Regulation of transcription factor Stat1

Interferons (IFN) are a group of cytokines crucially involved in the clearance of viral and bacterial infections. They are subdivided into two types, the alpha- and beta interferons (type I) and IFN-gamma (type II). In vivo analyses show that the predominant function of type I IFN is to establish an antiviral state and thus to provide innate resistance against viral pathogens. IFN-g can also contribute to antiviral immunity, but its major activity is that of a macrophage-activating factor enhancing the potential of the phagocytes to kill bacterial or protozoan pathogens. Both interferon types require de novo gene expression to produce a biological response. Although binding to distinct cell surface receptors, the two IFN types employ a similar mode of activating target genes through a Jak- Stat signal transduction path. However, in case of IFN-g the activated transcription factor is a dimer of Stat1 proteins. In case of type I IFNs the major transcription factor activated is ISGF3, a complex of Stat1, Stat2, and a third protein called IRF9. The importance of Stat1 as a subunit of the transcription factors induced by both IFN types was demonstared by targeted gene disruption, which caused a complete absence of innate resistance to both viruses and bacterial pathogens. Stat1 activation occurs through two distinct phosphorylation events on tyrosine 701 and serine 727. Particularly in case of S727, the implications of phosphorylation for immune responses are not understood. We hypothesize it may be critical for the biological activity of Stat1 and thus represent a crucial molecular event for the establishment of innate immunity. This will be tested in mice containing a mutation that causes an exchange of Stat1 serine 727 to alanine. These mice will be challenged with viral and bacterial pathogens and their ability to eradicate these infections in comparison to wt mice or Stat1-deficient mice will be investigated. Complementary experiments will be conducted in cell lines expressing phosphorylation site mutants of Stat1. Furthermore, we will assess the importance of Stat1 phosphoylation for target gene expression using DNA microarray technology. The ultimate goal is to define groups of genes whose expression requires only one of the phosphorylation events, and to distinguish them from those requiring both phosphorylation events. Finally, we seek to learn whether Stat1 causes changes in the chromatin structure of target genes, and whether these changes are influenced by its phosphorylation status.