Stat1 Depends on Transcriptional Synergy with Sp1

Abstract

STAT (signal transducer and activator of transcription) proteins combine with cytokine receptors and receptor-associated kinases in distinct protein/protein interactions that are critical for STAT-dependent signal transduction events, but the nature of any subsequent STAT interactions with DNA-binding proteins in the nucleus is less certain. Based on assays of DNA/protein binding and activity of transfected reporter plasmids, we determined that occupation of contiguous DNA-binding sites for Stat1 (the first member of the STAT family) and the transcriptional activator Sp1 are both required for full activation of the intercellular adhesion molecule-1 gene by interferon-␥. Thus, Stat1 binding to DNA cannot by itself be equated with biologic actions of Stat1. In co-immunoprecipitation experiments, we also obtained evidence of direct and selective Stat1/Sp1 interaction (in primary culture cells without overexpression), further indicating that Stat1/Sp1 synergy confers an element of specificity in the pathway leading to cytokine-activated transcription and cytokine-dependent immunity and inflammation. STAT1 proteins act as critical intermediates in cytokine-dependent gene activation based on their dual capacities for signal transduction (at the cell surface) and activation of transcription (in the nucleus) (1). Signal transduction depends on programmed assembly of cytokine receptors, receptor-associated JAK kinases, and in some cases serine kinases, that recruit and activate specific STAT proteins (2-4). Phosphorylated/activated STATs then dimerize, translocate to the nucleus, and direct transcription of specific target genes. For example, the first member of the STAT family (designated Stat1␣) undergoes tyrosine 701 and serine 727 phosphorylation in response to IFN-␥ (5). This activation step is triggered by IFN-␥-dependent oligomerization of the IFN-␥ receptor and consequent cross-phosphorylation of receptor-associated Jak1 and Jak2 kinases and the receptor ␣-chain (6). Receptor phosphorylation enables ␣-chain recruitment of Stat1 via its SH2 domain. Stat1 then undergoes phosphorylation and release from the receptor as a homodimer that can translocate to the nucleus and bind to a specific DNA element (7,8). Thus, distinct protein/protein interactions are critical for Stat1-dependent signal transduction events at the IFN-␥ receptor, but the nature of Stat1 interactions with other proteins (especially other transcription factors) in the nucleus is less certain. In the present report, we take advantage of a primary cell culture model with selective IFN-␥ responsiveness of the intercellular adhesion molecule-1 (ICAM-1) gene (9, 10) in order to study the basis for Stat1-dependent transcription. The results offer the first evidence that Stat1-mediated transactivation depends on synergistic interaction with another transcriptional activator (Sp1). EXPERIMENTAL PROCEDURESMaterials-Recombinant human IFN-␥ was from Genentech (San Francisco, CA); unlabeled dATP and dGTP were from Boehringer Mannheim; [␣-32 P]dCTP was from DuPo...

Interferon γ-dependent Induction of Human Intercellular Adhesion Molecule-1 Gene Expression Involves Activation of a Distinct STAT Protein Complex

“…In addition, studies using polyclonal antibodies to either the amino-terminal or the carboxyl-terminal portions of Stat1␣ appear to exclude Stat1␤ as a possible component of the GRF complex. These data are in agreement with the recently reported GAFlike factor binding to the IFN␥RE of ICAM-1 gene (39) and the recent finding of Stat1␣ involvement in the complex by immunoblotting using anti-Stat1 antibodies (40). Previous reports indicate that Stat1␣ is a common component of the IFN-activated trans-acting complexes GAF and ISGF3.…”

STAT3-mediated differentiation and survival of myeloid cells in response to granulocyte colony-stimulating factor: role for the cyclin-dependent kinase inhibitor p27Kip1

“…Other transcription factors known to bind to the p27 promoter are Sp1, NF-kB, and Myb (Kwon et al, 1996). Furthermore, it was shown that Sp1 and STAT1 synergize in interferon-g-induced responses (Look et al, 1995), while synergy between Sp1 and STAT3 was demonstrated in interleukin 6-induced transcription of the CCAAT/enhancer binding protein d (Cantwell et al, 1998). These factors therefore represent additional candidates for facilitating the observed transcriptional e ects of STAT3.…”

IL-2 and long-term T cell activation induce physical and functional interaction between STAT5 and ETS transcription factors in human T cells

“…In that respect, some Stat proteins bind to other transcription factors or coactivators to activate transcription (Bhattacharya et al, 1996;Look et al, 1995;Muhlethaler-Mottet et al, 1998;Qureshi et al, 1995;Schaefer et al, 1995;Shen and Stavnezer, 1998;Zhang et al, 1996). More speci®cally, MGF/Stat5a has only been reported to form a complex with the glucocorticoid receptor (GR) on the b-casein promoter devoid of GR binding site (GRE) (Cella et al, 1998;StoÈ cklin et al, 1996).…”