Molecular basis of Stat1 and PU.1 cooperation in cytokine-induced Fcγ receptor I promoter activation

Abstract

The high-affinity receptor for IgG (FcγRI) is a myeloid cell-specific and IFN-γ-induced gene, and thereby serves as a paradigm for cytokine-induced cell type-specific gene responses. The expression of FcγRI is regulated by PU.1 and Stat1 transcription factors. We established an experimental model to analyze the individual functions of Stat1 and PU.1 in cytokine-induced transcription of the natural FcγRI promoter in U3A cells lacking both factors. PU.1 was required for both the basal activity and for the IFN-γ-induced FcγRI promoter activation, while Stat1 alone could not initiate transcription. In contrast, in the context of a heterologous promoter, PU.1 inhibited the Stat1-mediated transcription. Systematic analysis of Stat1 and PU.1 mutants and FcγRI promoter elements revealed that activation of the promoter required the DNA binding, and the transactivation functions of both Stat1 and PU.1. PU.1 and Stat1 bound the promoter elements independently, and no physical interaction between the proteins was observed. The requirement of PU.1 for FcγRI promoter activity was supported by demonstration of in vitro interaction between PU.1 and components of the basal transcription machinery TBP and RNA polymerase II. Deletion of the acidic transactivation domain of PU.1 greatly diminished both the FcγRI promoter activity as well as the interaction with RNA polymerase II. In contrast, Stat1 did not interact with TBP or RNA polymerase II. These results define functional cooperativity between PU.1 and Stat1 in FcγRI promoter activation where PU.1 serves as an amplifier and bridging factor with the basal transcription machinery.