Casey E Romanoski

Interests

Research

Genetics, Genomics, Gene Regulation, Functional Genetics, Transcription, Non-Coding Functional Genetic Variation, Endothelial Cells, Atherosclerosis, Inflammation, Complex Disease Genetics, Gene-by-Environment Interactions, High-Throughput Sequencing Technologies, Bioinformatics, Regulatory Networks, Transcription Factors, Enhancers, Macrophages

Teaching

I am interested in teaching students about the underpinnings of molecular genetics, high-throughput 'omics', and computational approaches as they are being applied to better understand complex disease biology. I am dedicated to mentoring students to excel in both written and oral scientific communication. I am particularly interested in bolstering success of women and other underrepresented groups in the sciences.

Courses

Scholarly Contributions

Books

• Romanoski, C. E. (2010). Systems genetics of endothelial cell activation by oxidized phospholipids.

Chapters

• Romanoski, C. E. (2016). Genome-Wide Assessment Reveals Marked Differences In The Transcriptomes Of Mononuclear Phagocytes In The Developing Lung. In C21. APPLYING'OMICS TECHNOLOGIES TO REVEAL GENE REGULATORS AND BIOMARKERS IN LUNG DEVELOPMENT AND DISEASE.

Journals/Publications

• Li, J., Zhu, J., Gray, O., Sobreira, D. R., Wu, D., Huang, R. T., Miao, B., Sakabe, N. J., Krause, M. D., Kaikkonen, M. U., Romanoski, C. E., Nobrega, M. A., & Fang, Y. (2024). Mechanosensitive super-enhancers regulate genes linked to atherosclerosis in endothelial cells. The Journal of cell biology, 223(3).
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  Vascular homeostasis and pathophysiology are tightly regulated by mechanical forces generated by hemodynamics. Vascular disorders such as atherosclerotic diseases largely occur at curvatures and bifurcations where disturbed blood flow activates endothelial cells while unidirectional flow at the straight part of vessels promotes endothelial health. Integrated analysis of the endothelial transcriptome, the 3D epigenome, and human genetics systematically identified the SNP-enriched cistrome in vascular endothelium subjected to well-defined atherosclerosis-prone disturbed flow or atherosclerosis-protective unidirectional flow. Our results characterized the endothelial typical- and super-enhancers and underscored the critical regulatory role of flow-sensitive endothelial super-enhancers. CRISPR interference and activation validated the function of a previously unrecognized unidirectional flow-induced super-enhancer that upregulates antioxidant genes NQO1, CYB5B, and WWP2, and a disturbed flow-induced super-enhancer in endothelium which drives prothrombotic genes EDN1 and HIVEP in vascular endothelium. Our results employing multiomics identify the cis-regulatory architecture of the flow-sensitive endothelial epigenome related to atherosclerosis and highlight the regulatory role of super-enhancers in mechanotransduction mechanisms.
• Adelus, M. L., Ding, J., Tran, B. T., Conklin, A. C., Golebiewski, A. K., Stolze, L. K., Whalen, M. B., Cusanovich, D., & Romanoski, C. E. (2023). 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. bioRxiv : the preprint server for biology.
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  Endothelial cells (ECs), macrophages, and vascular smooth muscle cells (VSMCs) are major cell types in atherosclerosis progression, and heterogeneity in EC sub-phenotypes are becoming increasingly appreciated. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking.
• Afshar, Y., Ma, F., Quach, A., Jeong, A., Sunshine, H. L., Freitas, V., Jami-Alahmadi, Y., Helaers, R., Li, X., Pellegrini, M., Wohlschlegel, J. A., Romanoski, C. E., Vikkula, M., & Iruela-Arispe, M. L. (2023). Transcriptional drifts associated with environmental changes in endothelial cells. eLife, 12.
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  Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA sequencing and protein expression by liquid chromatography-mass spectrometry directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.
• Boutagy, N. E., Fowler, J. W., Grabinska, K. A., Cardone, R., Sun, Q., Vazquez, K. R., Whalen, M. B., Zhu, X., Chakraborty, R., Martin, K. A., Simons, M., Romanoski, C. E., Kibbey, R. G., & Sessa, W. C. (2023). TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes. Proceedings of the National Academy of Sciences of the United States of America, 120(38), e2218150120.
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  The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and C-glucose and C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α ( gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.
• Fowler, J. W., Boutagy, N. E., Zhang, R., Horikami, D., Whalen, M. B., Romanoski, C. E., & Sessa, W. C. (2023). SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6. Journal of lipid research, 64(8), 100411.
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  The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.
• Shin, J., Hong, J., Edwards-Glenn, J., Krukovets, I., Tkachenko, S., Adelus, M. L., Romanoski, C. E., Rajagopalan, S., Podrez, E., Byzova, T. V., Stenina-Adongravi, O., & Cherepanova, O. A. (2023). Unraveling the Role of Sex in Endothelial Cell Dysfunction: Evidence From Lineage Tracing Mice and Cultured Cells. Arteriosclerosis, thrombosis, and vascular biology, 44(1), 238-253.
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  Biological sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (ECs) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC.
• Civelek, M., Fogelman, A. M., Hedrick, C. C., Lusis, A. J., Romanoski, C. E., & Witztum, J. L. (2022). In memoriam: Judith A. Berliner, Ph.D. Journal of lipid research, 63(4), 100180.
• Kariotis, S., Jammeh, E., Swietlik, E. M., Pickworth, J. A., Rhodes, C. J., Otero, P., Wharton, J., Iremonger, J., Dunning, M. J., Pandya, D., Mascarenhas, T. S., Errington, N., Thompson, A. A., Romanoski, C. E., Rischard, F., Garcia, J. G., Yuan, J. X., An, T. S., Desai, A. A., , Coghlan, G., et al. (2022). Author Correction: Biological heterogeneity in idiopathic pulmonary arterial hypertension identified through unsupervised transcriptomic profiling of whole blood. Nature communications, 13(1), 7276.
• Shen, Z., Li, R. Z., Prohaska, T. A., Hoeksema, M. A., Spann, N. J., Tao, J., Fonseca, G. J., Le, T., Stolze, L. K., Sakai, M., Romanoski, C. E., & Glass, C. K. (2022). Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs. eLife, 11.
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  Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq. We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5-bp increase to >30-bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.
• Toropainen, A., Stolze, L. K., Örd, T., Whalen, M. B., Torrell, P. M., Link, V. M., Kaikkonen, M. U., & Romanoski, C. E. (2022). Functional noncoding SNPs in human endothelial cells fine-map vascular trait associations. Genome research, 32(3), 409-424.
