IRF1 supports DNA binding of STAT1 by promoting its phosphorylation

Abstract The signal transducer and activator of transcription 1 ( STAT 1), 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 ( IRF 1). In this study, we found that IRF 1 could induce STAT 1 phosphorylation and in turn STAT 1 activation. When IRF 1 was transiently expressed in HEK 293 cells, STAT 1 phosphorylated at Y701, dimerized and bound to an oligonucleotide containing a gamma‐activated sequence ( GAS ) derived from the IRF 1 promoter. IRF 1 expression also induced GAS ‐dependent promoter reporter activity, and phosphorylation of JAK 1, a kinase upstream of STAT 1. Although no direct interaction between IRF 1 and STAT 1 was observed, the transactivation domain of IRF 1 was required for IRF 1‐mediated STAT 1 activation, indicating the involvement of gene product(s) regulated by IRF 1. Moreover, supernatants from cells expressing IRF 1 induced phosphorylation of STAT 1 and JAK 1, and subsequent GAS binding by STAT 1 that could not be blocked by treatment with antibodies against IFN ‐β or IFN ‐γ. IFN ‐γ‐induced STAT 1 phosphorylation persisted for up to 30 h following stimulation of HEK 293, but declined in IRF 1 ‐deficient HEK 293 cells. IRF 1 ‐promoter activity induced by IFN ‐γ was also reduced in IRF 1 ‐deficient HEK 293 cells, which could be rescued by complementation with IRF 1. Together these results indicate that IRF 1 promotes DNA binding of STAT 1, which can in turn participate in a positive feedback loop of JAK – STAT signaling.