Darren A Cusanovich

Interests

Research

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

Teaching

Genomics, Bioinformatics, Complex disease biology ,Molecular biology

Courses

Scholarly Contributions

Journals/Publications

• 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., 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.
• 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.
• 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.
• 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
• 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, 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.
• 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.
• 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(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.
• 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.
• Cusanovich, D. A. (2017). Comprehensive single-cell transcriptional profiling of a multicellular organism. Science.
• 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(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(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.
• 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.
• 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. (2013). Primate transcript and protein expression levels evolve under compensatory selection pressures.. Science, 342(6162), 1100-1104. doi:10.1126/science.1242379
• 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. (2011). The effects of EBV transformation on gene expression levels and methylation profiles. Human Molecular Genetics.
• Cusanovich, D. A. (2009). Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q. Journal of Lipid Research.

Presentations

• Cusanovich, D. A. (2023). CC16 Induces Pulmonary Epithelial-Driven SPLUNC1 Expression by Signaling through VLA-2.. 2023 ERS International CongressERS.
• 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. (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).