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  Functional consequences of genetic variation in the noncoding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of functional noncoding SNPs with effects in human endothelial cells (ECs), we utilized our previous molecular quantitative trait locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional noncoding SNPs. Together with information from genome-wide association studies (GWASs) for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5711 variants validated, whose enriched attributes included: (1) mutations to TF binding motifs for ETS or AP-1 that are regulators of the EC state; (2) location in accessible and H3K27ac-marked EC chromatin; and (3) molQTL associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of noncoding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlighted evidence suggesting mechanisms by which noncoding SNPs at two loci affect risk for pulse pressure/large artery stroke and abdominal aortic aneurysm through respective effects on transcriptional regulation of and Together, we highlight the attributes and context dependence of functional noncoding SNPs and provide new mechanisms underlying vascular disease risk.
• Chella Krishnan, K., Vergnes, L., Acín-Pérez, R., Stiles, L., Shum, M., Ma, L., Mouisel, E., Pan, C., Moore, T. M., Péterfy, M., Romanoski, C. E., Reue, K., Björkegren, J. L., Laakso, M., Liesa, M., & Lusis, A. J. (2021). Sex-specific genetic regulation of adipose mitochondria and metabolic syndrome by Ndufv2. Nature metabolism, 3(11), 1552-1568.
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  We have previously suggested a central role for mitochondria in the observed sex differences in metabolic traits. However, the mechanisms by which sex differences affect adipose mitochondrial function and metabolic syndrome are unclear. Here we show that in both mice and humans, adipose mitochondrial functions are elevated in females and are strongly associated with adiposity, insulin resistance and plasma lipids. Using a panel of diverse inbred strains of mice, we identify a genetic locus on mouse chromosome 17 that controls mitochondrial mass and function in adipose tissue in a sex- and tissue-specific manner. This locus contains Ndufv2 and regulates the expression of at least 89 mitochondrial genes in females, including oxidative phosphorylation genes and those related to mitochondrial DNA content. Overexpression studies indicate that Ndufv2 mediates these effects by regulating supercomplex assembly and elevating mitochondrial reactive oxygen species production, which generates a signal that increases mitochondrial biogenesis.
• Conklin, A. C., Nishi, H., Schlamp, F., Örd, T., Õunap, K., Kaikkonen, M. U., Fisher, E. A., & Romanoski, C. E. (2021). Meta-Analysis of Smooth Muscle Lineage Transcriptomes in Atherosclerosis and Their Relationships to In Vitro Models. Immunometabolism, 3(3).
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  Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity in atherosclerotic plaques, and among other approaches, has been modeled in vitro by cholesterol loading.
• Hartiala, J. A., Han, Y., Jia, Q., Hilser, J. R., Huang, P., Gukasyan, J., Schwartzman, W. S., Cai, Z., Biswas, S., Trégouët, D. A., Smith, N. L., , I. C., , C. C., , G. C., Seldin, M., Pan, C., Mehrabian, M., Lusis, A. J., Bazeley, P., , Sun, Y. V., et al. (2021). Genome-wide analysis identifies novel susceptibility loci for myocardial infarction. European heart journal, 42(9), 919-933.
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  While most patients with myocardial infarction (MI) have underlying coronary atherosclerosis, not all patients with coronary artery disease (CAD) develop MI. We sought to address the hypothesis that some of the genetic factors which establish atherosclerosis may be distinct from those that predispose to vulnerable plaques and thrombus formation.
• Hartiala, J. A., Han, Y., Jia, Q., Huang, P., Woodward, N. C., Gukasyan, H., Kurt, Z., Stolze, L. K., Tregouet, D., Smith, N. L., Seldin, M., Mehrabian, M., Lusis, A. J., Wilson, W. H., Yang, X., Romanoski, C. E., Hazen, S. L., & Allayee, H. (2018). Genome-Wide Association Studies in >500,000 Individuals Identify Novel Loci for Coronary Artery Disease and Myocardial Infarction. Nature Communications.
• Kariotis, S., Jammeh, E., Swietlik, E. M., Pickworth, J. A., Rhodes, C. J., Otero, P., Wharton, J., Iremonger, J., Dunning, M. J., Pandya, D., Mascarenhas, T. S., Errington, N., Thompson, A. A., Romanoski, C. E., Rischard, F., Garcia, J. G., Yuan, J. X., An, T. S., Desai, A. A., , Coghlan, G., et al. (2021). Biological heterogeneity in idiopathic pulmonary arterial hypertension identified through unsupervised transcriptomic profiling of whole blood. Nature communications, 12(1), 7104.
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  Idiopathic pulmonary arterial hypertension (IPAH) is a rare but fatal disease diagnosed by right heart catheterisation and the exclusion of other forms of pulmonary arterial hypertension, producing a heterogeneous population with varied treatment response. Here we show unsupervised machine learning identification of three major patient subgroups that account for 92% of the cohort, each with unique whole blood transcriptomic and clinical feature signatures. These subgroups are associated with poor, moderate, and good prognosis. The poor prognosis subgroup is associated with upregulation of the ALAS2 and downregulation of several immunoglobulin genes, while the good prognosis subgroup is defined by upregulation of the bone morphogenetic protein signalling regulator NOG, and the C/C variant of HLA-DPA1/DPB1 (independently associated with survival). These findings independently validated provide evidence for the existence of 3 major subgroups (endophenotypes) within the IPAH classification, could improve risk stratification and provide molecular insights into the pathogenesis of IPAH.
• L, S. K., A, C. C., M, W. B., M, R. L., K, O., I, S., A, T., T, O., J, L., A, S., Y, F., M, K. U., & Romanoski, C. E. (2019). Molecular Quantitative Trait Locus Mapping In Human Endothelial Cells Identifies Regulatory SNPs Underlying Gene Expression and Complex Disease Traits. American Journal of Human Genetics.
• Padi, M., Mouneimne, G., Romanoski, C. E., & Cusanovich, D. A. (2019). Breast tumor stiffness instructs bone metastasis via mechanical memory. BioRxiv.
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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.
• Selvarajan, I., Toropainen, A., Garske, K. M., López Rodríguez, M., Ko, A., Miao, Z., Kaminska, D., Õunap, K., Örd, T., Ravindran, A., Liu, O. H., Moreau, P. R., Jawahar Deen, A., Männistö, V., Pan, C., Levonen, A. L., Lusis, A. J., Heikkinen, S., Romanoski, C. E., , Pihlajamäki, J., et al. (2021). Integrative analysis of liver-specific non-coding regulatory SNPs associated with the risk of coronary artery disease. American journal of human genetics, 108(3), 411-430.
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  Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function.
• 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(13), 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.
• Örd, T., Õunap, K., Stolze, L. K., Aherrahrou, R., Nurminen, V., Toropainen, A., Selvarajan, I., Lönnberg, T., Aavik, E., Ylä-Herttuala, S., Civelek, M., Romanoski, C. E., & Kaikkonen, M. U. (2021). Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci. Circulation research, 129(2), 240-258.
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  [Figure: see text].
