A Distal Regulatory Region Is Required for Constitutive and IFN-β-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-β 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 ∼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-β induction. In vivo genomic footprint assays revealed constitutive factor occupancy at the DRR and the PPR and an IFN-β-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-β treatment. AP-1 subunits, c-Jun and c-Fos, were responsible for the constitutive occupancy at the proximal region. Induction of TLR9 by IFN-β 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.

Phagosomal signaling byBorrelia burgdorferiin human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-β

“…S6). The regulatory effect of TLR8 stimulation on TLR2 is not at all unanticipated, given that IFN-β has been shown to up-regulate expression of TLR2 (36). In support of this idea, R848 induced transcription of both TLR8 and TLR2 (Table 1), whereas Pam 3 CSSNA only affected TLR2.…”

NS1 Protein of Influenza A Virus Inhibits the Function of Intracytoplasmic Pathogen Sensor, RIG-I

“…Western blot was performed as described previously (11). Antibodies against FLAG-tag and ␤-actin were purchased from Sigma-Aldrich, and c-myc-tag from Invitrogen.…”

“…Real-time RT-PCR was performed as described previously (11). Two sets of PCR assays were performed for each sample using primers specific for cDNA of the following genes: RIG-I, IFN-␤, TNF-␣, ISG15, MxA, and GAPDH.…”

Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells

“…TLR9 is predominantly expressed by DCs and macrophages (10,11) and can be induced by type I interferons (IFNs), signal transducers and activators of transcription 1, and two IFN regulatory factors. IRF-1 and IRF-2 up-regulate Tlr9 through directly binding to IFN regulatory factor element (IRF-E) within the TLR9 promoter (12). Collectively, these studies reveal the critical role of transcription factors in the regulation of TLR expression and function in cells of the innate immune system.…”

“…cDNA was prepared by oligo(dT) [12][13][14][15][16][17][18] and reverse transcriptase SuperScript II from Invitrogen with 2 g of DNase I-treated total RNA. One l of cDNA was amplified using primers shown in Table 1.…”