Michael Montgomery

All Publications

• Rewriting regulatory DNA to dissect and reprogram gene expression. bioRxiv : the preprint server for biology Martyn, G. E., Montgomery, M. T., Jones, H., Guo, K., Doughty, B. R., Linder, J., Chen, Z., Cochran, K., Lawrence, K. A., Munson, G., Pampari, A., Fulco, C. P., Kelley, D. R., Lander, E. S., Kundaje, A., Engreitz, J. M. 2023

  Abstract

  Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects and programmability of such DNA sequences remain difficult to map or predict because we have lacked scalable methods to precisely edit regulatory DNA and quantify the effects in an endogenous genomic context. Here we present an approach to measure the quantitative effects of hundreds of designed DNA sequence variants on gene expression, by combining pooled CRISPR prime editing with RNA fluorescence in situ hybridization and cell sorting (Variant-FlowFISH). We apply this method to mutagenize and rewrite regulatory DNA sequences in an enhancer and the promoter of PPIF in two immune cell lines. Of 672 variant-cell type pairs, we identify 497 that affect PPIF expression. These variants appear to act through a variety of mechanisms including disruption or optimization of existing transcription factor binding sites, as well as creation of de novo sites. Disrupting a single endogenous transcription factor binding site often led to large changes in expression (up to -40% in the enhancer, and -50% in the promoter). The same variant often had different effects across cell types and states, demonstrating a highly tunable regulatory landscape. We use these data to benchmark performance of sequence-based predictive models of gene regulation, and find that certain types of variants are not accurately predicted by existing models. Finally, we computationally design 185 small sequence variants (≤10 bp) and optimize them for specific effects on expression in silico. 84% of these rationally designed edits showed the intended direction of effect, and some had dramatic effects on expression (-100% to +202%). Variant-FlowFISH thus provides a powerful tool to map the effects of variants and transcription factor binding sites on gene expression, test and improve computational models of gene regulation, and reprogram regulatory DNA.

  View details for DOI 10.1101/2023.12.20.572268

  View details for PubMedID 38187584

  View details for PubMedCentralID PMC10769263

• Genome-Wide Analysis Reveals Mucociliary Remodeling of the Nasal Airway Epithelium Induced by Urban PM2.5 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Montgomery, M. T., Sajuthi, S. P., Cho, S., Everman, J. L., Rios, C. L., Goldfarbmuren, K. C., Jackson, N. D., Saef, B., Cromie, M., Eng, C., Medina, V., Elhawary, J. R., Oh, S. S., Rodriguez-Santana, J., Vladar, E. K., Burchard, E. G., Seibold, M. A. 2020; 63 (2): 172-184

  Abstract

  Air pollution particulate matter <2.5 μm (PM2.5) exposure is associated with poor respiratory outcomes. Mechanisms underlying PM2.5-induced lung pathobiology are poorly understood but likely involve cellular and molecular changes to the airway epithelium. We extracted and chemically characterized the organic and water-soluble components of air pollution PM2.5 samples, then determined the whole transcriptome response of human nasal mucociliary airway epithelial cultures to a dose series of PM2.5 extracts. We found that PM2.5 organic extract (OE), but not water-soluble extract, elicited a potent, dose-dependent transcriptomic response from the mucociliary epithelium. Exposure to a moderate OE dose modified the expression of 424 genes, including activation of aryl hydrocarbon receptor signaling and an IL-1 inflammatory program. We generated an OE-response gene network defined by eight functional enrichment groups, which exhibited high connectivity through CYP1A1, IL1A, and IL1B. This OE exposure also robustly activated a mucus secretory expression program (>100 genes), which included transcriptional drivers of mucus metaplasia (SPDEF and FOXA3). Exposure to a higher OE dose modified the expression of 1,240 genes and further exacerbated expression responses observed at the moderate dose, including the mucus secretory program. Moreover, the higher OE dose significantly increased the MUC5AC/MUC5B gel-forming mucin expression ratio and strongly downregulated ciliated cell expression programs, including key ciliating cell transcription factors (e.g., FOXJ1 and MCIDAS). Chronic OE stimulation induced mucus metaplasia-like remodeling characterized by increases in MUC5AC+ secretory cells and MUC5AC mucus secretions. This epithelial remodeling may underlie poor respiratory outcomes associated with high PM2.5 exposure.

