The interferon-inducible murine p48 (ISGF3g) gene is regulated by protooncogene c-myc (transcriptionycell growthycytotoxic drugsyresistance)

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-g-regulated element. In addition to the IFNregulated element, a myc–max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor. Transcription factor ISGF3 plays a crucial role (1) in induction of interferon (IFN)-stimulated genes (ISGs). This factor consists of a 48-kDa DNA binding protein (p48 or ISGF3g) and two signal transducing activator of transcription (STAT) proteins, STAT1 and STAT2. p48 belongs to a family of DNA binding proteins that includes IFN-regulatory factor (IRF) and oncoprotein myb (2, 3). Other members of this family are IRF-1, IRF-2, IRF-3, cIRF-3, IFN consensus sequence binding protein (ICSBP), Pip, and V-IRF of Kaposi sarcomaassociated herpesvirus (4–10). All these proteins possess structurally similar DNA binding domains in their N termini and nonconserved C termini (3). Despite such structural similarity, these proteins are diverse in their functions. IRF-1 induces the expression of IFN-b and certain ISGs (4, 11, 12). IRF-2 represses such induction. Cells lacking IRF-1 are readily transformed by activated oncogenes, indicating a tumor suppressor role for this protein (13). ICSBP is a negative regulator of ISG expression (14). Deletion of this gene in mice leads to a chronic myelogenous leukemia-like disease and immunodeficiency (15). Pip induces immunoglobulin gene expression but inhibits ISG expression (8, 9). Gene knock-out studies have shown that p48 plays an essential role in cellular antiviral responses (16). Thus, members of the IRF gene family are involved in regulation of various cell growth and defensive processes (3). Oncogenic transcription factor c-myc is implicated in the control of normal cellular proliferation and induction of neoplasia (17, 18). Its oncogenicity results from constitutive expression and does not require mutations in coding sequences. An Nterminal transactivation region and a C-terminal basic helix-loophelix leucine zipper (b-HLH-LZ) are the two major functional domains of c-myc (17). Even though Myc alone binds to its cognate sequence CACGTG at high concentrations in vitro, its functional activity in vivo is dependent on heterodimerization with another sequence specific DNA binding protein Max (19– 21). The b-HLH-LZ domain of Myc is essential for the formation of complexes with Max (20). Although MaxyMax dimers bind to the same sequence, they are not only transcriptionally inactive but also inhibit MycyMax induced transcription (22). Virtually all the Myc is found in complexes with Max in most cells (17, 18). Two other related proteins, Mad and Mxi1, competitively interact with Max, thereby disrupting MycyMax complexes (23, 24). MadyMax complexes are not known to induce gene expression, and Mad does not interact with Myc (23, 25). Consistent with this, cell proliferative and apoptotic actions of Myc are dependent on dimerization with Max (26). Thus, heteromeric complexes of Myc, Max, and Mad regulate the outcome of cell growth (17). Myc is a short-lived protein while Max is more stable (17). Levels of Myc transiently rise upon release of quiescent cells into growth permissive medium (G0yG1 to S) and return to basal level thereafter (19). Microinjection of Myc protein into the nuclei of quiescent cells stimulates DNA synthesis and promotes G1 to S phase transition. This ability of Myc to promote the cellular G1 to S phase transition likely contributes to its strong oncogenic potential (17). Down-regulation of Myc expression and withdrawal from cell cycle occur during phorbol 12-myristate 13acetate-induced differentiation of U937 cells (27). Because Myc is a transcriptional activator, its biological actions are mediated by gene products induced by it (17, 18). Very little is known about the target genes regulated by Myc. The involvement of IRF proteins in cell growth control prompted us to examine the role of p48 gene expression in the control of cell growth. In this report we show that the binding of MycyMax dimer induces gene expression via a Mycstimulated element (MSE) in the p48 promoter. Further, we show that the growth of cells lacking the p48 gene product is inhibited more readily by cytotoxic drugs in vitro, relative to wild-type cells. Based on these observations, we suggest that p48 may be a stress-alleviating factor for cycling cells. MATERIALS AND METHODS Cell Culture and Reagents. Murine macrophage RAW (RAW264.7) and L929 fibroblast cell lines were grown in RPMI 1640 medium and DMEM supplemented with 10% fetal bovine serum. Human fibrosarcoma cell lines 2fTGH, U3A (STAT1 deficient), U2A (p48 deficient), and U2AR (p48 rescued) were gifts from George Stark (Cleveland Clinic The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y947227-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: GATE, g-activated transcriptional element; ER, estrogen receptor; EMSA, electrophoretic mobility shift assay; IFN, interferon; ICSBP, IFN consensus sequence binding protein; ISRE, IFN-stimulated response element; ISG, IFN-stimulated gene; ISGF, ISG factor; MSE, Myc-stimulated element; OHT, 4-hydroxy tamoxifen; STAT, signal transducers and activators of transcription; IRF, interferon gene regulatory factor; b-HLH-LZ, basic helix-loop-helix leucine zipper; GST, glutathione S-transferase; SV40, simian virus 40. *To whom reprint requests should be addressed.