POU2AF1 Functions in the Human Airway Epithelium To Regulate Expression of Host Defense Genes This information is current as Haixia Zhou, Angelika Brekman, Wu-Lin Zuo, Xuemei Ou, of September 28, 2021. Renat Shaykhiev, Francisco J. Agosto-Perez, Rui Wang, Matthew S. Walters, Jacqueline Salit, Yael Strulovici-Barel, Michelle R. Staudt, Robert J. Kaner, Jason G. Mezey, Ronald G. Crystal and Guoqing Wang J Immunol 2016; 196:3159-3167; Prepublished online 29 Downloaded from February 2016; doi: 10.4049/jimmunol.1502400 http://www.jimmunol.org/content/196/7/3159 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2016/02/26/jimmunol.150240 Material 0.DCSupplemental References This article cites 43 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/196/7/3159.full#ref-list-1 Why The JI? Submit online. by guest on September 28, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology POU2AF1 Functions in the Human Airway Epithelium To Regulate Expression of Host Defense Genes Haixia Zhou,*,† Angelika Brekman,† Wu-Lin Zuo,† Xuemei Ou,† Renat Shaykhiev,† Francisco J. Agosto-Perez,† Rui Wang,† Matthew S. Walters,† Jacqueline Salit,† Yael Strulovici-Barel,† Michelle R. Staudt,† Robert J. Kaner,†,‡ Jason G. Mezey,†,x Ronald G. Crystal,†,‡ and Guoqing Wang† In the process of seeking novel lung host defense regulators by analyzing genome-wide RNA sequence data from normal human airway epithelium, we detected expression of POU domain class 2–associating factor 1 (POU2AF1), a known transcription cofactor previously thought to be expressed only in lymphocytes. Lymphocyte contamination of human airway epithelial samples obtained by bronchoscopy and brushing was excluded by immunohistochemistry staining, the observation of upregulation of POU2AF1 in Downloaded from purified airway basal stem/progenitor cells undergoing differentiation, and analysis of differentiating single basal cell clones. Lentivirus-mediated upregulation of POU2AF1 in airway basal cells induced upregulation of host defense genes, including MX1, IFIT3, IFITM, and known POU2AF1 downstream genes HLA-DRA, ID2, ID3, IL6, and BCL6. Interestingly, expression of these genes paralleled changes of POU2AF1 expression during airway epithelium differentiation in vitro, suggesting POU2AF1 helps to maintain a host defense tone even in pathogen-free condition. Cigarette smoke, a known risk factor for airway infection, sup- pressed POU2AF1 expression both in vivo in humans and in vitro in human airway epithelial cultures, accompanied by dereg- http://www.jimmunol.org/ ulation of POU2AF1 downstream genes. Finally, enhancing POU2AF1 expression in human airway epithelium attenuated the suppression of host defense genes by smoking. Together, these findings suggest a novel function of POU2AF1 as a potential regulator of host defense genes in the human airway epithelium. The Journal of Immunology, 2016, 196: 3159–3167. he airway epithelium functions as a complex barrier that regulating the interactions between epithelial cells and inflamma- serves as the front line of the host defense system of the lung tory cells, including cytokines, chemokines, MHC-II genes, com- T (1). In response to inhaled pathogens, airway epithelial plement, and adhesion proteins (1–3). Although interacting with cells generate two major types of host defense molecules, as fol- receptors such as the TLR family triggers activation of discrete parts lows: 1) intracellular or extracellular molecules with direct anti- of pulmonary host defense, the overall regulation of this diverse by guest on September 28, 2021 pathogen effects, such as reactive oxygen species, antimicrobial airway epithelial host defense system is not well understood. peptides, and proteins suppressing intracellular pathogen amplifi- In a genome-wide RNA sequencing analysis of human airway cation and spreading; and 2) surface or extracellular molecules epithelium gene expression, we observed expression of POU domain class 2–associating factor 1 (POU2AF1; also named OCA-B, OBF-1, *Department of Respiratory Medicine, West China Hospital, Sichuan University, and BOB.1), a gene previously considered to be expressed specifically Sichuan 610041, China; †Department of Genetic Medicine, Weill Cornell Medical in lymphocytes, where it functions as a coactivator of octamer-binding College, New York, NY 