  View details for DOI 10.1165/rcmb.2019-0454OC

  View details for Web of Science ID 000558637100007

  View details for PubMedID 32275839

  View details for PubMedCentralID PMC7397762

• A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma NATURE COMMUNICATIONS Everman, J. L., Sajuthi, S. P., Liegeois, M. A., Jackson, N. D., Collet, E. H., Peters, M. C., Chioccioli, M., Moore, C. M., Patel, B. B., Dyjack, N., Powell, R., Rios, C., Montgomery, M. T., Eng, C., Elhawary, J. R., Mak, A. Y., Hu, D., Huntsman, S., Salazar, S., Feriani, L., Fairbanks-Mahnke, A., Zinnen, G. L., Michel, C. R., Gomez, J., Zhang, X., Medina, V., Chu, H., Cicuta, P., Gordon, E. D., Zeitlin, P., Ortega, V. E., Reisdorph, N., Dunican, E. M., Tang, M., Elicker, B. M., Henry, T. S., Bleecker, E. R., Castro, M., Erzurum, S. C., Israel, E., Levy, B. D., Mauger, D. T., Meyers, D. A., Sumino, K., Gierada, D. S., Hastie, A. T., Moore, W. C., Denlinger, L. C., Jarjour, N. N., Schiebler, M. L., Wenzel, S. E., Woodruff, P. G., Rodriguez-Santana, J., Pearson, C. G., Burchard, E. G., Fahy, J. V., Seibold, M. A. 2024; 15 (1): 3900

  Abstract

  By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

  View details for DOI 10.1038/s41467-024-48034-5

  View details for Web of Science ID 001217774000027

  View details for PubMedID 38724552

  View details for PubMedCentralID PMC11082194

• IL-13-programmed airway tuft cells produce PGE(2), which promotes CFTR-dependent mucociliary function JCI INSIGHT Kotas, M. E., Moore, C. M., Gurrola, J. G., Pletcher, S. D., Goldberg, A. N., Alvarez, R., Yamato, S., Bratcher, P. E., Shaughnessy, C. A., Zeitlin, P. L., Zhang, I. H., Li, Y., Montgomery, M. T., Lee, K., Cope, E. K., Locksley, R. M., Seibold, M. A., Gordon, E. D. 2022; 7 (13)

  Abstract

  Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single-cell RNA sequencing of airway epithelium and mouse modeling, we found that IL-13 expanded and programmed airway tuft cells toward eicosanoid metabolism and that tuft cell deficiency led to a reduction in airway prostaglandin E2 (PGE2) concentration. Allergic airway epithelia bore a signature of PGE2 activation, and PGE2 activation led to cystic fibrosis transmembrane receptor-dependent ion and fluid secretion and accelerated mucociliary transport. These data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.

  View details for DOI 10.1172/jci.insight.159832

  View details for Web of Science ID 000824844700001

  View details for PubMedID 35608904

• Type 2 and interferon inflammation regulate SARS-CoV-2 entry factor expression in the airway epithelium NATURE COMMUNICATIONS Sajuthi, S. P., DeFord, P., Li, Y., Jackson, N. D., Montgomery, M. T., Everman, J. L., Rios, C. L., Pruesse, E., Nolin, J. D., Plender, E. G., Wechsler, M. E., Mak, A. Y., Eng, C., Salazar, S., Medina, V., Wohlford, E. M., Huntsman, S., Nickerson, D. A., Germer, S., Zody, M. C., Abecasis, G., Kang, H., Rice, K. M., Kumar, R., Oh, S., Rodriguez-Santana, J., Burchard, E. G., Seibold, M. A. 2020; 11 (1): 5139

  Abstract

  Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2, that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network, highly upregulated by type 2 (T2) inflammation through the action of interleukin-13, and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally, we define airway responses to common coronavirus infections in children, finding that these infections generate host responses similar to other viral species, including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

  View details for DOI 10.1038/s41467-020-18781-2

  View details for Web of Science ID 000583335300003

  View details for PubMedID 33046696

  View details for PubMedCentralID PMC7550582

• COVID-19-related Genes in Sputum Cells in Asthma Relationship to Demographic Features and Corticosteroids AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Peters, M. C., Sajuthi, S., Deford, P., Christenson, S., Rios, C. L., Montgomery, M. T., Woodruff, P. G., Mauger, D. T., Erzurum, S. C., Johansson, M. W., Denlinger, L. C., Jarjour, N. N., Castro, M., Hastie, A. T., Moore, W., Ortega, V. E., Bleecker, E. R., Wenzel, S. E., Israel, E., Levy, B. D., Seibold, M. A., Fahy, J., NHLBI Severe Asthma Res Program-3 2020; 202 (1): 83-90

