No abstract

The c-los serum response element (SRE) is a multifunctional regulatory region of the c-fos promoter that responds to a variety of inducers. Recently, we have demonstrated that the SRE binds the C/EBP-related transcription factor rat NFIL-6 (rNFIL-6). In this study we show that rNFIL-6 is regulated by the cAMP second messenger pathway in the rat pheochromocytoma PC12 cell line. Following forskolin treatment, rNFIL-6 binding to the SRE is increased, and the factor becomes phosphorylated and undergoes a trans-location to the nucleus. In transient cotransfection assays, rNFIL-6 is capable of trans-activating the c-fos promoter in a manner dependent on the SRE. These data show that rNFIL-6 undergoes a novel activation in which cAMP-induced nuclear trans-location allows rNFIL-6 to bind to the SRE and contribute to cofos activation. We propose that rNFIL-6 is an additional regulatory component of the c-los gene, which provides cAMP responsiveness to the multifunctional SRE.

Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-alpha, beta or gamma are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4-8 hrs and decreases after 16 hrs.

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral The c-Fos oncoprotein is the product of an immediate-early response gene which, together with c-Jun, can activate transcription of promoters bearing AP-1-binding sites (28). The c-Fos and c-Jun proteins have a homologous bZIP DNAbinding domain which allows them to form a complex via the leucine zipper and bind DNA via the basic motif (12,18,27,35,45,46,53). In addition, c-Fos and c-Jun have a number of independently acting homologous activation domains (1,2,5,7,50). One of these contains two homologous motifs (HOB1 and HOB2) which cannot activate transcription independently but do so cooperatively when combined (50). The HOBW motif contains a recognition site for MAP kinase whose presence is essential for the activity of HOB1. In c-Jun, this site is phosphorylated by a MAP kinase-like activity both in vitro and in vivo (6, 21, 37), suggesting that phosphorylation of HOB1 is essential for its function.The c-Fos protein is a member of a family of transcription factors which are intermittently similar along their lengths. They include Fra-1, Fra-2, FosB, and FosBSF. These c-Fosrelated proteins can all bind to c-Jun and related proteins JunB and JunD, forming heterodimers which can bind to AP-1 sites. It is unclear how these c-Fos-c-Jun-related heterodimers differ in function. Discrimination may come at the level of DNA binding (44) Initiation of transcription by RNA polymerase II can be mediated by a set of basal factors which assemble at cis-acting elements such as the TATA box and the initiator (Inr) element (41). Initiation of transcription from either one of these elements can be augmented by regulatory transcription factors bound to the promoter. These activators are thought to function by forming protein-protein interactions with basal transcription factors (14). The study of viral transactivators such as VP16, ElA, Zta, and IE2 has shown that contact with the general factors TATA-box-binding protein (TBP) and transcription factor IIB (TFIIB) can be an important step in activation (9,17,23,24,30,31,40). Several cellular transcription factors, PU.1, p53, c-Myc, and E2F, have domains which bind TBP in vitro (15,16,19,49), and recently, the c-Rel protein has been shown to form a functional contact with TBP in vivo (26,54). Direct contact with TBP is not, however, the only mechanism by which regulators activate transcription. The Spl protein, for example, can associate with other components of the basal trans...

By using a translation assay in oocytes, a 17S RNA fraction coding for the interferon-induced (2'-5')oligo(A) synthetase was purified from human cells. A cDNA library was prepared by cloning in Escherichia coli plasmid pBR322 and screened by positive hybridization-translation in oocytes. A cDNA clone corresponding to the (2'-5')oligo(A) synthetase mRNA was identified.In SV80 cells, this E cDNA recognizes three RNAs of 1.65, 1.85, and 3.6 kilobases, which are present only after interferon treatment of the cells. In Namalva cells, mainly one RNA of 1.8 kilobases is seen.All interferons (IFNs) induce in cells the synthesis of a certain number of proteins and their mRNAs (1-3). These induced proteins include translation regulatory enzymes (1-3), surface antigens (4-6), and unidentified proteins (7-9) and are probably involved in the complex antiviral, antimitogenic, and immunoregulatory effects of IFNs. Among the enzymes induced by IFNs, the (2'-5')oligo(A) synthetase has been best studied. This enzyme binds double-stranded (ds) RNA and polymerizes ATP into ppp(A2'p)nA dimers, trimers, and longer oligomers (10), which activate the latent RNase F (11-13), thereby inhibiting protein synthesis. The (2'-5')oligo(A) synthetase increases 10-to 10,000-fold in various vertebrate tissues and cell lines, exposed to IFN-a, -P, or -y (1-3, 14). Induction of the (2'-5')oligo(A) synthetase and activation of RNase F seem to be a central mechanism, although not unique (15), in IFN's antiviral action (1-3, 16) and in the decrease of cell proliferation (17,18 In SV80 cells, the E cDNA hybridizes to three cytoplasmic RNA species of 1.65, 1.85, and 3.6 kb, whereas in Namalva cells, the predominant RNA is 1.8 kb.MATERIALS AND METHODS Cell Cultures and IFNs. Human SV80 cells were grown to confluent monolayers of 5 x 107 cells and lymphoblastoid Namalva cells were grown in suspension to 1.5 x 106 cells per ml as reported earlier (6). Human IFN-(31 prepared (6, 25) and partially purified (26) to 107 units/mg of protein was used to treat cells at 200 units of IFN-j31 per ml for 12 hr, unless otherwise indicated. IFN-/31 (>2 x 108 units/mg) purified by monoclonal antibody affinity chromatography and IFN-a were obtained as described (27,28).mRNA Isolation and cDNA Cloning. Total cell RNA was prepared as reported earlier (6) with LiCl/urea (29) and purified on oligo(dT)-cellulose (yield, 0.4 mg per 109 SV80 cells). The 17S RNA fraction from IFN-treated cells was isolated (unpublished data) in a preparative gel electrophoresis apparatus in 1.5% agarose/6 M urea/25 mM sodium citrate, pH 3.5 (30), and repurified on oligo(dT)-cellulose. ds cDNA was made from 2 ,g of 17S RNA as detailed (9, 31). Both strands were made with avian myeloblastosis virus reverse transcriptase (J. Beard) by priming with (oligo)dT, the first strand being tailed with dATP prior to second-strand synthesis (32). Blunt ends were formed on the ds cDNA (9) and the largest fractions were tailed with dCTP and hybridized to dGTP-tailed, Pst I-cut pBR322 DNA. E. coli MM294...