Analysis of the subcellular distribution, assembly, and transcriptional activity of the trimeric, IFN-activated transcription complex ISGF3

The transduction of Type I interferon signals to the nucleus relies on activation of a protein complex, ISGF3, which is comprised of two STAT proteins, STAT1 and STAT2, and the IRF protein, IRF9 (also known as p48/ISGF3gamma). The STAT subunits are cytoplasmically localized in unstimulated cells and rapidly translocate to the nucleus of interferon stimulated cells, but the IRF9 protein is found in both the nucleus and the cytoplasm regardless of IFN stimulation. We have identified a novel bipartite nuclear retention signal within the IRF9 amino terminal DNA binding domain that efficiently and constitutively targets the protein into the nucleus. We also demonstrate that the nuclear distribution of IRF9 is dramatically altered by co-expression of the STAT2 protein, indicating that STAT2 forms a cytoplasmic complex with IRF9, overriding the intrinsic IRF9 nuclear targeting. Retention by STAT2 may serve to regulate the activity of free IRF9 and/or guarantee that cytoplasmic pools of pre-associated STAT2:IRF9 are available for rapid activation of the IFN response.; Because IRF9 is efficiently targeted into the nucleus in the absence of STAT protein activation, the influence of the nuclear IRF9 protein on gene regulation was then examined. We find that IRF9 lacks transcriptional activity in the absence of cellular STAT proteins. In contrast, increased expression of the native IRF9 protein can suppress IFN-responsive, ISRE-dependent transcription, suggesting a negative regulatory role in ISG transcription. The transcriptionally inert IRF9 protein was then readily converted to a constitutively-active ISG activator by fusion with the transcriptional activation domain (TAD) of STAT2. The IRF9-STAT2 hybrid is targeted to endogenous ISG loci and can activate their transcription. Using the IRF9-STAT2 fusion protein, the STAT2 TAD was subjected to a structure-function analysis that indicates that the STAT2 TAD is a modular transcriptional platform with several domains required for complete transcriptional activity, suggestive of multiple interaction surfaces for distinct transcriptional coactivators. Moreover, expression of the IRF9-STAT2 fusion can faithfully recapitulate the type I IFN biological response, producing a cellular antiviral state that inhibits virus replication.; Our studies confirm that STAT2 provides the fundamental and essential transcriptional activation signals. We then probed for novel functional interactions between STAT2 and the multimeric Mediator coactivator complex, and show that ISGF3-mediated transcription is dependent on STAT2 interactions with DRIP150, a highly conserved Mediator subunit. Endogenous DRIP150 and STAT2 could interact in solution, and DNA-affinity chromatography and chromatin immunoprecipitation assays demonstrate that DRIP150 binds to the mature, activated ISGF3:DNA complex and is recruited to target gene promoters in an IFN-dependent fashion. These findings indicate that the IFN-activated ISGF3 transcription factor regulates transcription through contact with DRIP150, implicating the Mediator coactivator complex in IFN-activated gene regulation.