• 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.
• Johnson, M. D., Younis, U. S., Menghani, S. V., Addison, K. J., Whalen, M., Pilon, A. L., Cress, A. E., Polverino, F., Romanoski, C., Kraft, M., Martinez, F. D., Guerra, S., & Ledford, J. G. (2020). CC16 Binding to α4β1 Integrin (VLA-4) Protects Against Infection. American journal of respiratory and critical care medicine.
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  Club cell secretory protein (CC16) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases: asthma and COPD. While exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating anti-inflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, Leucine-Valine-Aspartic Acid (LVD), a known integrin binding motif. Recombinant CC16 was generated with and without the putative integrin binding site. A Mycoplasma pneumoniae mouse model and a flourescent cellular adhesion assay were used to determine the impact of the LVD site in regards to CC16 function during live infection and on cellular adhesion during inflammatory conditions. CC16 bound to integrin alpha 4 and beta 1 (α4β1), also known as the adhesion molecule very late antigen-4 (VLA-4), dependent on the presence of the LVD integrin binding motif. During infection, rCC16 rescued lung function parameters both when administered to the lung and intravenously, but only when the LVD integrin binding site is intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.
• Merritt, E. F., Johnson, H. J., Wong, Z. S., Buntzman, A. S., Conklin, A. C., Cabral, C. M., Romanoski, C. E., Boyle, J. P., & Koshy, A. A. (2020). Transcriptional Profiling Suggests T Cells Cluster around Neurons Injected with Toxoplasma gondii Proteins. mSphere, 5(5).
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  's tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that causes in humans. Our recent work has shown that neurons are the primary CNS cell with which interacts and which it infects This predilection for neurons suggests that 's persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on -host cell interactions has been done and in nonneuronal cells. We address this gap by utilizing our -Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled -injected neurons (TINs), Bystander neurons (nearby non--injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8 T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8 T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8 T cells. Like other persistent intracellular pathogens, , a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding 's persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron- interaction By transcriptionally profiling neurons manipulated by , we unexpectedly revealed that immune cells, and specifically CD8 T cells, appear to cluster around these neurons, suggesting that CD8 T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8 T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.
• Romanoski, C. E., Qi, X., Sangam, S., Vanderpool, R. R., Stearman, R. S., Conklin, A., Gonzalez-Garay, M., Rischard, F., Ayon, R. J., Wang, J., Simonson, T., Babicheva, A., Shi, Y., Tang, H., Makino, A., Kanthi, Y., Geraci, M. W., Garcia, J. G., Yuan, J. X., & Desai, A. A. (2020). Transcriptomic profiles in pulmonary arterial hypertension associate with disease severity and identify novel candidate genes. Pulmonary circulation, 10(4), 2045894020968531.
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  Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of pulmonary arterial hypertension cases versus controls, highlighting potentially novel candidate genes involved in pulmonary arterial hypertension development.
• Sajti, E., Link, V. M., Ouyang, Z., Spann, N. J., Westin, E., Romanoski, C. E., Fonseca, G. J., Prince, L. S., & Glass, C. K. (2020). Transcriptomic and epigenetic mechanisms underlying myeloid diversity in the lung. Nature immunology.
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  The lung is inhabited by resident alveolar and interstitial macrophages as well as monocytic cells that survey lung tissues. Each cell type plays distinct functional roles under homeostatic and inflammatory conditions, but mechanisms establishing their molecular identities and functional potential remain poorly understood. In the present study, systematic evaluation of transcriptomes and open chromatin of alveolar macrophages (AMs), interstitial macrophages (IMs) and lung monocytes from two mouse strains enabled inference of common and cell-specific transcriptional regulators. We provide evidence that these factors drive selection of regulatory landscapes that specify distinct phenotypes of AMs and IMs and entrain qualitatively different responses to toll-like receptor 4 signaling in vivo. These studies reveal a striking divergence in a fundamental innate immune response pathway in AMs and establish a framework for further understanding macrophage diversity in the lung.
• Stolze, L. K., Conklin, A. C., Whalen, M. B., López Rodríguez, M., Õunap, K., Selvarajan, I., Toropainen, A., Örd, T., Li, J., Eshghi, A., Solomon, A. E., Fang, Y., Kaikkonen, M. U., & Romanoski, C. E. (2020). Systems Genetics in Human Endothelial Cells Identifies Non-coding Variants Modifying Enhancers, Expression, and Complex Disease Traits. American journal of human genetics, 106(6), 748-763.
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  The identification of causal variants and mechanisms underlying complex disease traits in humans is important for the progress of human disease genetics; this requires finding strategies to detect functional regulatory variants in disease-relevant cell types. To achieve this, we collected genetic and transcriptomic data from the aortic endothelial cells of up to 157 donors and four epigenomic phenotypes in up to 44 human donors representing individuals of both sexes and three major ancestries. We found thousands of expression quantitative trait loci (eQTLs) at all ranges of effect sizes not detected by the Gene-Tissue Expression Project (GTEx) in human tissues, showing that novel biological relationships unique to endothelial cells (ECs) are enriched in this dataset. Epigenetic profiling enabled discovery of over 3,000 regulatory elements whose activity is modulated by genetic variants that most frequently mutated ETS, AP-1, and NF-kB binding motifs, implicating these motifs as governors of EC regulation. Using CRISPR interference (CRISPRi), allele-specific reporter assays, and chromatin conformation capture, we validated candidate enhancer variants located up to 750 kb from their target genes, VEGFC, FGD6, and KIF26B. Regulatory SNPs identified were enriched in coronary artery disease (CAD) loci, and this result has specific implications for PECAM-1, FES, and AXL. We also found significant roles for EC regulatory variants in modifying the traits pulse pressure, blood protein levels, and monocyte count. Lastly, we present two unlinked SNPs in the promoter of MFAP2 that exhibit pleiotropic effects on human disease traits. Together, this supports the possibility that genetic predisposition for complex disease is manifested through the endothelium.
• 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, 60(4), 869-879.
