The immediate upstream region of the mouse interleukin 2 (Il‐2) gene harbors a strong transcriptional enhancer. This enhancer contains most, if not all of the sequence elements necessary for the T cell specific induction of the Il‐2 gene by the phorbol ester TPA and the plant lectin Concanavalin A. DNase I footprinting studies with fractionated extracts obtained from induced and uninduced E14 T cells revealed numerous recognition sites for potential trans‐acting factors. Five of these sites are also recognized by the TPA‐activated HeLa cell factors AP‐1 and AP‐3. Other sites including two TATA‐boxes, two purine‐rich sequence motifs and two copies of the GGGPuTTTCAA motif are recognized by lymphocyte specific factors. The latter motif is highly conserved between several lymphokine genes and is therefore designated as a T cell element (TCE). In E14 T cells, pentamers of the distal TCEd confer an activity similar to that of the entire Il‐2 enhancer, whereas in B and HeLa cells, the TCEd‐pentamer is inactive as is the Il‐2 enhancer. These data indicate the involvement of the TCEd and its recognition factor(s) in the cell type specific induction of the Il‐2 gene during T cell activation.

The T lymphocyte-specific enhancers of the murine and human Interleukin 2 (Il-2) genes harbour several binding sites for ubiquitous transcription factors. All these sites for the binding of AP-1, NF-kB or Oct-1 are non-canonical sites, i.e. they differ in one or a few base pairs from consensus sequences for the optimal binding of these factors. Although the factors bind weakly to these sites, the latter are functionally important because their mutation to non-binding sites results in a decrease of inducible activity of the Il-2 enhancer. Conversion of three sites to canonical binding sites of Octamer factors, AP-1 and NF-kB results in a drastic increase in enhancer activity and the induction of the Il-2 enhancer in non-T cells, such as B cell lines, murine L cells and human HeLa cells. The introduction of two or three canonical sites into the enhancer leads to a further increase of its activity. Il-2 enhancer induction is also observed in B cells when the concentration of AP-1 and Oct factors increases as a result of cotransfections with FosB and Octamer expression plasmids. When Il-2 enhancer constructs carrying canonical factor binding sites were injected into Xenopus oocytes the strong binding of ubiquitous factors substantially overcomes the silencing effect of negatively acting factors present in resting primary T lymphocytes. These results suggest a fine-tuned interplay between ubiquitous and lymphoid-specific factors binding to and transactivating the Il-2 enhancer and show that the binding affinity of ubiquitous factors to the enhancer contributes to its cell-type specific activity. Moreover, we believe that a dramatic increase of transcriptional activity brought about by single point mutations at strategic important factor binding sites may also have relevance to the activation of nuclear oncogenes.

Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.

The inducible, T cell-specific enhancers of murine and human Interleukin 2 (Il-2) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif. When cloned in multiple copies this so-called TCEd (distal T cell element) acts as an inducible proto-enhancer element in E14 T lymphoma cells, but not in HeLa cells. In extracts of induced, Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA. The binding of the most prominent factor, named TCF-1 (T cell factor 1), is correlated with the proto-enhancer activity of TCEd. TCF-1 consists of two polypeptides of about 50 kD and 105 kD; the former seems to be related to the 50 kD polypeptide of NF-kB. Purified NF-kB is also able to bind to the TCEd, but TCF-1 binds stronger than NF-kB to TCEd DNA. The conversion of the TCEd to a 'perfect' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and, as a functional consequence, to the activity of the 'converted' TCEd motifs in HeLa cells. Thus, the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells. These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene.

No abstract

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.

The promoters of IL-2 and IL-4 genes contain multiple binding sites for octamer factors. In peripheral T lymphocytes and several T cell lines, both the ubiquitous Oct factor Oct-1 and the lymphocyte-specific factor Oct-2 are expressed and bind to the IL-2 and IL-4 promoters. Prominent octamer binding sites of IL-2 and IL-4 promoters are their upstream promoter sites (UPS) which share 14 identical nucleotides. Multiple copies of the IL-2 and IL-4 UPS act as inducible enhancers in T cells, and their induction is inhibited by the immunosuppressant cyclosporin A (CsA). Closely linked to the octamer site, the IL-2 UPS contains a non-canonical AP-1 binding (TRE) site, and mutation in either site to a non-functional factor binding site impairs the induction of the IL-2 promoter. The binding of AP-1 and octamer factors to the IL-2 UPS DNA overlaps, and the tight association and functional cooperation of octamer with AP-1 factors is of crucial importance for the inducible IL-2 UPS activity. Introduction of five or ten spacer nucleotides between both IL-2 UPS sites results in a drastic reduction of inducible UPS activity, both in the loss of suppression by CsA and stimulation by the Ca(2+)-dependent phosphatase calcineurin. Within the IL-4 UPS the Oct and TRE-like motifs are separated by a binding site of nuclear factor of activated T cells (NF-AT). This site shares nine out of ten bp with an IL-2 NF-AT site. The strong binding of NF-ATp to the IL-4 UPS site suppresses the simultaneous binding of Oct factors to the IL-4 UPS. Because the two other Oct binding sites of IL-4 promoter show a similar sequence configuration, the binding of NF-AT seems to prevent the simultaneous binding of Oct factors to the IL-4 promoter. By contrast, both classes of factors bind simultaneously to the IL-2 promoter, and their tight association with AP-1 enhances the IL-2 promoter activity.