PU.1 and multiple IFN regulatory factor proteins synergize to mediate transcriptional activation of the human IL-1β gene

Both lymphoid and myeloid cells express two related members of the IFN regulatory factor (IRF) family of transcription factors, specifically IRF-4 and IFN consensus binding protein (ICSBP or IRF-8). We previously reported that macrophages express IRF-4 and in combination with the ETS-like protein PU.1 can synergistically activate a human IL-1 beta reporter gene. Here we report that this synergy is mediated by a composite PU.1/IRF element located within an upstream enhancer known to confer cytokine- and LPS-inducible expression. In macrophages, synergistic activation of IL-1 beta reporter gene expression was preferentially mediated by IRF-4, whereas IRF-4 and ICSBP were equally capable of synergizing with PU.1 when coexpressed in fibroblasts. Furthermore, coexpression of IRF-1 and IRF-2 dramatically increased the capacity of both PU.1/IRF-4 and PU.1/ICSBP to induce IL-1 beta reporter gene expression in fibroblasts. The additional synergy observed with IRF-1 and IRF-2 coexpression is mediated by a region of DNA distinct from either the IL-1 beta enhancer or promoter. We also assessed the capacity of these transcription factors to activate endogenous IL-1 beta gene when overexpressed in human embryonic kidney 293 cells. Although ectopic expression of PU.1 alone was sufficient to activate modest levels of IL-1 beta transcripts, endogenous IL-1 beta expression was markedly increased following coexpression of additional IRF proteins. Thus, maximal expression of both a human IL-1 beta reporter gene and the endogenous IL-1 beta gene was observed in cells that coexpressed PU.1, IRF-4 (or ICSBP), IRF1, and IRF2. Together, our observations suggest that these factors may function together as an enhanceosome.