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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 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, and it did not 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.
• Hitzel, J., Lee, E., Zhang, Y., Bibli, S. I., Li, X., Zukunft, S., Pflüger, B., Hu, J., Schürmann, C., Vasconez, A. E., Oo, J. A., Kratzer, A., Kumar, S., Rezende, F., Josipovic, I., Thomas, D., Giral, H., Schreiber, Y., Geisslinger, G., , Fork, C., et al. (2018). Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells. Nature communications, 9(1), 2292.
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  Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.
• Krause, M. D., Huang, R. T., Wu, D., Shentu, T. P., Harrison, D. L., Whalen, M. B., Stolze, L. K., Di Rienzo, A., Moskowitz, I. P., Civelek, M., Romanoski, C. E., & Fang, Y. (2018). Genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics. Proceedings of the National Academy of Sciences of the United States of America, 115(48), E11349-E11358.
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  Biomechanical cues dynamically control major cellular processes, but whether genetic variants actively participate in mechanosensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as strongly associated with CAD/IS; however, the causal mechanism related to this locus remains unknown. Using statistical analyses, assay of transposase accessible chromatin with whole-genome sequencing (ATAC-seq), H3K27ac/H3K4me2 ChIP with whole-genome sequencing (ChIP-seq), and CRISPR interference in human aortic endothelial cells (HAECs), our results demonstrate that rs17114036, a common noncoding polymorphism at 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR-Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional shear stress by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus (caQTL) mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays demonstrate that CAD/IS-protective allele at rs17114036 in PLPP3 intron 5 confers increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS-protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2 (KLF2), which increases the enhancer activity under unidirectional flow. These results demonstrate that a human SNP contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related -regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechanosensing mechanisms.
• Link, V. M., Duttke, S. H., Chun, H. B., Holtman, I. R., Westin, E., Hoeksema, M. A., Abe, Y., Skola, D., Romanoski, C. E., Tao, J., Fonseca, G. J., Troutman, T. D., Spann, N. J., Strid, T., Sakai, M., Yu, M., Hu, R., Fang, R., Metzler, D., , Ren, B., et al. (2018). Analysis of Genetically Diverse Macrophages Reveals Local and Domain-wide Mechanisms that Control Transcription Factor Binding and Function. Cell.
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  Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.
• Link, V. M., Romanoski, C. E., Metzler, D., & Glass, C. K. (2018). MMARGE: Motif Mutation Analysis for Regulatory Genomic Elements. Nucleic acids research, 46(14), 7006-7021.
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  Cell-specific patterns of gene expression are determined by combinatorial actions of sequence-specific transcription factors at cis-regulatory elements. Studies indicate that relatively simple combinations of lineage-determining transcription factors (LDTFs) play dominant roles in the selection of enhancers that establish cell identities and functions. LDTFs require collaborative interactions with additional transcription factors to mediate enhancer function, but the identities of these factors are often unknown. We have shown that natural genetic variation between individuals has great utility for discovering collaborative transcription factors. Here, we introduce MMARGE (Motif Mutation Analysis of Regulatory Genomic Elements), the first publicly available suite of software tools that integrates genome-wide genetic variation with epigenetic data to identify collaborative transcription factor pairs. MMARGE is optimized to work with chromatin accessibility assays (such as ATAC-seq or DNase I hypersensitivity), as well as transcription factor binding data collected by ChIP-seq. Herein, we provide investigators with rationale for each step in the MMARGE pipeline and key differences for analysis of datasets with different experimental designs. We demonstrate the utility of MMARGE using mouse peritoneal macrophages, liver cells, and human lymphoblastoid cells. MMARGE provides a powerful tool to identify combinations of cell type-specific transcription factors while simultaneously interpreting functional effects of non-coding genetic variation.
• Hogan, N. T., Whalen, M. B., Stolze, L. K., Hadeli, N. K., Lam, M. T., Springstead, J. R., Glass, C. K., & Romanoski, C. E. (2017). Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells. eLife, 6.
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  Endothelial cells (ECs) are critical determinants of vascular homeostasis and inflammation, but transcriptional mechanisms specifying their identities and functional states remain poorly understood. Here, we report a genome-wide assessment of regulatory landscapes of primary human aortic endothelial cells (HAECs) under basal and activated conditions, enabling inference of transcription factor networks that direct homeostatic and pro-inflammatory programs. We demonstrate that 43% of detected enhancers are EC-specific and contain SNPs associated to cardiovascular disease and hypertension. We provide evidence that AP1, ETS, and GATA transcription factors play key roles in HAEC transcription by co-binding enhancers associated with EC-specific genes. We further demonstrate that exposure of HAECs to oxidized phospholipids or pro-inflammatory cytokines results in signal-specific alterations in enhancer landscapes and associate with coordinated binding of CEBPD, IRF1, and NFκB. Collectively, these findings identify cis-regulatory elements and corresponding trans-acting factors that contribute to EC identity and their specific responses to pro-inflammatory stimuli.
• Romanoski, C. E. (2017). Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells. eLIFE.
• Romanoski, C. E. (2016). Deleting an Nr4a1 Super-Enhancer Subdomain Ablates Ly6C low Monocytes while Preserving Macrophage Gene Function. Immunity.
• Romanoski, C. E. (2016). Enhancer Transcription And Enhancer Function. The FASEB Journal.
• Romanoski, C. E. (2016). MAFG Is a Transcriptional Repressor of Bile Acid Synthesis and Metabolism (vol 21, pg 298, 2015). CELL METABOLISM.
• Romanoski, C. E. (2016). MAFG Is a Transcriptional Repressor of Bile Acid Synthesis and Metabolism. Cell Metabolism.
• Thomas, G. D., Hanna, R. N., Vasudevan, N. T., Hamers, A. A., Romanoski, C. E., McArdle, S., Ross, K. D., Blatchley, A., Yoakum, D., Hamilton, B. A., Mikulski, Z., Jain, M. K., Glass, C. K., & Hedrick, C. C. (2016). Deleting an Nr4a1 Super-Enhancer Subdomain Ablates Ly6C(low) Monocytes while Preserving Macrophage Gene Function. Immunity, 45(5), 975-987.
