mediating histone lactoylation and inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH and histone lactoylation with a corresponding potentiation of the inflammatory response when exposed to lipopolysaccharides. An analysis of chromatin accessibility shows that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state; upon stimulation, however, 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 the primary driving factor facilitating histone lactoylation and a major contributor to inflammatory signaling.
• Zhang, H., Mulqueen, R., Iannuzo, N., Farrera, D., Polverino, F., Galligan, J., Ledford, J., Adey, A., & Cusanovich, D. (2024). txci-ATAC-seq: a massive-scale single-cell technique to profile chromatin accessibility. Genome Biology, 25(1). doi:10.1186/s13059-023-03150-1
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  We develop a large-scale single-cell ATAC-seq method by combining Tn5-based pre-indexing with 10× Genomics barcoding, enabling the indexing of up to 200,000 nuclei across multiple samples in a single reaction. We profile 449,953 nuclei across diverse tissues, including the human cortex, mouse brain, human lung, mouse lung, mouse liver, and lung tissue from a club cell secretory protein knockout (CC16−/−) model. Our study of CC16−/− nuclei uncovers previously underappreciated technical artifacts derived from remnant 129 mouse strain genetic material, which cause profound cell-type-specific changes in regulatory elements near many genes, thereby confounding the interpretation of this commonly referenced mouse model.
• Adelus, M. L., Ding, J., Tran, B. T., Conklin, A. C., Golebiewski, A. K., Stolze, L. K., Whalen, M. B., Cusanovich, D. A., & Romanoski, C. E. (2023). Multiomics of human aortic endothelial cells reveals cell subtypes with heterogeneous responses to canonical endothelial-to-mesenchymal perturbations. BioRxiv. doi:10.1101/2023.04.03.535495
• Cusanovich, D. A. (2023). Ten (10) X-compatible Combinatorial Indexing ATAC sequencing (txci-ATAC-seq). protocols.io. doi:dx.doi.org/10.17504/protocols.io.dm6gp3o68vzp/v1
• Cusanovich, D. A., Ahmed, M. N., Martinez, F., Halonen, M., Kylathu, R., & Zaghloul, N. (2023). Single-Cell Profiling of Premature Neonate Airways Reveals a Continuum of Myeloid Differentiation. American Journal of Respiratory Cell and Molecular Biology.
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  Rationale: Single-cell genomic technologies hold great potential to advance our understanding of lung development and disease. A major limitation lies in accessing intact cells from primary lung tissues for profiling human airway health. Sampling methods, such as endotracheal aspirates, that are compatible with clinical interventions could enable longitudinal studies, the enrollment of large cohorts, and the development of novel diagnostics.Objectives: To explore single-cell RNA-seq (scRNA-seq) profiling of the cell types present at birth in the airway lumen of extremely premature neonates (
• Iannuzo, N., Welfley, H., Li, N. C., Johnson, M. D., Rojas-Quintero, J., Polverino, F., Guerra, S., Li, X., Cusanovich, D. A., Langlais, P. R., & Ledford, J. G. (2023). CC16 drives VLA-2-dependent SPLUNC1 expression. Frontiers in Immunology, 14(Issue). doi:10.3389/fimmu.2023.1277582
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  Rationale: CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated. Objectives: We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity. Methods: We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge. Measurements and main results: We identified 8 antimicrobial proteins significantly decreased by CC16-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden. Conclusion: Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.
• Iannuzo, N., Welfley, H., Li, N., Johnson, M. D., Guerra, S., Li, X., Cusanovich, D. A., Langlais, P. R., & Ledford, J. (2023). CC16 drives VLA-2-dependent SPLUNC1 expression. Frontiers in Immunology.
• Iannuzo, N., Welfley, H., Li, N., Johnson, M. G., Guerra, S., Li, X., Cusanovich, D. A., Langlais, P., & Ledford, J. G. (2023). CC16 Induces Pulmonary Epithelial-Driven SPLUNC1 Expression by Signaling through VLA-2.. European Respiratory Journal. doi:10.1183/13993003.congress-2023.oa4964
• Trujillo, M. N., Jennings, E. Q., Hoffman, E. A., Zhang, H., Phoebe, A. M., Mastin, G. E., Kitamura, N., Reisz, J. A., Megill, E., Kantner, D. S., Marcinkiewicz, M., Twardy, S. M., Lebario, F., Chapman, E., McCullough, R. L., D’Alessandro, A., Snyder, N. W., Cusanovich, D. A., & Galligan, J. J. (2023). Lactoylglutathione promotes inflammatory signaling in macrophages. Molecular Metabolism. doi:10.1101/2023.10.10.561739
• Welfley, H., Kylat, R., Zaghloul, N., Halonen, M., Martinez, F. D., Ahmed, M., & Cusanovich, D. A. (2023). Single-Cell Profiling of Premature Neonate Airways Reveals a Continuum of Myeloid Differentiation. American Journal of Respiratory Cell and Molecular Biology, 69(Issue 6). doi:10.1165/rcmb.2022-0293oc
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  Single-cell genomic technologies hold great potential to advance our understanding of lung development and disease. A major limitation lies in accessing intact cells from primary lung tissues for profiling human airway health. Sampling methods such as endotracheal aspiration that are compatible with clinical interventions could enable longitudinal studies, the enrollment of large cohorts, and the development of novel diagnostics. To explore single-cell RNA sequencing profiling of the cell types present at birth in the airway lumen of extremely premature neonates (,28 wk gestation), we isolated cells from endotracheal aspirates collected from intubated neonates within the first hour after birth. We generated data on 10 subjects, providing a rich view of airway luminal biology at a critical developmental period. Our results show that cells present in the airways of premature neonates primarily represent a continuum of myeloid differentiation, including fetal monocytes (25% of total), intermediate myeloid populations (48%), and macrophages (2.6%). Applying trajectory analysis to the myeloid populations, we identified two trajectories consistent with the developmental stages of interstitial and alveolar macrophages, as well as a third trajectory presenting an alternative pathway bridging the distinct macrophage precursors. The three trajectories share many dynamic genes (N = 5,451), but also have distinct transcriptional changes (259 alveolar-specific, 666 interstitial-specific, and 285 bridging-specific). Overall, our results define cells isolated within the so-called “golden hour of birth” in extremely premature neonate airways, representing complex lung biology, and can be used in studies of human development and disease.