10065; ‡Division of Pulmonary and Critical Care Medicine, Department of Medicine, Weill Cornell Medical College, New York, NY 10065; and OCT1 and OCT2 transcription factors to regulate Ig expression and xDepartment of Biological Statistics and Computational Biology, Cornell University, other host defense-related genes (4–6). POU2AF1 has no intrinsic Ithaca, New York, NY 14853 DNA-binding activity, but by recognizing the POU domain of OCT1 Received for publication November 17, 2015. Accepted for publication February 1, and OCT2, it plays an essential role in B cell responses to Ags and is 2016. required for the formation of germinal centers (7–9). This work was supported in part by National Institutes of Health Grants R01 HL107882, Because POU2AF1 function in B cells relates to host defense, with P20 HL113443, UL1 TR000457, and UL1 RR0204143. G.W., R.W., and A.B. were supported in part by National Institutes of Health Grant T32 HL094284. For Grant P20 the surprising observation that POU2AF1 is expressed in the airway HL113443, research reported in this publication was supported by the National Insti- epithelium, a cell population critical to pulmonary host defense, we tutes of Health and the Family Smoking Prevention and Tobacco Control Act. asked: 1) is POU2AF1 really expressed in the human airway epithe- The raw data presented in this article have been deposited in the National Center for lium, not in lymphocytes contaminating the airway epithelial samples; Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih. gov/geo/) under accession number GSE77659. and 2) if so, does POU2AF1 play a role in the airway epithelium by The content is solely the responsibility of the authors and does not necessarily broadly regulating the expression of genes relevant to host defense as it represent the official views of the National Institutes of Health or the Food and Drug does in B cells? Using in vivo and in vitro analyses, the data demonstrate Administration. that POU2AF1 is expressed in the human airway epithelium, it is Address correspondence and reprint requests to Dr. Ronald G. Crystal, Department of normally upregulated as airway basal stem/progenitor cells differentiate Genetic Medicine, Weill Cornell Medical College, 1300 York Avenue, Box 164, New York, NY 10065. E-mail address: [email protected] to a mucociliated epithelium, and the expression of POU2AF1 in the The online version of this article contains supplemental material. airway epithelium correlates with the expression of a large number of Abbreviations used in this article: ALI, air liquid interface; COPD, chronic obstructive genes relevant to airway epithelium host defense. Interestingly, cigarette pulmonary disease; CSE, cigarette smoke extract; GEO, Gene Expression Omnibus; MOI, smoking, a stress to the airway epithelium known to impair host de- multiplicity of infection; POU2AF1, POU domain class 2–associating factor 1; RFP, red fense, downregulates airway epithelial expression of POU2AF1, and fluorescent protein; SAE, small airway epithelium; SRA, Sequence Read Archive. upregulation of POU2AF1 in airway epithelium can attenuate smoking- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 related downregulation of airway epithelial host-defense genes. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502400 3160 POU2AF1 REGULATED AIRWAY EPITHELIUM HOST DEFENSE Materials and Methods onto an Illumina flowcell for paired-end sequencing reactions using the Human airway epithelial cells Illumina HiSEquation 2000, as previously detailed (15). The raw data have been deposited in the National Center for Biotechnology Information GEO Airway epithelial cells were collected by brushing the epithelium using database (GSE77659; http://www.ncbi.nlm.nih.gov/geo/). flexible bronchoscopy, as previously described (10). Subjects were re- To obtain comparable quantification measurements between the lenti- cruited under a protocol approved by the Weill Cornell Medical College virus vector and POU2AF1-ORF, a custom Python script was written to and Rockefeller University Institutional Review Boards, with written in- process the alignments using Samtools to extract the read accounts cor- formed consent obtained from each volunteer before enrollment in the responding to the POU2AF1-ORF and the lentivirus vector transcripts. study. Only