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  Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6C(low) monocytes, we present a proof-of-principle approach that addresses these limitations. Combining ChIP-seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6C(low) monocyte development. Mice lacking this enhancer lacked Ly6C(low) monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. Because Nr4a1 regulates inflammatory gene expression and differentiation of Ly6C(low) monocytes, decoupling these processes allows Ly6C(low) monocytes to be studied independently.
• Heinz, S., Romanoski, C. E., Benner, C., & Glass, C. K. (2015). The selection and function of cell type-specific enhancers. Nature reviews. Molecular cell biology, 16(3), 144-54.
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  The human body contains several hundred cell types, all of which share the same genome. In metazoans, much of the regulatory code that drives cell type-specific gene expression is located in distal elements called enhancers. Although mammalian genomes contain millions of potential enhancers, only a small subset of them is active in a given cell type. Cell type-specific enhancer selection involves the binding of lineage-determining transcription factors that prime enhancers. Signal-dependent transcription factors bind to primed enhancers, which enables these broadly expressed factors to regulate gene expression in a cell type-specific manner. The expression of genes that specify cell type identity and function is associated with densely spaced clusters of active enhancers known as super-enhancers. The functions of enhancers and super-enhancers are influenced by, and affect, higher-order genomic organization.
• Romanoski, C. E. (2015). AP-1 and ETS Family Transcription Factors Co-localize at Enhancers in Human Aortic Endothelial Cells. Arteriosclerosis, Thrombosis, and Vascular Biology.
• Romanoski, C. E. (2015). Differentiation enhanced. Nature.
• Romanoski, C. E. (2015). Environment drives selection and function of enhancers controlling tissue-specific macrophage identities. Cell.
• Romanoski, C. E. (2015). Epigenomics: Roadmap for regulation. Nature.
• Romanoski, C. E. (2015). Exploiting genomics and natural genetic variation to decode macrophage enhancers. Trends in immunology.
• Romanoski, C. E. (2015). Siglec receptors impact mammalian lifespan by modulating oxidative stress. Elife.
• Romanoski, C. E. (2015). The Transcriptional Repressor MafG Regulates Cholesterol Catabolism. Arteriosclerosis, Thrombosis, and Vascular Biology.
• Romanoski, C. E. (2015). The selection and function of cell type-specific enhancers. Nature Reviews Molecular Cell Biology.
• Romanoski, C. E., Glass, C. K., Stunnenberg, H. G., Wilson, L., & Almouzni, G. (2015). Epigenomics: Roadmap for regulation. Nature, 518(7539), 314-6.
• Romanoski, C. E., Link, V. M., Heinz, S., & Glass, C. K. (2015). Exploiting genomics and natural genetic variation to decode macrophage enhancers. Trends in immunology, 36(9), 507-18.
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  The mammalian genome contains on the order of a million enhancer-like regions that are required to establish the identities and functions of specific cell types. Here, we review recent studies in immune cells that have provided insight into the mechanisms that selectively activate certain enhancers in response to cell lineage and environmental signals. We describe a working model wherein distinct classes of transcription factors define the repertoire of active enhancers in macrophages through collaborative and hierarchical interactions, and discuss important challenges to this model, specifically providing examples from T cells. We conclude by discussing the use of natural genetic variation as a powerful approach for decoding transcription factor combinations that play dominant roles in establishing the enhancer landscapes, and the potential that these insights have for advancing our understanding of the molecular causes of human disease.
• Schwarz, F., Pearce, O. M., Wang, X., Samraj, A. N., Läubli, H., Garcia, J. O., Lin, H., Fu, X., Garcia-Bingman, A., Secrest, P., Romanoski, C. E., Heyser, C., Glass, C. K., Hazen, S. L., Varki, N., Varki, A., & Gagneux, P. (2015). Siglec receptors impact mammalian lifespan by modulating oxidative stress. eLife, 4.
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  Aging is a multifactorial process that includes the lifelong accumulation of molecular damage, leading to age-related frailty, disability and disease, and eventually death. In this study, we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors (CD33rSiglecs) and maximum lifespan in mammals. In keeping with this, we show that mice lacking Siglec-E, the main member of the CD33rSiglec family, exhibit reduced survival. Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular, structural, and cognitive level. We found that accelerated aging was related both to an unbalanced ROS metabolism, and to a secondary impairment in detoxification of reactive molecules, ultimately leading to increased damage to cellular DNA, proteins, and lipids. Taken together, our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation, which in turn reduces molecular damage and extends lifespan.
• de Aguiar Vallim, T. Q., Tarling, E. J., Ahn, H., Hagey, L. R., Romanoski, C. E., Lee, R. G., Graham, M. J., Motohashi, H., Yamamoto, M., & Edwards, P. A. (2015). MAFG is a transcriptional repressor of bile acid synthesis and metabolism. Cell metabolism, 21(2), 298-310.
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  Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.
• Gosselin, D., Link, V. M., Romanoski, C. E., Fonseca, G. J., Eichenfield, D. Z., Spann, N. J., Stender, J. D., Chun, H. B., Garner, H., Geissmann, F., & Glass, C. K. (2014). Environment drives selection and function of enhancers controlling tissue-specific macrophage identities. Cell, 159(6), 1327-40.
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  Macrophages reside in essentially all tissues of the body and play key roles in innate and adaptive immune responses. Distinct populations of tissue macrophages also acquire context-specific functions that are important for normal tissue homeostasis. To investigate mechanisms responsible for tissue-specific functions, we analyzed the transcriptomes and enhancer landscapes of brain microglia and resident macrophages of the peritoneal cavity. In addition, we exploited natural genetic variation as a genome-wide "mutagenesis" strategy to identify DNA recognition motifs for transcription factors that promote common or subset-specific binding of the macrophage lineage-determining factor PU.1. We find that distinct tissue environments drive divergent programs of gene expression by differentially activating a common enhancer repertoire and by inducing the expression of divergent secondary transcription factors that collaborate with PU.1 to establish tissue-specific enhancers. These findings provide insights into molecular mechanisms by which tissue environment influences macrophage phenotypes that are likely to be broadly applicable to other cell types.
• Kaikkonen, M. U., Niskanen, H., Romanoski, C. E., Kansanen, E., Kivelä, A. M., Laitalainen, J., Heinz, S., Benner, C., Glass, C. K., & Ylä-Herttuala, S. (2014). Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization. Nucleic acids research, 42(20), 12570-84.
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  Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells.
