title: A distal regulatory region is required for constitutive and IFN-beta-induced expression of murine TLR9 gene.
abstract: TLR9 is critical for the recognition of unmethylated CpG DNA in innate immunity. Accumulating evidence suggests distinct patterns of TLR9 expression in various types of cells. However, the molecular mechanism of TLR9 expression has received little attention. In the present study, we demonstrate that transcription of murine TLR9 is induced by IFN-beta in peritoneal macrophages and a murine macrophage cell line RAW264.7. TLR9 is regulated through two cis-acting regions, a distal regulatory region (DRR) and a proximal promoter region (PPR), which are separated by approximately 2.3 kbp of DNA. Two IFN-stimulated response element/IFN regulatory factor-element (ISRE/IRF-E) sites, ISRE/IRF-E1 and ISRE/IRF-E2, at the DRR and one AP-1 site at the PPR are required for constitutive expression of TLR9, while only the ISRE/IRF-E1 motif is essential for IFN-beta induction. In vivo genomic footprint assays revealed constitutive factor occupancy at the DRR and the PPR and an IFN-beta-induced occupancy only at the DRR. IRF-2 constitutively binds to the two ISRE/IRF-E sites at the DRR, while IRF-1 and STAT1 are induced to bind to the two ISRE/IRF-E sites and the ISRE/IRF-E1, respectively, only after IFN-beta treatment. AP-1 subunits, c-Jun and c-Fos, were responsible for the constitutive occupancy at the proximal region. Induction of TLR9 by IFN-beta was absent in STAT1-/- macrophages, while the level of TLR9 induction was decreased in IRF-1-/- cells. This study illustrates the crucial roles for AP-1, IRF-1, IRF-2, and STAT1 in the regulation of murine TLR9 expression.
authors.affiliation: Influenza Branch, Division of Viral and Rickettssial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Guo
authors.forename: Sanjay
authors.initials: S
authors.lastname: Garg
authors.forename: Karen M
authors.initials: KM
authors.lastname: Hill
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Jayashankar
authors.forename: Myesha R
authors.initials: MR
authors.lastname: Mooney
authors.forename: Mary
authors.initials: M
authors.lastname: Hoelscher
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Katz
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Boss
authors.forename: Suryaprakash
authors.initials: S
authors.lastname: Sambhara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018983:DNA Footprinting; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000276:immunology*; D050856:Interferon Regulatory Factor-1 / Q000235:genetics / Q000276:immunology; D050836:Interferon Regulatory Factor-2 / Q000235:genetics / Q000276:immunology; D016899:Interferon-beta / Q000276:immunology / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000276:immunology; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000276:immunology; D034622:RNA Interference; D012045:Regulatory Sequences, Nucleic Acid / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000276:immunology; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000276:immunology; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D017931:DNA Primers; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D050794:STAT1 Transcription Factor; C493486:Tlr2 protein, mouse; C495370:Tlr9 protein, mouse; D051195:Toll-Like Receptor 2; D051217:Toll-Like Receptor 9; D018808:Transcription Factor AP-1; D016899:Interferon-beta