p48/STAT-1α-Containing Complexes Play a Predominant Role in Induction of IFN-γ-Inducible Protein, 10 kDa (IP-10) by IFN-γ Alone or in Synergy with TNF-α

Abstract

Human IFN-γ-inducible protein, 10 kDa (hIP-10) and murine IP-10 (mIP-10) genes are induced by IFN-γ alone, and synergistically induced by TNF-α and IFN-γ. Upstream regions of the human and murine genes contain conserved regulatory motifs, including an IFN-stimulated response element (ISRE), which governs response of the mIP-10 gene to IFN-γ. Trans-acting factors mediating the IFN-γ response via ISRE remain incompletely defined. We examined ISRE-binding factors in the regulation of the hIP-10 gene. The requirement of p48 for hIP-10 induction by IFN-γ, with or without TNF-α, was demonstrated using p48-deficient U2A cells. An hIP-10 promoter-reporter mutant (mISRE3) that was relatively deficient for binding a related factor, IFN regulatory factor-1 (IRF-1) but competent for binding p48, was induced as well as the wild-type hIP-10 promoter, supporting the interpretation that p48 played a necessary and sufficient role in hIP-10 transcription. Genomic in vivo footprinting revealed IFN-γ/TNF-α-inducible binding at the ISRE consistent with the presence of p48 and associated factors, but not with IRF-1. Induction of hIP-10 by TNF-α/IFN-γ also required NFκB binding sites, which were protected in vivo and bound p65 homodimeric NFκB in vitro. These results documented the essential role of p48 (complexed with STAT-1α) for induction and sustained transcription of the IP-10 gene, strongly suggesting that IRF-1 is not required for IP-10 induction by these inflammatory cytokines.

Regulation of monocyte chemoattractant protein (MCP)‐1 transcription by interferon‐gamma (IFN‐γ) in human astrocytoma cells: postinduction refractory state of the gene, governed by its upstream elements

“…4). The transcription factor STAT-1␣ is required for the IFN-␥inducible expression of both hMCP-1 and IP-10 in CRT cells (22,30,32). Therefore, these data indicated that post-IFN-␥ receptor events, including activation of the STAT-1␣ transcription factor, were maintained during the refractory state and that their failure did not account for the unresponsive state of the hMCP-1 gene.…”

“…Construction of expression plasmids containing a series of deletion and substitution mutants of the MCP-1 promoter, directing either CAT or luciferase reporters, has been described in detail (22,29). GL-IP10 was generated by insertion of a 972 bp IP-10 genomic fragment into the promoterless pGL3-basic vector (30).…”

“…Initial studies established optimal time points for analyzing IFN-␥-induced occupancy of the hMCP-1 promoter by IVGF. In vivo methylation of cellular DNA and DNA preparation were performed as described (14,22,30). Ligation-mediated polymerase chain reaction was carried out according to the procedure of Mueller et al (31), as adapted for mMCP-1 by Ping et al and with modifications for hMCP-1, as we previously described (22,30).…”

“…In vivo methylation of cellular DNA and DNA preparation were performed as described (14,22,30). Ligation-mediated polymerase chain reaction was carried out according to the procedure of Mueller et al (31), as adapted for mMCP-1 by Ping et al and with modifications for hMCP-1, as we previously described (22,30). Both strands of the 213 bp promoter proximal region of the hMCP-1 gene were analyzed.…”

Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression

“…The GAS of the human GBP1 promoter binds a STAT1 dimer associated with the IRF9 (p48) protein (Seegert et al ., 1994). The same STAT1–IRF9 complex has also been found to provide for the IFN‐γ responses of other genes (Bluyssen et al ., 1995; Majumder et al ., 1998). Therefore, a second possibility would be that STAT1‐S727 phosphorylation is particularly important for inducibility in the context of a STAT1–IRF9 complex.…”