• Romanoski, C. E. (2014). Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization. Nucleic acids research.
• Erbilgin, A., Civelek, M., Romanoski, C. E., Pan, C., Hagopian, R., Berliner, J. A., & Lusis, A. J. (2013). Identification of CAD candidate genes in GWAS loci and their expression in vascular cells. Journal of lipid research, 54(7), 1894-905.
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  Recent genome-wide association studies (GWAS) have identified 35 loci that significantly associate with coronary artery disease (CAD) susceptibility. The majority of the genes represented in these loci have not previously been studied in the context of atherosclerosis. To characterize the roles of these candidate genes in the vessel wall, we determined their expression levels in endothelial, smooth muscle, and macrophage cells isolated from healthy, prelesioned, and lesioned mouse aortas. We also performed expression quantitative locus (eQTL) mapping of these genes in human endothelial cells under control and proatherogenic conditions. Of the 57 genes studied, 31 were differentially expressed in one or more cell types in disease state in mice, and the expression levels of 8 were significantly associated with the CAD SNPs in human cells, 7 of which were also differentially expressed in mice. By integrating human and mouse results, we predict that PPAP2B, GALNT4, MAPKAPK5, TCTN1, SRR, SNF8, and ICAM1 play a causal role in the susceptibility to atherosclerosis through a role in the vasculature. Additionally, we highlight the genetic complexity of a subset of CAD loci through the differential expression of multiple candidate genes per locus and the involvement of genes that lie outside linkage disequilibrium blocks.
• Heinz, S., Romanoski, C. E., Benner, C., Allison, K. A., Kaikkonen, M. U., Orozco, L. D., & Glass, C. K. (2013). Effect of natural genetic variation on enhancer selection and function. Nature, 503(7477), 487-92.
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  The mechanisms by which genetic variation affects transcription regulation and phenotypes at the nucleotide level are incompletely understood. Here we use natural genetic variation as an in vivo mutagenesis screen to assess the genome-wide effects of sequence variation on lineage-determining and signal-specific transcription factor binding, epigenomics and transcriptional outcomes in primary macrophages from different mouse strains. We find substantial genetic evidence to support the concept that lineage-determining transcription factors define epigenetic and transcriptomic states by selecting enhancer-like regions in the genome in a collaborative fashion and facilitating binding of signal-dependent factors. This hierarchical model of transcription factor function suggests that limited sets of genomic data for lineage-determining transcription factors and informative histone modifications can be used for the prioritization of disease-associated regulatory variants.
• Kaikkonen, M. U., Spann, N. J., Heinz, S., Romanoski, C. E., Allison, K. A., Stender, J. D., Chun, H. B., Tough, D. F., Prinjha, R. K., Benner, C., & Glass, C. K. (2013). Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Molecular cell, 51(3), 310-25.
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  Recent studies suggest a hierarchical model in which lineage-determining factors act in a collaborative manner to select and prime cell-specific enhancers, thereby enabling signal-dependent transcription factors to bind and function in a cell-type-specific manner. Consistent with this model, TLR4 signaling primarily regulates macrophage gene expression through a pre-existing enhancer landscape. However, TLR4 signaling also induces priming of ∼3,000 enhancer-like regions de novo, enabling visualization of intermediates in enhancer selection and activation. Unexpectedly, we find that enhancer transcription precedes local mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). H3K4 methylation at de novo enhancers is primarily dependent on the histone methyltransferases Mll1, Mll2/4, and Mll3 and is significantly reduced by inhibition of RNA polymerase II elongation. Collectively, these findings suggest an essential role of enhancer transcription in H3K4me1/2 deposition at de novo enhancers that is independent of potential functions of the resulting eRNA transcripts.
• Maubaret, C. G., Salpea, K. D., Romanoski, C. E., Folkersen, L., Cooper, J. A., Stephanou, C., Li, K. W., Palmen, J., Hamsten, A., Neil, A., Stephens, J. W., Lusis, A. J., Eriksson, P., Talmud, P. J., Humphries, S. E., , S. B., & , E. c. (2013). Association of TERC and OBFC1 haplotypes with mean leukocyte telomere length and risk for coronary heart disease. PloS one, 8(12), e83122.
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  To replicate the associations of leukocyte telomere length (LTL) with variants at four loci and to investigate their associations with coronary heart disease (CHD) and type II diabetes (T2D), in order to examine possible causal effects of telomere maintenance machinery on disease aetiology.
• Romanoski, C. E. (2013). 25-Hydroxycholesterol activates the integrated stress response to reprogram transcription and translation in macrophages. Journal of Biological Chemistry.
• Romanoski, C. E. (2013). Association of TERC and OBFC1 haplotypes with mean leukocyte telomere length and risk for coronary heart disease. PloS one.
• Romanoski, C. E. (2013). Effect of natural genetic variation on enhancer selection and function. Nature.
• Romanoski, C. E. (2013). Identification of CAD candidate genes in GWAS loci and their expression in vascular cells. Journal of lipid research.
• Romanoski, C. E. (2013). Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Molecular cell.
• Shibata, N., Carlin, A. F., Spann, N. J., Saijo, K., Morello, C. S., McDonald, J. G., Romanoski, C. E., Maurya, M. R., Kaikkonen, M. U., Lam, M. T., Crotti, A., Reichart, D., Fox, J. N., Quehenberger, O., Raetz, C. R., Sullards, M. C., Murphy, R. C., Merrill, A. H., Brown, H. A., , Dennis, E. A., et al. (2013). 25-Hydroxycholesterol activates the integrated stress response to reprogram transcription and translation in macrophages. The Journal of biological chemistry, 288(50), 35812-23.
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  25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.
• Lee, S., Birukov, K. G., Romanoski, C. E., Springstead, J. R., Lusis, A. J., & Berliner, J. A. (2012). Role of phospholipid oxidation products in atherosclerosis. Circulation research, 111(6), 778-99.
