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.
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 x 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 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 x IRF8 x 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.
Unlu
Sebnem
S
University of Pittsburgh, School of Medicine, Department of Molecular Genetics and Biochemistry, Pittsburgh, PA 15261, USA.
Kumar
Arvind
A
Waterman
Wayne R
WR
Tsukada
Junichi
J
Wang
Kent Z Q
KZ
Galson
Deborah L
DL
Auron
Philip E
PE
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D050835:Interferon Regulatory Factors / Q000378:metabolism* / Q000502:physiology; D053583:Interleukin-1beta / Q000096:biosynthesis* / Q000235:genetics*; D008070:Lipopolysaccharides / Q000276:immunology*; D051379:Mice; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology; D020218:Response Elements; D050794:STAT1 Transcription Factor / Q000378:metabolism / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism* / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D015533:Transcriptional Activation / Q000235:genetics / Q000276:immunology*; D014443:Tyrosine / Q000378:metabolism; D000099185:Interferon Regulatory Factor-8; D000099232:Proto-Oncogene Protein Spi-1
D050835:Interferon Regulatory Factors; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D014443:Tyrosine; D000099185:Interferon Regulatory Factor-8; D000099232:Proto-Oncogene Protein Spi-1; C114046:LIL-Stat transcription factor; C494079:STAT1 protein, human