• Ahmed, M. H., Cusanovich, D. A., Halonen, M., Kylat, R. I., Martinez, F. J., Welfley, H., & Zaghloul, N. (2022). Mapping fetal myeloid differentiation in airway samples from premature neonates with single-cell profiling. BioRxiv. doi:10.1101/2022.07.08.499395
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  Single-cell genomic technologies hold great potential to advance our understanding of development and disease. A major limitation lies in isolating intact cells from primary tissues for profiling. Sampling methods compatible with current clinical interventions could enable longitudinal studies, the enrollment of large cohorts, and even the development of novel diagnostics. To explore single-cell RNA-seq (scRNA-seq) profiling of the cell types present at birth in the airway lumen of extremely premature (
• Cusanovich, D. A., Carr, T. F., & Halonen, M. (2022). Evaluation of Swab-Seq as a scalable, sensitive assay for community surveillance of SARS-CoV-2 Infection. Scientific Reports.
• Cusanovich, D. A., Johnson, M. D., & Galligan, J. J. (2022). Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei. BMC Genomics.
• Kang, H. J., Allison, S., Spangenberg, A., Carr, T., Sprissler, R., Halonen, M., & Cusanovich, D. A. (2022). Evaluation of Swab-Seq as a scalable, sensitive assay for community surveillance of SARS-CoV-2 infection. Scientific Reports, 12(Issue 1). doi:10.1038/s41598-022-06901-5
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  The ongoing SARS-CoV-2 pandemic and subsequent demand for viral testing has led to issues in scaling diagnostic lab efforts and in securing basic supplies for collection and processing of samples. This has motivated efforts by the scientific community to establish improved protocols that are more scalable, less resource intensive, and less expensive. One such developmental effort has resulted in an assay called “Swab-Seq”, so named because it was originally developed to work with dry nasal swab samples. The existing gold standard test consists of RNA extracted from a nasopharyngeal (NP) swab that is subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR). Swab-Seq adapts this method to a next-generation sequencing readout. By pairing this modification with extraction-free sampling techniques, Swab-Seq achieves high scalability, low cost per sample, and a reasonable turnaround time. We evaluated the effectiveness of this assay in a community surveillance setting by testing samples collected from both symptomatic and asymptomatic individuals using the traditional NP swab. In addition, we evaluated extraction-free sampling techniques (both saliva and saline mouth gargle samples). We found the assay to be as clinically sensitive as the qRT-PCR assay, adaptable to multiple sample types, and able to easily accommodate hundreds of samples at a time. We thus provide independent validation of Swab-Seq and extend its utility regarding sample type and sample stability. Assays of this type greatly expand the possibility of routine, noninvasive, repeated testing of asymptomatic individuals suitable for current and potential future needs.
• Zhang, H., Rice, M. E., Alvin, J. W., Farrera-Gaffney, D., Galligan, J. J., Johnson, M. D., & Cusanovich, D. A. (2022). Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei. BMC Genomics, 23(Issue 1). doi:10.1186/s12864-021-08266-x
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  Background: The “Assay for Transposase Accessible Chromatin sequencing” (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. With all possible combinations of components, we created 24 experimental conditions with four replicates for each (a total of 96 samples). In addition, we tested the 12 native conditions in a primary sample type (mouse lung tissue) with two different input amounts. Through these extensive comparisons, we were able to observe the effect of different ATAC-seq conditions on data quality and to examine the utility and potential redundancy of various quality metrics. Results: In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. Conclusions: We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens.
• Cusanovich, D. A. (2021). Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes. Genome Research.
• Cusanovich, D. A., Romanoski, C. E., Mouneimne, G., & Padi, M. (2021). Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning. Cell Reports.
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  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.
• Durham, T. J., Daza, R. M., Gevirtzman, L., Cusanovich, D. A., Bolonduro, O., Noble, W. S., Shendure, J., & Waterston, R. H. (2021). Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes. Genome Research, 31(Issue 10). doi:10.1101/gr.271791.120
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  Recently developed single-cell technologies allow researchers to characterize cell states at ever greater resolution and scale. Caenorhabditis 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 give insight about gene function and into the biochemical state of different cell types; recent advances in other single-cell genomics technologies can now also characterize the regulatory context of the genome that gives rise to these gene expression levels at a single-cell resolution. To explore the regulatoryDNA of individual cell types in C. elegans, we collected single-cell chromatin accessibility data using the sci-ATAC-seq assay in L2 larvae to match the available single-cell RNA-seq data set. By using a novel implementation of the latent Dirichlet allocation algorithm, we identify 37 clusters of cells that correspond to different cell types in the worm, providing new maps of putative cell type-specific gene regulatory sites, with promise for better understanding of cellular differentiation and gene regulation.
• Watson, A. W., Grant, A. D., Parker, S. S., Hill, S., Whalen, M. B., Chakrabarti, J., Harman, M. W., Roman, M. R., Forte, B. L., Gowan, C. C., Castro-Portuguez, R., Stolze, L. K., Franck, C., Cusanovich, D. A., Zavros, Y., Padi, M., Romanoski, C. E., & Mouneimne, G. (2021). Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning. Cell Reports, 35(Issue 13). doi:10.1016/j.celrep.2021.109293
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  While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.
• Chang, E. H., Willis, A. L., Romanoski, C. E., Cusanovich, D. A., Pouladi, N., Li, J., Lussier, Y. A., & Martinez, F. D. (2020). Rhinovirus Infections in Individuals with Asthma Increase ACE2 Expression and Cytokine Pathways Implicated in COVID-19. American Journal of Respiratory and Critical Care Medicine, 202(Issue 5). doi:10.1164/rccm.202004-1343le
• Chang, E. H., Willis, A. L., Romanoski, C. E., Cusanovich, D. A., Pouladi, N., Li, J., Lussier, Y. A., & Martinez, F. D. (2020). Rhinovirus Infections in Individuals with Asthma Increase ACE2 Expression and Cytokine Pathways Implicated in COVID-19. American journal of respiratory and critical care medicine, 202(5), 753-755.
• Cusanovich, D. A. (2020). sci-ATAC-seq3. protocols.io. doi:dx.doi.org/10.17504/protocols.io.be8mjhu6
• Cusanovich, D. A., Daza, R. M., Durham, T. B., Gevirtzman, L., Noble, W. S., Shendure, J., & Waterston, R. H. (2020). Comprehensive characterization of tissue-specific chromatin accessibility in L2Caenorhabditis elegansnematodes. bioRxiv (Cold Spring Harbor Laboratory). doi:10.1101/2020.09.15.299123
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  Abstract 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.