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  There is increasing clinical evidence that phospholipid oxidation products (Ox-PL) play a role in atherosclerosis. This review focuses on the mechanisms by which Ox-PL interact with endothelial cells, monocyte/macrophages, platelets, smooth muscle cells, and HDL to promote atherogenesis. In the past few years major progress has been made in identifying these mechanisms. It has been recognized that Ox-PL promote phenotypic changes in these cell types that have long-term consequences for the vessel wall. Individual Ox-PL responsible for specific cellular effects have been identified. A model of the configuration of bioactive truncated Ox-PL within membranes has been developed that demonstrates that the oxidized fatty acid moiety protrudes into the aqueous phase, rendering it accessible for receptor recognition. Receptors and signaling pathways for individual Ox-PL species are now determined and receptor independent signaling pathways identified. The effects of Ox-PL are mediated both by gene regulation and transcription independent processes. It has now become apparent that Ox-PL affects multiple genes and pathways, some of which are proatherogenic and some are protective. However, at concentrations that are likely present in the vessel wall in atherosclerotic lesions, the effects promote atherogenesis. There have also been new insights on enzymes that metabolize Ox-PL and the significance of these enzymes for atherosclerosis. With the knowledge we now have of the regulation and effects of Ox-PL in different vascular cell types, it should be possible to design experiments to test the role of specific Ox-PL on the development of atherosclerosis.
• Lee, S., Springstead, J. R., Parks, B. W., Romanoski, C. E., Palvolgyi, R., Ho, T., Nguyen, P., Lusis, A. J., & Berliner, J. A. (2012). Metalloproteinase processing of HBEGF is a proximal event in the response of human aortic endothelial cells to oxidized phospholipids. Arteriosclerosis, thrombosis, and vascular biology, 32(5), 1246-54.
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  Atherosclerosis is a chronic inflammatory disease initiated by monocyte recruitment and retention in the vessel wall. An important mediator of monocyte endothelial interaction is the chemokine interleukin (IL)-8. The oxidation products of phospholipids, including oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC), accumulate in atherosclerotic lesions and strongly induce IL-8 in human aortic endothelial cells (HAECs). The goal of this study was to identify the proximal events leading to induction of IL-8 by Ox-PAPC in vascular endothelial cells.
• Romanoski, C. E. (2012). Metalloproteinase processing of HBEGF is a proximal event in the response of human aortic endothelial cells to oxidized phospholipids. Arteriosclerosis, thrombosis, and vascular biology.
• Romanoski, C. E. (2012). Role of phospholipid oxidation products in atherosclerosis. Circulation research.
• Kim, J. B., Xia, Y., Romanoski, C. E., Lee, S., Meng, Y., Shi, Y., Bourquard, N., Gong, K. W., Port, Z., Grijalva, V., Reddy, S. T., Berliner, J. A., Lusis, A. J., & Shih, D. M. (2011). Paraoxonase-2 modulates stress response of endothelial cells to oxidized phospholipids and a bacterial quorum-sensing molecule. Arteriosclerosis, thrombosis, and vascular biology, 31(11), 2624-33.
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  Chronic infection has long been postulated as a stimulus for atherogenesis. Pseudomonas aeruginosa infection has been associated with increased atherosclerosis in rats, and these bacteria produce a quorum-sensing molecule 3-oxo-dodecynoyl-homoserine lactone (3OC12-HSL) that is critical for colonization and virulence. Paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL and also protects against the effects of oxidized phospholipids thought to contribute to atherosclerosis. We now report the response of human aortic endothelial cells (HAECs) to 3OC12-HSL and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) in relation to PON2 expression.
• Romanoski, C. E. (2011). Network for Activation of Human Endothelial Cells by Oxidized Phospholipids A Critical Role of Heme Oxygenase 1. Circulation research.
• Romanoski, C. E. (2011). Paraoxonase-2 Modulates Stress Response of Endothelial Cells to Oxidized Phospholipids and a Bacterial Quorum--Sensing Molecule. Arteriosclerosis, thrombosis, and vascular biology.
• Romanoski, C. E., Che, N., Yin, F., Mai, N., Pouldar, D., Civelek, M., Pan, C., Lee, S., Vakili, L., Yang, W., Kayne, P., Mungrue, I. N., Araujo, J. A., Berliner, J. A., & Lusis, A. J. (2011). Network for activation of human endothelial cells by oxidized phospholipids: a critical role of heme oxygenase 1. Circulation research, 109(5), e27-41.
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  Oxidized palmitoyl arachidonyl phosphatidylcholine (Ox-PAPC) accumulates in atherosclerotic lesions, is proatherogenic, and influences the expression of more than 1000 genes in endothelial cells.
• Romanoski, C. E. (2010). Systems genetics analysis of gene-by-environment interactions in human cells. The American Journal of Human Genetics.
• Romanoski, C. E., Lee, S., Kim, M. J., Ingram-Drake, L., Plaisier, C. L., Yordanova, R., Tilford, C., Guan, B., He, A., Gargalovic, P. S., Kirchgessner, T. G., Berliner, J. A., & Lusis, A. J. (2010). Systems genetics analysis of gene-by-environment interactions in human cells. American journal of human genetics, 86(3), 399-410.
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  Gene by environment (GxE) interactions are clearly important in many human diseases, but they have proven to be difficult to study on a molecular level. We report genetic analysis of thousands of transcript abundance traits in human primary endothelial cell (EC) lines in response to proinflammatory oxidized phospholipids implicated in cardiovascular disease. Of the 59 most regulated transcripts, approximately one-third showed evidence of GxE interactions. The interactions resulted primarily from effects of distal-, trans-acting loci, but a striking example of a local-GxE interaction was also observed for FGD6. Some of the distal interactions were validated by siRNA knockdown experiments, including a locus involved in the regulation of multiple transcripts involved in the ER stress pathway. Our findings add to the understanding of the overall architecture of complex human traits and are consistent with the possibility that GxE interactions are responsible, in part, for the failure of association studies to more fully explain common disease variation.
• Park, M. S., Romanoski, C. E., & Pryor, B. M. (2008). A re-examination of the phylogenetic relationship between the causal agents of carrot black rot, Alternaria radicina and A. carotiincultae. Mycologia, 100(3), 511-27.
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  The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], beta-tubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, beta-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.
• Bennett, B. J., Romanoski, C. E., & Lusis, A. J. (2007). Network-centered view of coronary artery disease. Expert review of cardiovascular therapy, 5(6), 1095-103.
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  Systems biology is an emerging field that attempts to examine multiple elements of a biologic system using techniques such as expression microarrays or proteomics. The goal of systems biology is to detect all the elements of a system and create an interaction network between these so that the system can be explained under specified conditions. In this review we discuss how systems-based approaches are being applied to cardiovascular disease, illustrating concepts, such as the integration of orthogonal datasets, coexpression networks and emergent properties.
• Romanoski, C. E. (2007). Network-centered view of coronary artery disease. Expert review of cardiovascular therapy.

