PU.1, INTERFERON REGULATORY FACTOR-1, AND INTERFERON CONSENSUS SEQUENCE-BINDING PROTEIN COOPERATE TO INCREASE GP91(PHOX) EXPRESSION

gp91(phox) is a subunit of the phagocyte respiratory burst oxidase cat alytic unit. Transcription of CYBB, the gene encoding gp91(phox), is r estricted to terminally differentiated phagocytic cells. An element in the proximal CYBB promoter binds a protein complex, referred to as he matopoiesis-associated factor (HAF1), that is necessary for interferon -gamma (IFN gamma)-induced gp91(phox) expression. In these investigati ons, we determined that HAF1 was a multiprotein complex, cross-immunor eactive with the transcription factors PU.1, interferon regulatory fac tor 1 (IRF-1), and interferon consensus sequence-binding protein (ICSB P), In electrophoretic mobility shift assay, the HAF1 complex was reco nstituted by either in vitro translated PU,1 with IRF-1 or PU.1 with I CSBP, but not by IRF-1 with ICSBP, HAF1a, a slower mobility complex wi th the same binding site specificity as HAF1, was also investigated. S imilar to the HAF1 complex, the HAF1a complex was cross-immunoreactive with PU,1, IRF-1, and ICSBP, Unlike the HAF1 complex, reconstitution of the HAF1a complex required in vitro translated PU,1 with both IRF-1 and ICSBP, An artificial promoter construct containing the HAF1/HAF1a binding site was modestly activated in the myelomonocytic cell line U 937 by co-transfection either with PU,1 and IRF-1 or with PU,1 and ICS BP, but it was strongly activated by co-transfection with PU,1, IRF-1, and ICSBP, This activation required serine 148-phosphorylated PU.1, T hese studies describe a novel mechanism for PU,1 transcriptional activ ation via interaction with both IRF-1 and ICSBP, a target gene for the interaction of IRF-1 with ICSBP, and a novel activation function for ICSBP as a component of a multiprotein complex.