• Domcke, S., Hill, A. J., Daza, R. M., Cao, J., O'Day, D. R., Pliner, H. A., Aldinger, K. A., Pokholok, D., Zhang, F., Milbank, J. H., Zager, M. A., Glass, I. A., Steemers, F. J., Doherty, D., Trapnel, C., Cusanovich, D. A., & Shendure, J. (2020). A human cell atlas of fetal chromatin accessibility. Science, 370(Issue 6518). doi:10.1126/science.aba7612
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  The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells. We leveraged cell types defined by gene expression to annotate these data and cataloged hundreds of thousands of candidate regulatory elements that exhibit cell type-specific chromatin accessibility. We investigated the properties of lineage-specific transcription factors (such as POU2F1 in neurons), organ-specific specializations of broadly distributed cell types (such as blood and endothelial), and cell type-specific enrichments of complex trait heritability. These data represent a rich resource for the exploration of in vivo human gene regulation in diverse tissues and cell types.
• Domcke, S., Hill, A. J., Daza, R. M., Cao, J., O'Day, D. R., Pliner, H. A., Aldinger, K. A., Pokholok, D., Zhang, F., Milbank, J. H., Zager, M. A., Glass, I. A., Steemers, F. J., Doherty, D., Trapnell, C., Cusanovich, D. A., & Shendure, J. (2020). A human cell atlas of fetal chromatin accessibility. Science, 370(6518).
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  The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells. We leveraged cell types defined by gene expression to annotate these data and cataloged hundreds of thousands of candidate regulatory elements that exhibit cell type-specific chromatin accessibility. We investigated the properties of lineage-specific transcription factors (such as POU2F1 in neurons), organ-specific specializations of broadly distributed cell types (such as blood and endothelial), and cell type-specific enrichments of complex trait heritability. These data represent a rich resource for the exploration of in vivo human gene regulation in diverse tissues and cell types.
• Rai, V., Quang, D. X., Erdos, M. R., Cusanovich, D. A., Daza, R. M., Narisu, N., Zou, L. S., Didion, J. P., Guan, Y., Shendure, J., Parker, S. C., & Collins, F. S. (2020). Single-cell ATAC-Seq in human pancreatic islets and deep learning upscaling of rare cells reveals cell-specific type 2 diabetes regulatory signatures. Molecular Metabolism, 32(Issue). doi:10.1016/j.molmet.2019.12.006
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  Objective: Type 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome-wide association studies (GWAS) have identified > 400 independent signals that encode genetic predisposition. More than 90% of associated single-nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity. Methods: We present genome-wide single-cell chromatin accessibility profiles in >1,600 cells derived from a human pancreatic islet sample using single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq). We also developed a deep learning model based on U-Net architecture to accurately predict open chromatin peak calls in rare cell populations. Results: We show that sci-ATAC-seq profiles allow us to deconvolve alpha, beta, and delta cell populations and identify cell-type-specific regulatory signatures underlying T2D. Particularly, T2D GWAS SNPs are significantly enriched in beta cell-specific and across cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. We also demonstrate, using less abundant delta cells, that deep learning models can improve signal recovery and feature reconstruction of rarer cell populations. Finally, we use co-accessibility measures to nominate the cell-specific target genes at 104 non-coding T2D GWAS signals. Conclusions: Collectively, we identify the islet cell type of action across genetic signals of T2D predisposition and provide higher-resolution mechanistic insights into genetically encoded risk pathways.
• Rai, V., Quang, D. X., Erdos, M. R., Cusanovich, D. A., Daza, R. M., Narisu, N., Zou, L. S., Didion, J. P., Guan, Y., Shendure, J., Parker, S. C., & Collins, F. S. (2020). Single-cell ATAC-Seq in human pancreatic islets and deep learning upscaling of rare cells reveals cell-specific type 2 diabetes regulatory signatures. Molecular metabolism, 32, 109-121.
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  Type 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome-wide association studies (GWAS) have identified > 400 independent signals that encode genetic predisposition. More than 90% of associated single-nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity.
• Watson, A. W., Grant, A., Parker, S. S., Harman, M. W., Roman, M. R., Uhlorn, B. L., Gowan, C., Castro-Portuguez, R., Stolze, L. K., Franck, C., Cusanovich, D. A., Padi, M., Romanoski, C. E., & Mouneimne, G. (2020). Breast Tumor Stiffness Instructs Bone Metastasis Via Mechanical Memory. Cell reports. doi:10.2139/ssrn.3696766
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  The mechanical microenvironment of primary breast tumors plays a substantial role in promoting tumor progression1. While the transitory response of cancer cells to pathological stiffness in their native microenvironment has been well described2, 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 driver3,4. Expanding our conceptual model, we reveal for the first time that a mechanically sensitive transcription factor can maintain a temporal profile of chromatin accessibility in vitro and in vivo. The combination of these distinctive 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 could 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 phenotypes5–9, 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.
• Allan, C. M., Heizer, P. J., Tu, Y., Sandoval, N. P., Jung, R. S., Morales, J. E., Sajti, E., Troutman, T. D., Saunders, T. L., Cusanovich, D. A., Beigneux, A. P., Romanoski, C. E., Fong, L. G., & Young, S. G. (2019). An upstream enhancer regulates expression in a tissue-specific manner. Journal of lipid research.
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  GPIHBP1, the protein that shuttles lipoprotein lipase (LPL) to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ~3.6 kb upstream from exon 1 of mouse To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (). Removing the enhancer reduced expression by >90% in the liver and by ~50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, nor did it lead to hypertriglyceridemia--even when mice were fed a high-fat diet. Compound heterozygotes ( mice) displayed further reductions in expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling expression.
• Allan, C. M., Heizer, P. J., Tu, Y., Sandoval, N. P., Jung, R. S., Morales, J., Sajti, E., Troutman, T. D., Saunders, T. L., Cusanovich, D. A., Beigneux, A. P., Romanoski, C. E., Fong, L. G., & Young, S. G. (2019). An upstream enhancer regulates Gpihbp1 expression in a tissue-specific manner. Journal of Lipid Research. doi:10.1194/jlr.m091322
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  Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia-even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1Enh/- mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.
