IL-1 Transcriptional Responses to Lipopolysaccharides Are Regulated by a Complex of RNA Binding Proteins This information is current as Lihua Shi, Li Song, Kelly Maurer, Ying Dou, Vishesh R. of October 1, 2021. Patel, Chun Su, Michelle E. Leonard, Sumei Lu, Kenyaita M. Hodge, Annabel Torres, Alessandra Chesi, Struan F. A. Grant, Andrew D. Wells, Zhe Zhang, Michelle A. Petri and Kathleen E. Sullivan J Immunol published online 17 January 2020 http://www.jimmunol.org/content/early/2020/01/16/jimmun Downloaded from ol.1900650 Supplementary http://www.jimmunol.org/content/suppl/2020/01/16/jimmunol.190065 http://www.jimmunol.org/ Material 0.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published January 17, 2020, doi:10.4049/jimmunol.1900650 The Journal of Immunology IL-1 Transcriptional Responses to Lipopolysaccharides Are Regulated by a Complex of RNA Binding Proteins Lihua Shi,* Li Song,* Kelly Maurer,* Ying Dou,*,1 Vishesh R. Patel,* Chun Su,† Michelle E. Leonard,† Sumei Lu,† Kenyaita M. Hodge,† Annabel Torres,‡,x,{,‖ Alessandra Chesi,† Struan F. A. Grant,†,‡,x,{ Andrew D. Wells,†,‖ Zhe Zhang,# Michelle A. Petri,** and Kathleen E. Sullivan* The IL1A and IL1B genes lie in close proximity on chromosome 2 near the gene for their natural inhibitor, IL1RN. Despite diverse functions, they are all three inducible through TLR4 signaling but with distinct kinetics. This study analyzed transcriptional induction kinetics, chromosome looping, and enhancer RNA production to understand the distinct regulation of these three genes in human cells. IL1A, IL1B, and IL1RN were rapidly induced after stimulation with LPS; however, IL1B mRNA production was less inhibitable by iBET151, suggesting it does not use pause-release regulation. Surprisingly, chromatin looping contacts between Downloaded from IL1A and IL1B were highly intermingled, although those of IL1RN were distinct, and we focused on comparing IL1A and IL1B transcriptional pathways. Our studies demonstrated that enhancer RNAs were produced from a subset of the regulatory regions, that they were critical for production of the mRNAs, and that they bound a diverse array of RNA binding proteins, including p300 but not CBP. We, furthermore, demonstrated that recruitment of p300 was dependent on MAPKs. Integrator is another RNA binding protein recruited to the promoters and enhancers, and its recruitment was more dependent on NF-kB than MAPKs. We found that integrator and NELF, an RNA polymerase II pausing protein, were associated with RNA in a manner that facilitated http://www.jimmunol.org/ interaction. We conclude that IL1A and IL1B share many regulatory contacts, signaling pathways, and interactions with enhancer RNAs. A complex of protein interactions with enhancer RNAs emphasize the role of enhancer RNAs and the overall structural aspects of transcriptional regulation. The Journal of Immunology, 2020, 204: 000–000. he IL-1 family of cytokines is comprised of 11 members the genes for which are located on chromosome 2q14 in close that all function in the regulation of inflammation (1). apposition. IL-1a is a dual function cytokine regulating tran- T Among the IL-1 family members are IL-1a and IL-1b, scription directly through a DNA binding function as well as regulating the inflammatory response through the binding of a by guest on October 1, 2021 cell membrane receptor (2). IL-1a in the nucleus binds chromatin *Division of Allergy Immunology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; †Center for Spatial and Functional Genomics, Children’s Hospital of Phil- and is generally induced through proapoptotic signals. Necrotic adelphia, Philadelphia, PA 19104; ‡Division of Human Genetics, Children’s Hospital of signals, in contrast, cause IL-1a to distribute primarily in the Philadelphia, Philadelphia, PA 19104; xDivision of Endocrinology and Diabetes, Child- { a ren’s Hospital of Philadelphia, Philadelphia, PA, 19104; Department of Pediatrics, cytoplasm (3). When released from the cell, IL-1 stimulates a Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of robust inflammatory response, driving chemokine expression ‖ Medicine, Philadelphia, PA, 19104; Department of Pathology and Laboratory Medi- that regulates the influx of neutrophils and monocytes. cine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA # b 19104; Department of Biomedical and Health Informatics, Children’s Hospital of IL-1 has similar but nonoverlapping functions, and it functions Philadelphia, Philadelphia, PA 19104; and **Division of Rheumatology, Johns Hopkins as a cytokine. It was discovered as a key inflammatory agent University School of Medicine, Baltimore, MD 21205 driving fever in rabbits (4). IL-1b is transcriptionally and 1 Current address: Division of Hematology and Oncology, Children’s Hospital of posttranslationally regulated and is primarily induced after Chongqing Medical University, Chongqing, China. recognition of microbial patterns (5, 6). Monocytes appear to be ORCIDs: 0000-0001-6388-8666 (C.S.); 0000-0002-5490-3884 (S.L.); 0000-0002- b 0954-7446 (A.C.); 0000-0003-2025-5302 (S.F.A.G.); 0000-0003-1441-5373 (M.A.P.); the main cell that produces IL-1 following stimulation with LPS. 0000-0003-4018-1646 (K.E.S.). The transcriptional induction of IL-1b leads to the accumulation Received for publication June 11, 2019. Accepted for publication December 5, 2019. of an inactive protein precursor in the cytoplasm (pro–IL-1b that This work was supported by National Institutes of Health (NIH) Grant R01 ES017627 is processed by the activation of nucleotide binding domain and and the Wallace Chair of Pediatrics (K.E.S.). S.F.A.G. is funded by the Daniel B. leucine-rich repeat pyrin containing protein-3) to active IL-1b (7). Burke Endowed Chair for Diabetes Research, Children’s Hospital of Philadelphia, Near the IL1B gene, encoding IL-1b, lies the gene IL1RN that and NIH Grant R01 HG010067. A.D.W. is supported by NIH Grants R01 AI130115 and R01 AI123539. encodes the IL-1R antagonist (IL-1ra). IL-1ra is a potent inhibitor a b Address correspondence and reprint requests to Dr. Kathleen E. Sullivan, Division of of both IL-1 and IL-1 (8) by blocking the IL-1R1. This agent is Allergy Immunology, Children’s Hospital of Philadelphia, 34th Street and Civic Center used to treat inflammatory conditions such as arthritis and dis- Boulevard, Philadelphia, PA 19104. E-mail address: [email protected] orders of the inflammasome (9). Despite the fact that IL-1ra The online version of this article contains supplemental material. functions as an antagonist of IL-1a and IL-1b, it too is induced Abbreviations used in this article: ASO, antisense oligonucleotide; ATAC-seq, assay for primarily by microbial byproducts. The close approximation of transposase-accessible chromatin using sequencing; ChIP, chromatin immunoprecipi- tation; eRNA, enhancer-derived RNA; FWD, forward; IL-1ra, IL-1R antagonist; these three genes with similar regulatory patterns offers an oppor- INTS11, integrator 11 complex; OCR, open coding region; PRB, probe; qRT-PCR, tunity to dissect commonalities and differences in the transcriptional quantitative RT-PCR; REV, reverse; RNA IP, RNA immunoprecipitation; SLE, sys- regulatory pattern of these three genes. Understanding the regu- temic lupus erythematosus. lation of these three genes is of critical importance in chronic Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 inflammatory conditions in which increased expression of the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900650 2 IL-1 TRANSCRIPTIONAL RESPONSES TO LPS antagonist might provide clinical benefit. We noted increased ex- 59-TAGAGCTAGCGAATTATACAAAATTAGCTGGGCATGG-39; CD831A299- pression of all three mRNAs in cells from patients with systemic lupus FWD, 59-TAGAGCTAGCGAATTCCCAAAGCTGGACGTGCTG-39; 9 erythematosus (SLE), and the use of the therapeutic IL-1ra antagonist CD831A299-reverse (REV), 5 -ACGCGGCCGCGGATCAATTGCAGTG- TAAAGTGTAAAACAC-39; and CD831A327-REV, 59-ACGCGGCCGCG- has shown promise in SLE (10). Thus, there is a clinical rationale for GATCGATGAACTTTCCAATAAGACAAACC-39. the examination of transcriptional pathways regulating this locus. Inhibitors and stimuli There is also a strong scientific rationale for the study of enhancer– promoter interactions at this locus. Much of what is understood The inhibitors included 20 mM C646 (Santa Cruz Biotechnology, Dallas, regarding the mechanism by which enhancers regulate transcription TX). It is an inhibitor of p300-induced histone acetylation (29, 30). Twenty has come from genome-wide studies of developmentally regulated micromolars of iBET151 (Life Science Research,