Phosphorylation of IRF8 in a pre-associated complex with Spi-1/PU.1 and non-phosphorylated Stat1 is critical for LPS induction of the IL1B gene
Phosphorylation of IRF8 in a pre-associated complex with Spi-1/PU.1 and non-phosphorylated Stat1 is critical for LPS induction of the IL1B gene
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DOI：
10.1016/j.molimm.2007.02.016
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发表时间：
2007-07-01
影响因子：
3.6
通讯作者：
Auron, Philip E.
中科院分区：
文献类型：
--
作者：
Unlu, Sebnem;Kumar, Arvind;Auron, Philip E.
已知转录的快速诱导是由翻译后修饰后结合 DNA 的因子介导的。我们在此报告，非酪氨酸磷酸化 (NTP)-Stat1 参与与 Spi-1/PU.1 和 IRF8 的协同相互作用，形成用于 IL1B 基因诱导的预关联、平衡复合物。假定的 STAT 结合位点（与位于 LPS 和 IL-1 反应元件 (LILRE) 中的复合 Spi-1-IRF8 位点重叠）处的双点突变将人 IL1B LPS 依赖性报告基因活性抑制为对照野生型载体的约 10%。染色质免疫沉淀显示 IRF8、Spi-1 和 NTP-Stat1 在 LILRE 处存在刺激独立的组成型结合，而 C/EBP β 的结合在 LPS 刺激后在相邻的 C/EBP β 位点被激活。与统计数据 1 相比，IRF8 在 LPS 处理后发生酪氨酸磷酸化。 NTP-Stat I Y701F 的异位表达增强了单核细胞中包含 LPS 的 IL1B 报告基因活性，支持 NTP-Stat1 的参与。相比之下，Y211F IRF8 突变蛋白的共表达起到 LPS 包含的 IL1B 报告活性的显性失活抑制剂的作用。使用 LPS 处理的单核细胞提取物进行的体外 DNA 结合证实，LILRE 增强剂组成性结合含有 IRF8、Spi-1 和 NTP-Stat 1 的三分子复合物。使用体外表达蛋白的结合研究表明，NTP-Stat I 增强了 Spi-1 和 IRF8 与 LILRE 的结合。 TRAF6（一种 LIPS 替代物）与 Spi-1 和 IRF8 的共表达增强了 HEK293R 细胞中的 IL1B 报告基因活性，而当共表达 Y211F IRF8 时，该活性显着降低。这些结果表明，重要炎症基因的快速转录诱导依赖于Spi-1(.)IRF8.NTP-Stat1复合物与LILRE的组成型协同结合，这为IRF8磷酸化后立即诱导基因做好准备。染色质前相关因子（如 IRF8）的磷酸化可能是先天免疫相关基因快速转录激活的重要策略。 (c) 2007 Elsevier Ltd. 保留所有权利。
Rapid induction of transcription is known to be mediated by factors which bind DNA following post-translational modification. We report here that non-tyrosine phosphorylated (NTP)-Stat1 is involved in a cooperative interaction with Spi-1/PU.1 and IRF8 to form a pre-associated, poised complex for IL1B gene induction. A double point mutation at a putative STAT binding site, which overlaps this composite Spi-1-IRF8 site located in the LPS and IL-1 response element (LILRE), inhibited human IL1B LPS-dependent reporter activity to about 10 percent of the control wild type vector. Chromatin immunoprecipitation revealed stimulation-independent constitutive binding of IRF8, Spi-1 and NTP-Stat1 at the LILRE, while binding of C/EBP beta was activated at an adjacent C/EBP beta site after LPS stimulation. In contrast to Stat 1, IRF8 was tyrosine phosphorylated following LPS treatment. Supporting the involvement of NTP-Stat1, LPS-incluced IL1B reporter activity in monocytes was enhanced by ectopic expression of NTP-Stat I Y701F. In contrast, co-expression of a Y211F IRF8 mutein functioned as a dominant-negative inhibitor of LPS-incluced IL1B reporter activity. hi vitro DNA binding using extracts from LPS-treated monocytes confirmed that the LILRE enhancer constitutively binds a trimolecular complex containing IRF8, Spi-1 and NTP-Stat 1. Binding studies using in vitro-expressed proteins revealed that NTP-Stat I enhanced the binding of Spi-1 and IRF8 to the LILRE. Co-expression of TRAF6, an LIPS surrogate, with Spi-1 and IRF8 enhanced IL1B reporter activity in HEK293R cells, which was dramatically reduced when Y211F IRF8 was co-expressed. These results suggest that the rapid transcriptional induction of an important inflammatory gene is dependent upon constitutive cooperative binding of a Spi-1(.)IRF8.NTP-Statl complex to the LILRE, which primes the gene for immediate induction following IRF8 phosphorylation. Phosphorylation of chromatin pre-associated factors like IRF8 may be an important strategy for the rapid transcriptional activation of genes involved in innate immunity. (c) 2007 Elsevier Ltd. All rights reserved.