• Castro-Portuguez, R., Cusanovich, D. A., Forte, B. L., Franck, C., Gowan, C. C., Grant, A. D., Harman, M. W., Mouneimne, G., Padi, M., Parker, S. S., Roman, M. R., Romanoski, C. E., & Watson, A. (2019). Breast tumor stiffness instructs bone metastasis via mechanical memory. bioRxiv. doi:10.1101/847699
• Collins, F. S., Cusanovich, D. A., Daza, R. M., Didion, J. P., Erdos, M. R., Guan, Y., Narisu, N., Parker, S. C., Quang, D., Rai, V., Shendure, J., & Zou, L. S. (2019). Single cell ATAC-seq in human pancreatic islets and deep learning upscaling of rare cells reveals cell-specific type 2 diabetes regulatory signatures. bioRxiv. doi:10.1101/749283
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  ABSTRACT Objective Type 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome wide association studies (GWAS) have identified >400 independent signals that encode genetic predisposition. More than 90% of the associated single nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, and so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity. Methods We present genome-wide single cell chromatin accessibility profiles in >1,600 cells derived from a human pancreatic islet sample using single-cell-combinatorial-indexing ATAC-seq (sci-ATAC-seq). We also developed a deep learning model based on the U-Net architecture to accurately predict open chromatin peak calls in rare cell populations. Results We show that sci-ATAC-seq profiles allow us to deconvolve alpha, beta, and delta cell populations and identify cell-type-specific regulatory signatures underlying T2D. Particularly, we find that T2D GWAS SNPs are significantly enriched in beta cell-specific and cross cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. We also demonstrate, using less abundant delta cells, that deep-learning models can improve signal recovery and feature reconstruction of rarer cell populations. Finally, we use co-accessibility measures to nominate the cell-specific target genes at 104 non-coding T2D GWAS signals. Conclusions Collectively, we identify the islet cell type of action across genetic signals of T2D predisposition and provide higher-resolution mechanistic insights into genetically encoded risk pathways.
• Cao, J., Cusanovich, D. A., Ramani, V., Aghamirzaie, D., Pliner, H. A., Hill, A. J., Daza, R. M., McFaline-Figueroa, J. L., Packer, J. S., Christiansen, L., Steemers, F. J., Adey, A. C., Trapnell, C., & Shendure, J. (2018). Joint profiling of chromatin accessibility and gene expression in thousands of single cells. Science, 361(Issue 6409). doi:10.1126/science.aau0730
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  Although we can increasingly measure transcription, chromatin, methylation, and other aspects of molecular biology at single-cell resolution, most assays survey only one aspect of cellular biology. Here we describe sci-CAR, a combinatorial indexing-based coassay that jointly profiles chromatin accessibility and mRNA (CAR) in each of thousands of single cells. As a proof of concept, we apply sci-CAR to 4825 cells, including a time series of dexamethasone treatment, as well as to 11,296 cells from the adult mouse kidney. With the resulting data, we compare the pseudotemporal dynamics of chromatin accessibility and gene expression, reconstruct the chromatin accessibility profiles of cell types defined by RNA profiles, and link cis-regulatory sites to their target genes on the basis of the covariance of chromatin accessibility and transcription across large numbers of single cells.
• Cusanovich, D. A. (2018). Joint profiling of chromatin accessibility and gene expression in thousands of single cells. Science.
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  Although we can increasingly measure transcription, chromatin, methylation, and other aspects of molecular biology at single-cell resolution, most assays survey only one aspect of cellular biology. Here we describe sci-CAR, a combinatorial indexing–based coassay that jointly profiles chromatin accessibility and mRNA (CAR) in each of thousands of single cells. As a proof of concept, we apply sci-CAR to 4825 cells, including a time series of dexamethasone treatment, as well as to 11,296 cells from the adult mouse kidney. With the resulting data, we compare the pseudotemporal dynamics of chromatin accessibility and gene expression, reconstruct the chromatin accessibility profiles of cell types defined by RNA profiles, and link cis-regulatory sites to their target genes on the basis of the covariance of chromatin accessibility and transcription across large numbers of single cells.
• Cusanovich, D. A. (2018). The cis-regulatory dynamics of embryonic development at single cell resolution. Nature, 555(7697), 538-542. doi:10.1038/nature25981
• Cusanovich, D. A., Hill, A. J., Aghamirzaie, D., Daza, R. M., Pliner, H. A., Berletch, J. B., Filippova, G. N., Huang, X., Christiansen, L., DeWitt, W. S., Lee, C., Regalado, S. G., Read, D. F., Steemers, F. J., Disteche, C. M., Trapnell, C., & Shendure, J. (2018). A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility. Cell, 174(Issue 5). doi:10.1016/j.cell.2018.06.052
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  We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in ∼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and ∼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types. We develop a technique for mapping single cell gene expression data to single-cell chromatin accessibility data, facilitating the comparison of atlases. By intersecting mouse chromatin accessibility with human genome-wide association summary statistics, we identify cell-type-specific enrichments of the heritability signal for hundreds of complex traits. These data define the in vivo landscape of the regulatory genome for common mammalian cell types at single-cell resolution. Profiling chromatin accessibility at single-cell resolution across 13 tissues in mice identifies 85 distinct patterns and a catalog of ∼400,000 potential regulatory elements, presenting a resource for understanding the chromatin regulatory landscape of diverse mammalian cell types and for interpreting human genome-wide association studies.
• Cusanovich, D. A., Hill, A. J., Aghamirzaie, D., Daza, R. M., Pliner, H. A., Berletch, J. B., Filippova, G. N., Huang, X., Christiansen, L., DeWitt, W. S., Lee, C., Regalado, S. G., Read, D. F., Steemers, F. J., Disteche, C. M., Trapnell, C., & Shendure, J. (2018). A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility. Cell, 174(5), 1309-1324.e18.
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  We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in ∼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and ∼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types. We develop a technique for mapping single cell gene expression data to single-cell chromatin accessibility data, facilitating the comparison of atlases. By intersecting mouse chromatin accessibility with human genome-wide association summary statistics, we identify cell-type-specific enrichments of the heritability signal for hundreds of complex traits. These data define the in vivo landscape of the regulatory genome for common mammalian cell types at single-cell resolution.
• Cusanovich, D. A., Reddington, J. P., Garfield, D. A., Daza, R. M., Aghamirzaie, D., Marco-Ferreres, R., Pliner, H. A., Christiansen, L., Qiu, X., Steemers, F. J., Trapnell, C., Shendure, J., & Furlong, E. E. (2018). The cis-regulatory dynamics of embryonic development at single-cell resolution. Nature, 555(Issue 7697). doi:10.1038/nature25981
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  Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila melanogaster embryos spanning three landmark embryonic stages: 2-4 h after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises around 6,000 multipotent cells; 6-8 h after egg laying (predominantly stage 10-11), to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying (predominantly stage 13), when each of the embryo's more than 20,000 cells are undergoing terminal differentiation. Our results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. We identify 30,075 distal regulatory elements that exhibit tissue-specific accessibility. We validated the germ-layer specificity of a subset of these predicted enhancers in transgenic embryos, achieving an accuracy of 90%. Overall, our results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types.
