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

• Published in: Molecular immunology Vol. 44; no. 13; pp. 3364 - 3379
• Main Authors: Unlu, Sebnem, Kumar, Arvind, Waterman, Wayne R., Tsukada, Junichi, Wang, Kent Z.Q., Galson, Deborah L., Auron, Philip E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2007
• Subjects: Animals; CCAAT/enhancer-binding protein; Cell Line; Cell Line, Tumor; Chromatin; DNA; DNA-binding proteins; Enhancers; ETS; Humans; Immunity; Immunoprecipitation; Inflammation; Interferon; Interferon Regulatory Factors - metabolism; Interferon Regulatory Factors - physiology; Interleukin 1; Interleukin-1beta - biosynthesis; Interleukin-1beta - genetics; IRF; LIL-Stat; Lipopolysaccharides; Lipopolysaccharides - immunology; Macrophages/immunology; Mice; Monocytes; Phosphorylation; Point mutation; Post-translation; Protein Interaction Mapping; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins - physiology; PU.1 protein; Regulatory sequences; Response Elements; Stat1 protein; STAT1 Transcription Factor - metabolism; STAT1 Transcription Factor - physiology; TLR signal transduction; TRAF6 protein; Trans-Activators - metabolism; Trans-Activators - physiology; Transcription; Transcription activation; Transcription factors; Transcription Factors - genetics; Transcription Factors - physiology; Transcriptional Activation - genetics; Transcriptional Activation - immunology; Tyrosine; Tyrosine - metabolism; LIL-Stat; C/EBP; Transcription factors; ETS; DBD; Macrophages/immunology; ChIP; P-Tyr; LPS; EMSA; NF-IL6; Interleukin 1; IFNGR; ISGF3; IAD; STAT; LILRE; IL1B; TLR signal transduction; GAS; NTP-Stat1; Interferon; IRF; SH2; DNA-binding proteins; ISRE
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2007.02.016

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/EBPβ was activated at an adjacent C/EBPβ site after LPS stimulation. In contrast to Stat1, IRF8 was tyrosine phosphorylated following LPS treatment. Supporting the involvement of NTP-Stat1, LPS-induced IL1B reporter activity in monocytes was enhanced by ectopic expression of NTP-Stat1 Y701F. In contrast, co-expression of a Y211F IRF8 mutein functioned as a dominant-negative inhibitor of LPS-induced IL1B reporter activity. In 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-Stat1. Binding studies using in vitro-expressed proteins revealed that NTP-Stat1 enhanced the binding of Spi-1 and IRF8 to the LILRE. Co-expression of TRAF6, an LPS 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-Stat1 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.