  Abstract

  Rationale: Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 (angiotensin-converting enzyme 2), and TMPRSS2 (transmembrane protease serine 2) mediate viral infection of host cells. We reasoned that differences in ACE2 or TMPRSS2 gene expression in sputum cells among patients with asthma may identify subgroups at risk for COVID-19 morbidity.Objectives: To determine the relationship between demographic features and sputum ACE2 and TMPRSS2 gene expression in asthma.Methods: We analyzed gene expression for ACE2 and TMPRSS2, and for ICAM-1 (intercellular adhesion molecule 1) (rhinovirus receptor as a comparator) in sputum cells from 330 participants in SARP-3 (Severe Asthma Research Program-3) and 79 healthy control subjects.Measurements and Main Results: Gene expression of ACE2 was lower than TMPRSS2, and expression levels of both genes were similar in asthma and health. Among patients with asthma, male sex, African American race, and history of diabetes mellitus were associated with higher expression of ACE2 and TMPRSS2. Use of inhaled corticosteroids (ICS) was associated with lower expression of ACE2 and TMPRSS2, but treatment with triamcinolone acetonide did not decrease expression of either gene. These findings differed from those for ICAM-1, where gene expression was increased in asthma and less consistent differences were observed related to sex, race, and use of ICS.Conclusions: Higher expression of ACE2 and TMPRSS2 in males, African Americans, and patients with diabetes mellitus provides rationale for monitoring these asthma subgroups for poor COVID-19 outcomes. The lower expression of ACE2 and TMPRSS2 with ICS use warrants prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID-19 morbidity.

  View details for DOI 10.1164/rccm.202003-0821OC

  View details for Web of Science ID 000546619900017

  View details for PubMedID 32348692

  View details for PubMedCentralID PMC7328313

• Dissecting the cellular specificity of smoking effects and reconstructing lineages in the human airway epithelium NATURE COMMUNICATIONS Goldfarbmuren, K. C., Jackson, N. D., Sajuthi, S. P., Dyjack, N., Li, K. S., Rios, C. L., Plender, E. G., Montgomery, M. T., Everman, J. L., Bratcher, P. E., Vladar, E. K., Seibold, M. A. 2020; 11 (1): 2485

  Abstract

  Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and non-smokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium.

  View details for DOI 10.1038/s41467-020-16239-z

  View details for Web of Science ID 000537281800001

  View details for PubMedID 32427931

  View details for PubMedCentralID PMC7237663

• The nonlesional skin surface distinguishes atopic dermatitis with food allergy as a unique endotype SCIENCE TRANSLATIONAL MEDICINE Leung, D. M., Calatroni, A., Zaramela, L. S., LeBeau, P. K., Dyjack, N., Brar, K., David, G., Johnson, K., Leung, S., Ramirez-Gama, M., Liang, B., Rios, C., Montgomery, M. T., Richers, B. N., Hall, C. F., Norquest, K. A., Jung, J., Bronova, I., Kreimer, S., Talbot, C., Crumrine, D., Cole, R. N., Elias, P., Zengler, K., Seibold, M. A., Berdyshev, E., Goleva, E. 2019; 11 (480)

  Abstract

  Skin barrier dysfunction has been reported in both atopic dermatitis (AD) and food allergy (FA). However, only one-third of patients with AD have FA. The purpose of this study was to use a minimally invasive skin tape strip sampling method and a multiomics approach to determine whether children with AD and FA (AD FA+) have stratum corneum (SC) abnormalities that distinguish them from AD without FA (AD FA-) and nonatopic (NA) controls. Transepidermal water loss was found to be increased in AD FA+. Filaggrin and the proportion of ω-hydroxy fatty acid sphingosine ceramide content in nonlesional skin of children with AD FA+ were substantially lower than in AD FA- and NA skin. These abnormalities correlated with morphologic changes in epidermal lamellar bilayer architecture responsible for barrier homeostasis. Shotgun metagenomic studies revealed that the nonlesional skin of AD FA+ had increased abundance of Staphylococcus aureus compared to NA. Increased expression of keratins 5, 14, and 16 indicative of hyperproliferative keratinocytes was observed in the SC of AD FA+. The skin transcriptome of AD FA+ had increased gene expression for dendritic cells and type 2 immune pathways. A network analysis revealed keratins 5, 14, and 16 were positively correlated with AD FA+, whereas filaggrin breakdown products were negatively correlated with AD FA+. These data suggest that the most superficial compartment of nonlesional skin in AD FA+ has unique properties associated with an immature skin barrier and type 2 immune activation.

  View details for DOI 10.1126/scitranslmed.aav2685

  View details for Web of Science ID 000459180900006

  View details for PubMedID 30787169

  View details for PubMedCentralID PMC7676854