• Pliner, H. A., Packer, J. S., McFaline-Figueroa, J. L., Cusanovich, D. A., Daza, R. M., Aghamirzaie, D., Srivatsan, S., Qiu, X., Jackson, D., Minkina, A., Adey, A. C., Steemers, F. J., Shendure, J., & Trapnell, C. (2018). Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data. Molecular Cell, 71(Issue 5). doi:10.1016/j.molcel.2018.06.044
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  Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of “chromatin hubs”—they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale. Pliner et al. introduce Cicero, a software program to connect distal regulatory elements with target genes using single-cell ATAC-seq data. They find evidence that groups of co-accessible elements form chromatin hubs and undergo coordinated changes in histone marks that are predictive of changes in gene expression in skeletal muscle development.
• Pliner, H. A., Packer, J. S., McFaline-Figueroa, J. L., Cusanovich, D. A., Daza, R. M., Aghamirzaie, D., Srivatsan, S., Qiu, X., Jackson, D., Minkina, A., Adey, A. C., Steemers, F. J., Shendure, J., & Trapnell, C. (2018). Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data. Molecular cell, 71(5), 858-871.e8.
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  Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.
• Adey, A., Cusanovich, D. A., Daza, R. M., Jackson, D., McFaline-Figueroa, J. L., Minkina, A., Packer, J. S., Pliner, H. A., Qiu, X., Shendure, J., Srivatsan, S., Steemers, F. J., & Trapnell, C. (2017). Chromatin accessibility dynamics of myogenesis at single cell resolution. bioRxiv (Cold Spring Harbor Laboratory). doi:10.1101/155473
• Cao, J., Packer, J. S., Ramani, V., Cusanovich, D. A., Huynh, C., Daza, R., Qiu, X., Lee, C., Furlan, S. N., Steemers, F. J., Adey, A., Waterston, R. H., Trapnell, C., & Shendure, J. (2017). Comprehensive single-cell transcriptional profiling of a multicellular organism. Science, 357(Issue 6352). doi:10.1126/science.aam8940
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  To resolve cellular heterogeneity, we developed a combinatorial indexing strategy to profile the transcriptomes of single cells or nuclei, termed sci-RNA-seq (single-cell combinatorial indexing RNA sequencing). We applied sci-RNA-seq to profile nearly 50,000 cells from the nematode Caenorhabditis elegans at the L2 larval stage, which provided >50-fold “shotgun” cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm. We integrated these profiles with whole-animal chromatin immunoprecipitation sequencing data to deconvolve the cell type–specific effects of transcription factors. The data generated by sci-RNA-seq constitute a powerful resource for nematode biology and foreshadow similar atlases for other organisms.
• Cusanovich, D. A. (2017). Comprehensive single-cell transcriptional profiling of a multicellular organism. Science.
• Cusanovich, D. A., Reddington, J. P., Garfield, D., Daza, R. M., Marco-Ferreres, R., Christiansen, L., Qiu, X., Steemers, F. J., Trapnell, C., Shendure, J., & Furlong, E. E. (2017). The cis-regulatory dynamics of embryonic development at single cell resolution. BioRxiv. doi:10.1101/166066
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  ABSTRACT Single cell measurements of gene expression are providing new insights into lineage commitment, yet the regulatory changes underlying individual cell trajectories remain elusive. Here, we profiled chromatin accessibility in over 20,000 single nuclei across multiple stages of Drosophila embryogenesis. Our data reveal heterogeneity in the regulatory landscape prior to gastrulation that reflects anatomical position, a feature that aligns with future cell fate. During mid embryogenesis, tissue granularity emerges such that cell types can be inferred by their chromatin accessibility, while maintaining a signature of their germ layer of origin. We identify over 30,000 distal elements with tissue-specific accessibility. Using transgenic embryos, we tested the germ layer specificity of a subset of predicted enhancers, achieving near-perfect accuracy. Overall, these data demonstrate the power of shotgun single cell profiling of embryos to resolve dynamic changes in open chromatin during development, and to uncover the cis -regulatory programs of germ layers and cell types.
• Gasperini, M., Findlay, G. M., McKenna, A., Milbank, J. H., Lee, C., Zhang, M. D., Cusanovich, D. A., & Shendure, J. (2017). CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions. American Journal of Human Genetics, 101(Issue 2). doi:10.1016/j.ajhg.2017.06.010
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  The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion (“ScanDel”). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.
• Gasperini, M., Findlay, G. M., McKenna, A., Milbank, J. H., Lee, C., Zhang, M. D., Cusanovich, D. A., & Shendure, J. (2017). CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions. American journal of human genetics, 101(2), 192-205.
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  The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.
• Cusanovich, D. A., Caliskan, M., Billstrand, C., Michelini, K., Chavarria, C., De Leon, S., Mitrano, A., Lewellyn, N., Elias, J. A., Chupp, G. L., Lang, R. M., Shah, S. J., Decara, J. M., Gilad, Y., & Ober, C. (2016). Integrated analyses of gene expression and genetic association studies in a founder population. Human Molecular Genetics, 25(Issue 10). doi:10.1093/hmg/ddw061
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  Genome-wide association studies (GWASs) have become a standard tool for dissecting genetic contributions to disease risk. However, these studies typically require extraordinarily large sample sizes to be adequately powered. Strategies that incorporate functional information alongside genetic associations have proved successful in increasing GWAS power. Following this paradigm, we present the results of 20 different genetic association studies for quantitative traits related to complex diseases, conducted in the Hutterites of South Dakota. To boost the power of these association studies, we collected RNA-sequencing data from lymphoblastoid cell lines for 431 Hutterite individuals. We then used Sherlock, a tool that integrates GWAS and expression quantitative trait locus (eQTL) data, to identify weak GWAS signals that are also supported by eQTL data. Using this approach, we found novel associations with quantitative phenotypes related to cardiovascular disease, including carotid intima-media thickness, left atrial volume index, monocyte count and serum YKL-40 levels.
• Cusanovich, D. A., Caliskan, M., Billstrand, C., Michelini, K., Chavarria, C., De Leon, S., Mitrano, A., Lewellyn, N., Elias, J. A., Chupp, G. L., Lang, R. M., Shah, S. J., Decara, J. M., Gilad, Y., & Ober, C. (2016). Integrated analyses of gene expression and genetic association studies in a founder population. Human molecular genetics, 25(10), 2104-2112.
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  Genome-wide association studies (GWASs) have become a standard tool for dissecting genetic contributions to disease risk. However, these studies typically require extraordinarily large sample sizes to be adequately powered. Strategies that incorporate functional information alongside genetic associations have proved successful in increasing GWAS power. Following this paradigm, we present the results of 20 different genetic association studies for quantitative traits related to complex diseases, conducted in the Hutterites of South Dakota. To boost the power of these association studies, we collected RNA-sequencing data from lymphoblastoid cell lines for 431 Hutterite individuals. We then used Sherlock, a tool that integrates GWAS and expression quantitative trait locus (eQTL) data, to identify weak GWAS signals that are also supported by eQTL data. Using this approach, we found novel associations with quantitative phenotypes related to cardiovascular disease, including carotid intima-media thickness, left atrial volume index, monocyte count and serum YKL-40 levels.
