Guo Zhu, Garg Sanjay, Hill Karen M, Jayashankar Lakshmi, Mooney Myesha R, Hoelscher Mary, Katz Jacqueline M, Boss Jeremy M, Sambhara Suryaprakash
Influenza Branch, Division of Viral and Rickettssial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
J Immunol. 2005 Dec 1;175(11):7407-18. doi: 10.4049/jimmunol.175.11.7407.
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.
Toll样受体9（TLR9）在天然免疫中识别未甲基化的CpG DNA至关重要。越来越多的证据表明，TLR9在各种类型细胞中的表达模式各异。然而，TLR9表达的分子机制却很少受到关注。在本研究中，我们证明了小鼠TLR9的转录在腹腔巨噬细胞和小鼠巨噬细胞系RAW264.7中可被干扰素-β（IFN-β）诱导。TLR9通过两个顺式作用区域进行调控，一个是远端调控区域（DRR），另一个是近端启动子区域（PPR），它们被约2.3千碱基对（kbp）的DNA隔开。DRR处的两个干扰素刺激反应元件/干扰素调节因子元件（ISRE/IRF-E）位点，即ISRE/IRF-E1和ISRE/IRF-E2，以及PPR处的一个激活蛋白-1（AP-1）位点是TLR9组成型表达所必需的，而只有ISRE/IRF-E1基序对IFN-β诱导至关重要。体内基因组足迹分析显示，DRR和PPR处存在组成型因子占据，而IFN-β诱导的占据仅发生在DRR处。干扰素调节因子2（IRF-2）组成型结合DRR处的两个ISRE/IRF-E位点，而只有在IFN-β处理后，干扰素调节因子1（IRF-1）和信号转导子和转录激活子1（STAT1）才分别被诱导结合到两个ISRE/IRF-E位点和ISRE/IRF-E1位点。AP-1亚基c-Jun和c-Fos负责近端区域的组成型占据。在STAT1基因敲除的巨噬细胞中，IFN-β不能诱导TLR9表达，而在IRF-1基因敲除的细胞中，TLR9的诱导水平降低。本研究阐明了AP-1、IRF-1、IRF-2和STAT1在调节小鼠TLR9表达中的关键作用。
