The activities of interferons (IFNs) 1 is mediated mainly via successive phosphorylation events of IFN receptors through specific receptor-associated tyrosine kinases that belong to the Jak family and the subsequent phosphorylation of transcription factors that translocate from the cytoplasm to the nucleus. The transcription factors belong to the signal transducers and activators of transcription (STAT) family of proteins (for review see Refs. 1-3). In the case of IFN-␣/ signaling, mainly STAT1␣ (p91), STAT1 (p84), and STAT2 (p113) are phosphorylated on specific Tyr residues allowing their association and translocation to the nucleus. This complex, ISGF3␣, associates in the nucleus with another polypeptide termed ISGF3␥ (p48) that belongs to a different family of transcription factors termed interferon regulatory factors (IRFs). The activated factor, ISGF3, binds interferon-stimulated response element (ISRE) containing promoters and serves as a transcriptional activator. In the case of IFN-␥ signaling, similar events occur on the receptor, yet mainly STAT1␣ is recruited which upon phosphorylation translocates to the nucleus where it binds to gamma activation sequence (for review see Refs. 1, 2, 4). Both Tyr phosphorylation and serine/threonine (Ser/Thr) phosphorylation, mediated by the mitogen-activated protein kinase, are crucial for IFN signaling. Tyr phosphorylation is critical for the translocation and the binding to the DNA, and Ser/Thr phosphorylation is crucial for maximal transcriptional activation (5,6). This signaling cascade is down-regulated upon activation of a specific set of Tyr phosphatases that inactivate the receptors, the kinases, and the transcription factors (7-9).The delayed response to IFN is mediated mainly by the IRF family of proteins that at least in part are induced by STATs. The members of this family of factors share significant homology in the first 115 amino acids (aa) that comprise the DNA binding domain (DBD) and are reflected by the ability to bind a similar DNA motif termed ISRE or, alternatively, interferon consensus sequence
Interferon (IFN) consensus sequence binding protein (ICSBP) is a member of a family of transcription factors termed IFN regulatory factors (IRF) and is also called IRF-8. Its expression is restricted mainly to cells of the immune system, and it plays a key role in the maturation of macrophages. ICSBP exerts its activity through the formation of different DNA-binding heterocomplexes. The interacting partner dictates a specific DNA recognition sequence, thus rendering ICSBP dual transcriptional activity, that is, repression or activation. Accordingly, such DNA elements were identified at the promoter regions of target genes that manifest macrophage action. A specific module (IRF association domain [IAD]) within ICSBP and a PEST domain located on the interacting partners mediate this association. Thus, ICSBP serves as an excellent prototype, demonstrating how a small subset of transcription factors can regulate gene expression in a spatial, temporal, and delicate tuning through combinatorial protein-protein interactions on different enhanceasomes.
Type I IFNs cause the induction of a subset of genes termed IFN-stimulated genes (ISGs), which harbor a specific DNA element, IFN-stimulated response element (ISRE). This ISRE confers the responsiveness to the IFN signal through the binding of a family of transcription factors designated IFN regulatory factors (IRFs). Some IRFs can bind to the DNA alone, such as IRF-1, which elicits transcriptional activation, or IRF-2, which leads to transcriptional repression. In addition, these factors associate with IRF-8/IFN consensus sequence binding protein (ICSBP), an immune cell-restricted IRF, and the assembled heterocomplexes lead to synergistic repression of ISRE elements. ISG15 is a prototype ISG that contains a well-characterized ISRE. Here we show that PU.1, an ETS member essential for myeloid/lymphoid cell differentiation, forms heterocomplexes with the immune-restricted IRFs, IRF-8\/ICSBP and IRF-4, which lead to transcriptional activation of ISG15. These data allowed the characterization of a subset of ISREs designated ETS/IRF response element (EIRE), which are differentially regulated in immune cells. EIREs are unique in their ability to recruit different factors to an assembled enhanceosomes. In nonimmune cells the factors will mainly include IRF members, while cell type-restricted factors, such as PU.1, IRF-8\/ICSBP, and IRF-4, will be recruited in immune cells. IRF heterocomplex formation leads to transcriptional repression, and conversely, PU.1/IRFs heterocomplex formation leads to transcriptional activation. The fact that IRF-8\/ICSBP is an IFN-γ-induced factor explains why some of the EIREs are also induced by type II IFN. Our results lay the molecular basis for the unique regulation of ISGs, harboring EIRE, in immune cells.
Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells. ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements. These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain). To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library. Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells. Trip15 was recently identified as a component of the COP9/ signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2. This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1. This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP. Interferon (IFN)1 regulatory factors (IRFs) constitute a family of nine cellular transcription factors that share high homology at the first 115 amino acids which comprise the DNAbinding domain and therefore bind to similar DNA elements. These factors mediate numerous biological activities such as, anti-viral activity, IFN signaling, and immunomodulation (1, 2). IRFs are expressed in many cell types and tissues except for IFN consensus sequence-binding protein (ICSBP, also termed IRF-8) and IRF-4, which are expressed specifically in immune cells. IRFs act as transcriptional repressor or activator and the current data suggest that they harbor dual function. This dual functionality is in part due to interactions with different transcription factors resulting in the ability to interact with various promoters leading to alteration in transcriptional activities, i.e. repression or activation (for review, see Ref.
IFN consensus sequence-binding protein (ICSBP) is a member of the IFN-regulatory factors (IRF) and is thus also called IRF-8. Its expression is restricted to hematopoietic cells and IRF-8\ICSBP(-/-) mice are defective in myeloid cell differentiation. This factor exerts its transcriptional activity through interaction with other transcription factors, which leads to either repression or activation. In this paper, we describe the use of a dominant-negative (DN) mutant of IRF-8\ICSBP designed to serve as a molecular tool to dissociate the role of the various protein-protein interactions. This DN-ICSBP is truncated at the DNA-binding domain and can still associate with other factors, but the heterocomplexes produced are incapable of binding to the DNA. We show that the DN-ICSBP is able to compete for the interaction of IRF-8\ICSBP with either IRF or non-IRF members such as PU.1. Accordingly, this DN construct was able to inhibit the PU.1-dependent expression of the IgLlambda in the plasmacytoma cell line J558L. However, stable expression of this DN-ICSBP led to apoptosis of only hematopoietic cells. The data suggests that DN-ICSBP can form heterocomplexes with an as-yet unidentified survival factor for hematopoietic cells.
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