• Ramani, V., Cusanovich, D. A., Hause, R. J., Ma, W., Qiu, R., Deng, X., Blau, C. A., Disteche, C. M., Noble, W. S., Shendure, J., & Duan, Z. (2016). Mapping 3D genome architecture through in situ DNase Hi-C. Nature Protocols, 11(Issue 11). doi:10.1038/nprot.2016.126
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  With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor. The resulting ligation products are optionally sheared, affinity-purified via streptavidin bead immobilization, and subjected to traditional next-generation library preparation for Illumina paired-end sequencing. Importantly, in situ DNase Hi-C obviates the dependence on a restriction enzyme to digest chromatin, instead relying on the endonuclease DNase I. Libraries generated by in situ DNase Hi-C have a higher effective resolution than traditional Hi-C libraries, which makes them valuable in cases in which high sequencing depth is allowed for, or when hybrid capture technologies are expected to be used. The protocol described here, which involves â 1/44 d of bench work, is optimized for the study of mammalian cells, but it can be broadly applicable to any cell or tissue of interest, given experimental parameter optimization.
• Ramani, V., Cusanovich, D. A., Hause, R. J., Ma, W., Qiu, R., Deng, X., Blau, C. A., Disteche, C. M., Noble, W. S., Shendure, J., & Duan, Z. (2016). Mapping 3D genome architecture through in situ DNase Hi-C. Nature protocols, 11(11), 2104-21.
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  With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor. The resulting ligation products are optionally sheared, affinity-purified via streptavidin bead immobilization, and subjected to traditional next-generation library preparation for Illumina paired-end sequencing. Importantly, in situ DNase Hi-C obviates the dependence on a restriction enzyme to digest chromatin, instead relying on the endonuclease DNase I. Libraries generated by in situ DNase Hi-C have a higher effective resolution than traditional Hi-C libraries, which makes them valuable in cases in which high sequencing depth is allowed for, or when hybrid capture technologies are expected to be used. The protocol described here, which involves ∼4 d of bench work, is optimized for the study of mammalian cells, but it can be broadly applicable to any cell or tissue of interest, given experimental parameter optimization.
• Cusanovich, D. A., Daza, R., Adey, A., Pliner, H. A., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C., & Shendure, J. (2015). Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science, 348(6237), 910-4.
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  Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from more than 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress toward a human cell atlas.
• Cusanovich, D. A., Daza, R., Adey, A., Pliner, H. A., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C., & Shendure, J. (2015). Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science, 348(Issue 6237). doi:10.1126/science.aab1601
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  Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from more than 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress toward a human cell atlas.
• Davenport, E. R., Cusanovich, D. A., Michelini, K., Barreiro, L. B., Ober, C., & Gilad, Y. (2015). Genome-Wide Association Studies of the Human Gut Microbiota. PloS one, 10(11), e0140301.
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  The bacterial composition of the human fecal microbiome is influenced by many lifestyle factors, notably diet. It is less clear, however, what role host genetics plays in dictating the composition of bacteria living in the gut. In this study, we examined the association of ~200K host genotypes with the relative abundance of fecal bacterial taxa in a founder population, the Hutterites, during two seasons (n = 91 summer, n = 93 winter, n = 57 individuals collected in both). These individuals live and eat communally, minimizing variation due to environmental exposures, including diet, which could potentially mask small genetic effects. Using a GWAS approach that takes into account the relatedness between subjects, we identified at least 8 bacterial taxa whose abundances were associated with single nucleotide polymorphisms in the host genome in each season (at genome-wide FDR of 20%). For example, we identified an association between a taxon known to affect obesity (genus Akkermansia) and a variant near PLD1, a gene previously associated with body mass index. Moreover, we replicate a previously reported association from a quantitative trait locus (QTL) mapping study of fecal microbiome abundance in mice (genus Lactococcus, rs3747113, P = 3.13 x 10-7). Finally, based on the significance distribution of the associated microbiome QTLs in our study with respect to chromatin accessibility profiles, we identified tissues in which host genetic variation may be acting to influence bacterial abundance in the gut.
• Davenport, E. R., Cusanovich, D. A., Michelini, K., Barreiro, L. B., Ober, C., & Gilad, Y. (2015). Genome-wide association studies of the human gut microbiota. PLoS ONE, 10(Issue 11). doi:10.1371/journal.pone.0140301
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  The bacterial composition of the human fecal microbiome is influenced by many lifestyle factors, notably diet. It is less clear, however, what role host genetics plays in dictating the composition of bacteria living in the gut. In this study, we examined the association of ∼200K host genotypes with the relative abundance of fecal bacterial taxa in a founder population, the Hutterites, during two seasons (n = 91 summer, n = 93 winter, n = 57 individuals collected in both). These individuals live and eat communally, minimizing variation due to environmental exposures, including diet, which could potentially mask small genetic effects. Using a GWAS approach that takes into account the relatedness between subjects, we identified at least 8 bacterial taxa whose abundances were associated with single nucleotide polymorphisms in the host genome in each season (at genome-wide FDR of 20%). For example, we identified an association between a taxon known to affect obesity (genus Akkermansia) and a variant near PLD1, a gene previously associated with body mass index. Moreover, we replicate a previously reported association from a quantitative trait locus (QTL) mapping study of fecal microbiome abundance in mice (genus Lactococcus, rs3747113, P = 3.13 × 10-7). Finally, based on the significance distribution of the associated microbiome QTLs in our study with respect to chromatin accessibility profiles, we identified tissues in which host genetic variation may be acting to influence bacterial abundance in the gut.
• Cusanovich, D. A. (2014). The Functional Consequences of Variation in Transcription Factor Binding. PLoS Genetics, 10(3), e1004226. doi:10.1371/journal.pgen.1004226
• Cusanovich, D. A., Pavlovic, B., Pritchard, J. K., & Gilad, Y. (2014). The Functional Consequences of Variation in Transcription Factor Binding. PLoS Genetics, 10(Issue 3). doi:10.1371/journal.pgen.1004226
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  One goal of human genetics is to understand how the information for precise and dynamic gene expression programs is encoded in the genome. The interactions of transcription factors (TFs) with DNA regulatory elements clearly play an important role in determining gene expression outputs, yet the regulatory logic underlying functional transcription factor binding is poorly understood. Many studies have focused on characterizing the genomic locations of TF binding, yet it is unclear to what extent TF binding at any specific locus has functional consequences with respect to gene expression output. To evaluate the context of functional TF binding we knocked down 59 TFs and chromatin modifiers in one HapMap lymphoblastoid cell line. We then identified genes whose expression was affected by the knockdowns. We intersected the gene expression data with transcription factor binding data (based on ChIP-seq and DNase-seq) within 10 kb of the transcription start sites of expressed genes. This combination of data allowed us to infer functional TF binding. Using this approach, we found that only a small subset of genes bound by a factor were differentially expressed following the knockdown of that factor, suggesting that most interactions between TF and chromatin do not result in measurable changes in gene expression levels of putative target genes. We found that functional TF binding is enriched in regulatory elements that harbor a large number of TF binding sites, at sites with predicted higher binding affinity, and at sites that are enriched in genomic regions annotated as "active enhancers.". © 2014 Cusanovich et al.
