Human ~-and 13-interferons (IFNs) stimulate rapid but transient increases in transcription from a set of previously quiescent genes. Protein synthesis is not required for initial stimulation, but duration of the response is limited to a few hours by a process requiring synthesis of new proteins. An IFN-stimulated response element (ISRE) was identified 5' to an inducible gene by deletion analysis and point mutagenesis, and sequence comparisons with other promoters defined the consensus element YAGTTTC(A/T)YTTTYCC. Two classes of IFN-inducible nuclear factors were found that bind to the ISRE. The most rapidly induced factor appeared without new protein synthesis, whereas a second factor required active protein synthesis for its appearance and maintenance. The kinetics of appearance and loss of these binding activities correlate with the activation and repression of IFN-stimulated genes. These different IFN-activated or induced factors may bind sequentially to the same essential promoter element to first increase and then repress transcription.

The signal transduction pathway through which interferon-a (IFNa) stimulates transcription of a defined set of genes involves activation of DNA-binding factors specific for the IFNa-stimulated response element (ISRE). IFN-stimulated gene factor-3 (ISGF3), the positive regulator of transcription, was derived in response to IFNa treatment from preexisting protein components that were activated first in the cell cytoplasm prior to appearance in the nucleus. Nuclear translocation of ISGF3 required several minutes and could be inhibited by NaF. Formation of active ISGF3 was mimicked in vitro by mixing cytoplasmic extracts from IFNa-stimulated cells with extracts of cells treated to contain high amounts of the unactivated factor. Active ISGF3 was found to be formed from association of two latent polypeptide precursors that were distinguished biochemically by differential sensitivity to N-ethyl maleimide. One precursor was modified in response to IFNa occupation of its cell-surface receptor, thus enabling association with the second subunit. The resulting complex then was competent for nuclear translocation and binding to ISRE. Cytoplasmically localized transcription factor precursors thus serve as second messengers to translate directly an extracellular signal into specific transcriptional activity in the nucleus.

Interferon-stimulated gene factor 3 (ISGF3) is the ligand-dependent transcriptional activator that, in response to interferon treatment, is assembled in the cell cytoplasm, is translocated to the nucleus, and binds the consensus DNA site, the interferon-stimulated response element. We have purified ISGF3 and identified its constituent proteins: a DNAbinding protein of 48 kDa and three larger polypeptides (84, 91, and 113 kDa), which themselves do not have DNA-binding activity. The multisubunit structure of ISGF3 most likely reflects its participation in receiving a ligand-dependent signal, translocating to the nucleus, and binding to DNA to activate transcription.Many genes are subject to immediate transcriptional activation by attachment of specific protein ligands to cell surface receptors. However, different polypeptide ligands activate the transcription of different sets of genes (1-8). How this specificity is achieved remains unknown. We began the study of genes that were transcriptionally responsive to interferon a (IFNa-stimulated genes, or ISGs; refs. 2, 3) with the goal of identifying the nuclear transcriptional regulators and how they might be modified after the IFNa receptor had been occupied. We identified the interferon-stimulated response element (ISRE; refs. 9-13), the required binding site for IFNa activation to which presumed transcription factors bind (11). Then ISRE-binding proteins and the conditions required for their presence in cells were determined (11). One IFNa-induced factor, ISG factor 3 (ISGF3), has the necessary characteristics to be the transcriptional activator of the ISGs: it rises rapidly without ongoing protein synthesis (11,12), requires the same nucleotides for binding as are required for IFN-dependent gene activation (13,28), and is absolutely dependent on IFNa treatment (3, 14) and its level in cells under a variety of conditions parallels the rate of ISG transcription (15, 16). For example, IFNy does not induce ISGF3, but cells pretreated with IFNy make 10 times more ISGF3 when subsequently treated with IFNa and induced transcription of ISGs is 10 times higher (16).We showed that ISGF3 activation occurs in the cytoplasm and furthermore can be produced in vitro by mixing cytoplasm of IFN-treated cells. Our results accord with the report of Dale et al. (17) that an IFN-dependent DNA binding factor that is probably ISGF3 could be formed in cytoplasts (enucleated cells) exposed to IFNa. It is possible that the "waiting" cytoplasmic proteins directly receive a signal generated by receptor occupation and then move to the nucleus to activate transcription. In this report we describe the purification of ISGF3 and identification of the constituent polypeptides of this heteromeric transcriptional activator. MATERIALS AND METHODSCell Culture, IFN Induction, and Preparation of Nuclear Extract. HeLa cells (ATCC clone S3) in suspension were treated for 15 hr with IFNy (1 ng/ml) and for 1 hr with IFNa (500 units/ml). Soluble proteins from isolated nuclei were prepared as describe...

