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-1β 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-1β 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-1β 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-1β enhancer or promoter. We also assessed the capacity of these transcription factors to activate endogenous IL-1β gene when overexpressed in human embryonic kidney 293 cells. Although ectopic expression of PU.1 alone was sufficient to activate modest levels of IL-1β transcripts, endogenous IL-1β expression was markedly increased following coexpression of additional IRF proteins. Thus, maximal expression of both a human IL-1β reporter gene and the endogenous IL-1β 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.

Cytokine transcription is usually regulated by transcription factor binding and chromatin remodeling following an inducing signal. By contrast, these data showed the interleukin (IL)-1␤ promoter assembles into a "poised" structure, as evidenced by nuclease accessibility and loss of core histones immediately surrounding the transcription start site. Strikingly, these properties do not change upon transcriptional activation by lipopolysaccharide. Furthermore, association of two key transcriptional activators, PU.1 and C/EBP␤, is robust pre-and post-stimulation indicating the IL-1␤ promoter is packaged into a nontranscribed but poised promoter architecture in cells capable of rapidly inducing IL-1␤. Monocyte stimulation causes recruitment of a third factor, IRF-4, to the IL-1␤ enhancer. PU.1 phosphorylation at a CK2 kinase consensus element is required for this recruitment. We showed that CK2 phosphorylates PU.1, CK2 inhibitors abrogate IL-1␤ induction, and CK2 inducibly associates with the IL-1␤ enhancer. Taken together, these data indicate a novel two-step mechanism for IL-1␤ transcription: 1) formation of a poised chromatin architecture, and 2) phosphorylation of an enhancer-bound factor that recruits other activators. We propose that this poised structure may generally characterize rapidly activated genes.

The complexity of eukaryotic gene regulation is slowly being resolved. What has become clear is that transcriptional regulation is a multi-step process that involves the assembly of macromolecular complexes. This review will discuss the biology of the ETS family transcription factor PU.1, with emphasis on its interactions with two members of the Interferon Regulatory Factor (IRF) family, interferon consensus sequence binding protein (ICSBP), and IRF-4. The role of these interactions in transcriptional regulation is discussed, with respect to DNA binding motifs, protein-protein interaction and phosphorylation states that modulate PU.1/IRF interactions. Furthermore, potential transcriptional mechanisms for several genes are discussed, focusing on genes involved in innate immunity. Data from these studies suggest at least four distinct paradigms for transcriptional regulation by an ETS protein in conjunction with either ICSBP or IRF-4. These paradigms may describe regulatory mechanisms common to many distinct transcription factor families. Last, recent data from several laboratories have now documented the expression of ICSBP and IRF-4 in a range of cell types. These data suggest that ICSBP and IRF-4 may serve functions within these cell types that are distinct from their previously recognized functions.

Gene expression is a tightly regulated process involving multiple levels of control spanning histone acetylation to protein turnover. One of the first events in this cascade is transcription, which itself is a multistep process involving protein-protein interaction and macromolecular assembly. Here we review the role of the interferon (IFN) regulatory factor (IRF) transcription factor family member IRF-4 in transcriptional regulation. IRF-4 was initially characterized in lymphocytes and was shown to function as both a transcriptional repressor and activator. More recently, IRF-4 expression and function have been reported in macrophages. The ability of IRF-4 to serve as both a transcriptional activator and repressor is determined, in part, by binding to distinct DNA-binding motifs and through interaction with various additional transcription factors, most notably with the Ets family member PU.1. The details governing these functional differences are the focus of this review. Importantly, the role of posttranslational modification and nuclear translocation of IRF-4 in transcriptional regulation are addressed. Several possible paradigms of transcriptional regulation by IRF-4 are proposed, where these paradigms may describe regulatory mechanisms common to many distinct transcription factor families.

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