• Cusanovich, D. A. (2013). Integrative genomics approaches to understanding the role of gene regulation in human evolution, disease, and cellular networks: A triptych. Doctoral Thesis.
• Cusanovich, D. A. (2013). Primate transcript and protein expression levels evolve under compensatory selection pressures.. Science, 342(6162), 1100-1104. doi:10.1126/science.1242379
• Khan, Z., Ford, M. J., Cusanovich, D. A., Mitrano, A., Pritchard, J. K., & Gilad, Y. (2013). Primate transcript and protein expression levels evolve under compensatory selection pressures. Science, 342(Issue 6162). doi:10.1126/science.1242379
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  Changes in gene regulation have likely played an important role in the evolution of primates. Differences in messenger RNA (mRNA) expression levels across primates have often been documented; however, it is not yet known to what extent measurements of divergence in mRNA levels reflect divergence in protein expression levels, which are probably more important in determining phenotypic differences. We used high-resolution, quantitative mass spectrometry to collect protein expression measurements from human, chimpanzee, and rhesus macaque lymphoblastoid cell lines and compared them to transcript expression data from the same samples. We found dozens of genes with significant expression differences between species at the mRNA level yet little or no difference in protein expression. Overall, our data suggest that protein expression levels evolve under stronger evolutionary constraint than mRNA levels.
• Caliskan, M., Cusanovich, D. A., Ober, C., & Gilad, Y. (2012). The effects of EBV transformation on gene expression levels and methylation profiles. Human molecular genetics. doi:10.1093/hmg/dds027
• Cusanovich, D. A. (2012). Regulatory element copy number differences shape primate expression profiles. Proceedings of the National Academy of Sciences of the United States of America.
• Cusanovich, D. A. (2012). The combination of a genome-wide association study of lymphocyte count and analysis of gene expression data reveals novel asthma candidate genes. Human Molecular Genetics.
• Cusanovich, D. A., Billstrand, C., Zhou, X., Chavarria, C., De Leon, S., Michelini, K., Pai, A. A., Ober, C., & Gilad, Y. (2012). The combination of a genome-wide association study of lymphocyte count and analysis of gene expression data reveals novel asthma candidate genes. Human Molecular Genetics, 21(Issue 9). doi:10.1093/hmg/dds021
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  Recent genome-wide association studies (GWAS) have identified a number of novel genetic associations with complex human diseases. In spite of these successes, results from GWAS generally explain only a small proportion of disease heritability, an observation termed the 'missing heritability problem'. Several sources for the missing heritability have been proposed, including the contribution of many common variants with small individual effect sizes, which cannot be reliably found using the standard GWAS approach. The goal of our study was to explore a complimentary approach, which combines GWAS results with functional data in order to identify novel genetic associations with small effect sizes. To do so, we conducted a GWAS for lymphocyte count, a physiologic quantitative trait associated with asthma, in 462 Hutterites. In parallel, we performed a genome-wide gene expression study in lymphoblastoid cell lines from 96 Hutterites. We found significant support for genetic associations using the GWAS data when we considered variants near the 193 genes whose expression levels across individuals were most correlated with lymphocyte counts. Interestingly, these variants are also enriched with signatures of an association with asthma susceptibility, an observation we were able to replicate. The associated loci include genes previously implicated in asthma susceptibility as well as novel candidate genes enriched for functions related to T cell receptor signaling and adenosine triphosphate synthesis. Our results, therefore, establish a new set of asthma susceptibility candidate genes. More generally, our observations support the notion that many loci of small effects influence variation in lymphocyte count and asthma susceptibility. © The Author 2012. Published by Oxford University Press. All rights reserved.
• Iskow, R. C., Gokcumen, O., Abyzov, A., Malukiewicz, J., Zhu, Q., Sukumar, A. T., Pai, A. A., Mills, R. E., Habegger, L., Cusanovich, D. A., Rubel, M. A., Perry, G. H., Gerstein, M., Stone, A. C., Gilad, Y., & Lee, C. (2012). Regulatory element copy number differences shape primate expression profiles. Proceedings of the National Academy of Sciences of the United States of America, 109(Issue 31). doi:10.1073/pnas.1205199109
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  Gene expression differences are shaped by selective pressures and contribute to phenotypic differences between species. We identified 964 copy number differences (CNDs) of conserved sequences across three primate species and examined their potential effects on gene expression profiles. Samples with copy number different genes had significantly different expression than samples with neutral copy number. Genes encoding regulatory molecules differed in copy number and were associated with significant expression differences. Additionally, we identified 127 CNDs that were processed pseudogenes and some of which were expressed. Furthermore, there were copy number-different regulatory regions such as ultraconserved elements and long intergenic noncoding RNAs with the potential to affect expression. We postulate that CNDs of these conserved sequences fine-tune developmental pathways by altering the levels of RNA.
• Cusanovich, D. A. (2011). The effects of EBV transformation on gene expression levels and methylation profiles. Human Molecular Genetics.
• Çalişkan, M., Cusanovich, D. A., Ober, C., & Gilad, Y. (2011). The effects of EBV transformation on gene expression levels and methylation profiles. Human Molecular Genetics, 20(Issue 8). doi:10.1093/hmg/ddr041
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  Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) provide a conveniently accessible and renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. A lingering concern, however, is that the changes associated with EBV transformation of B cells reduce the usefulness of LCLs as a surrogate model for primary tissues. To evaluate the validity of this concern, we compared global gene expression and methylation profiles between CD20+ primary B cells sampled from six individuals and six independent replicates of transformed LCLs derived from each sample. These data allowed us to obtain a detailed catalog of the genes and pathways whose regulation is affected by EBV transformation. We found that the expression levels and promoter methylation profiles of more than half of the studied genes were affected by the EBV transformation, including enrichments of genes involved in transcription regulation, cell cycle and immune response. However, we show that most of the differences in gene expression levels between LCLs and B cells are of small magnitude, and that LCLs can often recapitulate the naturally occurring gene expression variation in primary B cells. Thus, our observations suggest that inference of the genetic architecture that underlies regulatory variation in LCLs can typically be generalized to primary B cells. In contrast, inference based on functional studies in LCLs may be more limited to the cell lines. © The Author 2011. Published by Oxford University Press. All rights reserved.
