ABSTRACT
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.
Subject(s)
Apoptosis , B-Lymphocytes/metabolism , Repressor Proteins/genetics , Animals , B-Lymphocytes/immunology , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression , Genetic Vectors , Humans , Immunoglobulin lambda-Chains/immunology , Immunoglobulin lambda-Chains/metabolism , Interferon Regulatory Factors , Mice , Plasmacytoma , Repressor Proteins/metabolism , Retroviridae/genetics , Sequence Deletion , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
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-gamma-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.
Subject(s)
Cytokines/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation/immunology , Interferon Type I/pharmacology , Interferons/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Response Elements/immunology , Trans-Activators/metabolism , Transcription Factors/metabolism , Ubiquitins/analogs & derivatives , 3T3 Cells , Animals , Consensus Sequence/immunology , Cytokines/antagonists & inhibitors , Cytokines/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Drug Synergism , Hematopoietic Stem Cells/chemistry , Hematopoietic Stem Cells/metabolism , Humans , Interferon Regulatory Factor-1 , Interferon Regulatory Factors , K562 Cells/chemistry , K562 Cells/metabolism , Mice , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/physiology , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/physiology , Repressor Proteins/biosynthesis , Repressor Proteins/physiology , Trans-Activators/biosynthesis , Trans-Activators/physiology , Transcription Factors/biosynthesis , Transcription Factors/physiology , Transcriptional Activation/immunology , U937 Cells/chemistry , U937 Cells/metabolismABSTRACT
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.
