IRF1 supports DNA binding of STAT1 by promoting its phosphorylation

The signal transducer and activator of transcription 1 (STAT1), a pivotal transcription factor in Janus kinase (JAK)-STAT signaling, regulates the expression of a wide range of immune-related genes, including interferon (IFN) regulatory factor 1 (IRF1). In this study, we found that IRF1 could induce STAT1 phosphorylation and in turn STAT1 activation. When IRF1 was transiently expressed in HEK293 cells, STAT1 phosphorylated at Y701, dimerized and bound to an oligonucleotide containing a gamma-activated sequence (GAS) derived from the IRF1 promoter. IRF1 expression also induced GAS-dependent promoter reporter activity, and phosphorylation of JAK1, a kinase upstream of STAT1. Although no direct interaction between IRF1 and STAT1 was observed, the transactivation domain of IRF1 was required for IRF1-mediated STAT1 activation, indicating the involvement of gene product(s) regulated by IRF1. Moreover, supernatants from cells expressing IRF1 induced phosphorylation of STAT1 and JAK1, and subsequent GAS binding by STAT1 that could not be blocked by treatment with antibodies against IFN-β or IFN-γ. IFN-γ-induced STAT1 phosphorylation persisted for up to 30 h following stimulation of HEK293, but declined in IRF1-deficient HEK293 cells. IRF1-promoter activity induced by IFN-γ was also reduced in IRF1-deficient HEK293 cells, which could be rescued by complementation with IRF1. Together these results indicate that IRF1 promotes DNA binding of STAT1, which can in turn participate in a positive feedback loop of JAK-STAT signaling.