• Cusanovich, D. A. (2009). Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q. Journal of Lipid Research.
• Ober, C., Nord, A. S., Thompson, E. E., Pan, L., Tan, Z., Cusanovich, D., Sun, Y., Nicolae, R., Edelstein, C., Schneider, D. H., Billstrand, C., Pfaffinger, D., Phillips, N., Anderson, R. L., Philips, B., Rajagopalan, R., Hatsukami, T. S., Rieder, M. J., Heagerty, P. J., , Nickerson, D. A., et al. (2009). Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q. Journal of Lipid Research, 50(Issue 5). doi:10.1194/jlr.m800515-jlr200
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  Plasma lipoprotein(a) (Lp[a]) level is an independent risk factor of cardiovascular disease that is under strong genetic control. We conducted a genome-wide association study of plasma Lp(a) in 386 members of a founder population that adheres to a communal lifestyle, proscribes cigarette smoking, and prepares and eats meals communally. We identified associations with 77 single nucleotide polymorphisms (SNPs) spanning 12.5 Mb on chromosome 6q26-q27 that met criteria for genome-wide significance (P ≤ 1.3 × 10-7) and were within or flanking nine genes, including LPA. We show that variation in at least six genes in addition to LPA are significantly associated with Lp(a) levels independent of each other and of the kringle IV repeat polymorphism in the LPA gene. One novel SNP in intron 37 of the LPA gene was also associated with Lp(a) levels and carotid artery disease number in unrelated Caucasians (P = 7.3 × 10-12 and 0.024, respectively), also independent of kringle IV number. This study suggests a complex genetic architecture of Lp(a) levels that may involve multiple loci on chromosome 6q26-q27. Copyright © 2009 by the American Society for Biochemistry and Molecular Biology, Inc.

Proceedings Publications

• Galligan, J. J., Trujillo, M. N., Jennings, E. Q., Zhang, H., Kitamura, N., Hoffman, E. A., Snyder, N. W., & Cusanovich, D. A. (2023). Lactoylglutathione mediates protein lactoylation and inflammatory signaling.. In Free Radical Biology and Medicine.
• Ahmed, M. H., Allison, S., Anderson, D., Carr, T. F., Cusanovich, D. A., Halonen, M., Kang, H., Lopez, S. G., Martinez, F. J., Spangenberg, A., & Welfley, H. (2021). Airway Sampling Techniques for Single-Cell Profiling of Lung Biology. In American Thoracic Society.
• Cusanovich, D. A., Pavlovic, B. J., Pritchard, J. K., & Gilad, Y. (2014). ASHG 2013 Poster - The Functional Consequences of Variation in Transcription Factor Binding. In American Society of Human Genetics.

Presentations

• Cusanovich, D. A. (2024). Preliminary molecular support for insulin-associated asthma in CRS using single-cell multiomics. A2DRC Seminar Series.
• Cusanovich, D. A. (2024, March). Dynamic Gene Regulation in Development and Disease: Using Single-Cell (and Multi-Cell) Genomics to Search for "Crouching Tigers" in the Human Genome. UTMB Department of Biochemsitry and Molecular Biology Seminar. Galveston, TX: University of Texas Medical Branch.
• Cusanovich, D. A. (2023). Lactoylglutathione mediates protein lactoylation and inflammatory signaling.. Free Radical Biology and Medicine.
• Cusanovich, D. A. (2022, May). Analysis strategies for single-cell ATAC-seq. Aegis Consortium Bioinformatics Workshop.
• Cusanovich, D. A. (2022, May). Building Tools to Understand Single-cell Genomics at Scale. UA Research Day. HSIB: COM.
• Cusanovich, D. A. (2020, April). Novel Strategies for SARS-CoV-2 testing. A2DRC Biweekly Reesearch Update Seminar. University of Arizona: Ashtma and Airway Disease Research Center.
• Cusanovich, D. A. (2020, February). Single-cell epigenomics yields insights into dynamic gene regulation in development and disease. Gut Group Monthly Meeting. University of Arizona: Gut Group.
• Cusanovich, D. A. (2020, January). Single-cell Epigenomics:Towards an Understanding of Chromatin Dynamics in Development & Disease. Genetics Graduate Interdisciplinary Program Seminar Series Invited Speaker. University of Arizona: Genetics GIDP.
• Cusanovich, D. A. (2020, July). Cusanovich Lab COVID-19 EffortsDeveloping Sequencing-based Diagnostics. CMM Weekly COVID-19 Update Seminar. University of Arizona: Cellular and Molecular Medicine.
• Cusanovich, D. A. (2020, July). Swab-Seq in the Southwest: The path to TaqPath, Saliva, CIV-seq. Swab-Seq Development Working Group 2020.
• Cusanovich, D. A. (2019, April). Single-cell Genomics via Combinatorial Indexing:A Scalable and Adaptable Framework. Molecular, Cellular and Tissue Bioengineering Symposium. ASU: ASU.
• Cusanovich, D. A. (2019, March). Single-cell Genomics via Combinatorial Indexing: A Scalable and Adaptable Framework. Single Cell Genomics Day. New York, NY: New York Genome Center.
• Cusanovich, D. A. (2019, October). Single-cell Genomic Technologies - A Call to Arms. Innovations and Inventions. University of Arizona: College of Medicine.
• Cusanovich, D. A. (2019, September). Single-cell Epigenomics:Towards an Understanding of Chromatin Dynamics in Development & Disease. Cancer Biology Seminar Series Invited Speaker. University of Arizona: CBIO.
• Cusanovich, D. A. (2019, September). Single-cell Genomics: from the bench to the lung. Joint Biology Research Retreat. Biosphere 2: University of Arizona.
• Cusanovich, D. A. (2018, October). A discussion of the potential value of single-cell genomic approaches to asthma disease models. A2DRC Biweekly Reesearch Update Seminar. University of Arizona: Ashtma and Airway Disease Research Center.

Poster Presentations

• Cusanovich, D. A. (2024, November). Advancing Single-Cell Multiomics Profiling with mTEA-seq: In-sights into Early-Life Insulin-Featured Asthma. American Society of Human Genetics. Denver, CO.
• Cusanovich, D. A. (2023). Expanding the scalability and flexibility of a microfluidic single-cell chromatin accessibility assay with transposase-based pre-indexing. The conceptual power of single cell biology (Cell Symposium).