Proceedings Publications

• Romanoski, C. E. (2013). 'Enhancer Therapy': emerging strategy targeting cell-specific enhancer RNAs. In HUMAN GENE THERAPY.
• Romanoski, C. E. (2013). Examination of the roles of cholesterol depletion and thiol interactions in gene regulation by Ox-PAPC in endothelial cells using systems approaches. In ANGIOGENESIS.
• Romanoski, C. E. (2013). Systems genetics analyses of human aortic endothelial cell transcript expression predict causal genes associated with complex disorders of the vascular wall. In ANGIOGENESIS.
• Romanoski, C. E. (2010). What Molecules Regulate the Response to Oxidized Phospholipids?. In ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY.

Presentations

• Romanoski, C. E. (2023, June). Systems genetics of endothelial cells for vascular disease. . Gordon Research Conference in Atherosclerosis.. Barcelona, Spain: Gordon Research Conferences.
• Romanoski, C. E. (2023, May). Identifying Effects of Common Genetic Variation in Human Endothelial Cells. . Vascular Discovery 2023 Meeting -- Conference of the American Heart Association. Boston, MA: American Heart Association.
• Romanoski, C. E. (2023, May). Systems Genetics to Understand Endothelial Cells in Human Health and Disease. UCLA Bioinformatics Interdepartmental Graduate Program Seminar Series.. UCLA Los Angeles California: UCLA.
• Romanoski, C. E. (2023, Sept.). Systems Genetics of Human Endothelial Cells: From Variant to Function.. Cleveland Clinic's Dept. of Cardiovascular & Metabolic Sciences Speaker Series.. Cleveland Clinic, Cleveland Ohio: Cleveland Clinic.
• Romanoski, C. E. (2020, October). A Systems Genetics Analysis of Human Endothelial Cells. The Biological and Biomedical Joint Seminar Series (MCB and other UA departments). UA (Virtual during Quarentine).
• Romanoski, C. E. (2020, October). Invited Presentation, "A Systems Genetics Analysis of Human Endothelial Cells". North American Vascular Biology Organization 2020. Virtual (was to be in Rhode Island): North American Vascular Biology Organization (NAVBO).
• Romanoski, C. E. (2018, March). Identification of Transcriptional Networks for Vascular Disease. University of Virginia Cardiovascular Research Center Seminar Series. Charlottesville, VA.
• Romanoski, C. E. (2018, March). Systems genetics of human aortic endothelial cells. Cleveland Clinic Annual Page Lecture Series. Cleveland, OH *note: cancelled at last minute due to 'Nor'easter' weather*.

Poster Presentations

• Golebiewski, A. K., Stolze, L. K., & Romanoski, C. E. (2023, Nov.). Alternative promoter usage by NFKb tunes transcript diversity during the endothelial inflammatory response. American Society of Human Genetics (ASHG) 2023 Meeting. Washington DC: American Society of Human Genetics.
• Hartiala, J. A., Han, Y., Jia, Q., Huang, P., Woodward, N. C., Gukasyan, H., Kurt, Z., Stolze, L. K., Tregouet, D., Smith, N. L., Seldin, M., Mehrabian, M., Lusis, A. J., Wilson, W. H., Hazen, S. L., Yang, X., Romanoski, C. E., & Allayee, H. (2018, October). Genome-Wide Association Studies in >500,000 Individuals Identify Novel Loci for Coronary Artery Disease and Myocardial Infarction. American Society of Human Genetics Annual Conference. San Diego, CA: American Society of Human Genetics.
• Selvarajan, I., Ravindran, A., Polonen, P., Toropainen, A., Laakso, M., Heinaniemi, M., Romanoski, C. E., Civelek, M., & Kaikkonen, M. U. (2018, October). CAD susceptibility enhancers: From association to mechanism. American Society of Human Genetics Annual Conference. San Diego, CA: American Society of Human Genetics.
• Stolze, L. K., Whalen, M. B., Hadeli, N. K., Eshghi, A., Conklin, A., Victor, R., & Romanoski, C. E. (2018, October). Using endothelial cell molecular QTLs to dissect coronary artery disease risk. American Society of Human Genetics Annual Conference. San Diego, CA: American Society of Human Genetics.

Others

• Romanoski, C. E. (2020, June). Inside AJHG: A Chat with Casey Romanoski. American Society of Human Genetics (ASHG) Website. https://www.ashg.org/careers-learning/career-interviews/inside-ajhg-with-casey-romanoski/
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  From the website: Each month, the editors of The American Journal of Human Genetics interview an author of a recently published paper. This month, we check in with Casey Romanoski to discuss her paper, “Systems Genetics in Human Endothelial Cells Identifies Non-coding Variants Modifying Enhancers, Expression, and Complex Disease Traits”.
• Romanoski, C. E. (2018, February). MARGE: Mutation Analysis for Regulatory Genomic Elements. https://doi.org/10.1101/268839