Critical to our understanding of the developmental potential of stem cells and subsequent control of their differentiation in vitro and in vivo is a thorough understanding of the genes that control stem cell fate. Here, we report that Foxd3, a member of the forkhead family of transcriptional regulators, is required for maintenance of embryonic cells of the early mouse embryo. Foxd3−/− embryos die after implantation at approximately 6.5 days postcoitum with a loss of epiblast cells, expansion of proximal extraembryonic tissues, and a distal, mislocalized anterior organizing center. Moreover, it has not been possible to establish Foxd3−/− ES cell lines or to generate Foxd3−/− teratocarcinomas. Chimera analysis reveals that Foxd3 function is required in the epiblast and that Foxd3−/− embryos can be rescued by a small number of wild-type cells. Foxd3−/− mutant blastocysts appear morphologically normal and express Oct4, Sox2, and Fgf4, but when placed in vitro the inner cell mass initially proliferates and then fails to expand even when Fgf4 is added. These results establish Foxd3 as a factor required for the maintenance of progenitor cells in the mammalian embryo.[Keywords: mouse embryogenesis; stem cells; Foxd3; winged helix gene] Multipotent progenitor cells exist transiently in the mammalian embryo in several tissues including the preimplantation stage blastocyst, the gastrulating epiblast, and the neural crest. Each of these cell types can be cultured in vitro to generate multipotent stem cell lines, and when grafted ectopically, blastocysts and epiblast tissue will produce teratocarcinomas containing multipotent stem cells. Understanding the common molecular regulatory mechanisms of different stem cell types is critical to an understanding of how multipotency is maintained in vivo and how differentiation is controlled.Mammalian embryonic development initiates with a series of cleavage divisions, and at 2.5 days postcoitum (dpc) the free-floating mouse embryo undergoes compaction: cell boundaries become tightly apposed to one another and cells are no longer equivalent. Inner cells contribute to the inner cell mass (ICM) and embryo proper, whereas outer cells contribute to the trophectoderm, a tissue essential for implantation. After implantation, the ICM proliferates and extends distally to form the cuplike egg cylinder (at ∼ 5.5 dpc) consisting of an inner layer of epiblast cells and an outer layer of extraembryonic visceral endoderm (for review, see Hogan et al. 1994).Few genes have been identified that are required for the maintenance of the epiblast cell population and the establishment of ICM-derived embryonic stem (ES) cells in vitro. Oct4−/− embryos, mutant for this POU family transcription factor, die around 5.0 dpc, shortly after implantation but before the egg cylinder is formed. Oct4−/− ES cells cannot be established, as the ICM cells fail to survive (Nichols et al. 1998). Survival of multipotent cells of the ICM is highly sensitive to Oct4 expression levels; low Oct4 levels result in the differentiat...

Inteferon-stimulated gene factor 2 (ISGF2) was purified from HeLa cells treated with alpha interferon. The factor, a single polypeptide of 56 kilodaltons (kDa), bound both to the central 9 base pairs of the 15-base-pair interferon-stimulated response element (ISRE) that is required for transcriptional activation of interferonstimulated genes and to the PRD-I regulatory element of the beta interferon gene. ISGF2 was a phosphoprotein, and dephosphorylation in vitro reduced its DNA-binding activity. However, conditions that changed the amount of ISGF2 did not change the phosphorylated isoforms in vivo. ISGF2 in unstimulated cells existed in trace amounts and was induced by both alpha interferon and gamma interferon as well as by virus infection. Plasmid-bearing Escherichia coli clones encoding ISGF2 were selected with antibody against purified ISGF2. Sequence analysis revealed that the ISGF2 protein was the same as that encoded by the cDNA clone IRF-1, which has been claimed to activate transcription of interferon genes. We show that transcription of the ISGF2 gene was induced by alpha interferon, gamma interferon, and double-stranded RNA. However, ISGF2 was neither necessary nor sufficient for induced transcription of the beta interferon gene, while the factor NFKB was clearly involved.Interferon-stimulated genes (ISGs) undergo a rapid transcriptional activation of greater than 100-fold in fibroblasts or HeLa cells treated with type I interferon (alpha interferon [IFN-a] or beta interferon [IFN-P]) (4,13,29,47,48). The induction peaks at about 1 h after treatment and declines to essentially baseline levels after as little as 6 h. If protein synthesis is prevented, the decline is considerably more gradual (13, 30). Most ISGs do not respond to gamma interferon (IFN--y), but a few exhibit an immediate transcriptional response to either IFN-a or IFN-y treatment (9, 50). The larger biological consequences of interferon treatment, slowing or cessation of cell growth and attainment of the antiviral state, occur within 24 h after treatment (for reviews, see references 42 and 53). In view of this signal-response program, two general kinds of induced proteins need to be examined: those connected with the original transcriptional response, and those that ultimately lead to the antiviral state or growth inhibition.To begin to study the first group of proteins, we cloned the promoters of two ISGs (33, 47) and identified the interferonstimulated response element (ISRE) (34, 47), a 15-base-pair (bp) highly conserved enhancer element within a larger, less-conserved region of homology found in some ISGs (14). The ISRE was shown to be necessary and sufficient for ISG transcriptional induction by 34,47,49). The ISRE region from one IFN-a-stimulated gene also contained sequences required for IFN--y-stimulated transcription (50); although we have recently found that the IFN-y-responsive element is not limited to the IFN-a ISRE (D. J. Lew, T. Decker, and J. E. Darnell, Jr., unpublished data).Three with nuclear extracts from IFN-a-trea...