The human IL-3 locus is regulated cooperatively by two NFAT-dependent enhancers that have distinct tissue-specific activities

The human IL-3 gene is expressed by activated T cells, mast cells, and eosinophils. We previously identified an enhancer 14 kb upstream of the IL-3 gene, but this element only functioned in a subset of T cells and not in mast cells. To identify additional mechanisms governing IL-3 gene expression, we mapped DNase I hypersensitive (DH) sites and evolutionarily conserved DNA sequences in the IL-3 locus. The most conserved sequence lies 4.5 kb upstream of the IL-3 gene and it encompassed an inducible cyclosporin A-sensitive DH site. A 245-bp fragment spanning this DH site functioned as a cyclosporin A-sensitive enhancer, and was induced by calcium and kinase signaling pathways in both T cells and mast cells via an array of three NFAT sites. The enhancer also encompassed AML1, AP-1, and Sp1 binding sites that potentially mediate function in both T and myeloid lineage cells, but these sites were not required for in vitro enhancer function in T cells. In stably transfected T cells, the -4.5-kb enhancer cooperated with the -14-kb enhancer to activate the IL-3 promoter. Hence, the IL-3 gene is regulated by two enhancers that have distinct but overlapping tissue specificities. We also identified a prominent constitutive DH site at -4.1 kb in T cells, mast cells, and CD34(+) myeloid cells. This element lacked in vitro enhancer function, but may have a developmental role because it appears to be the first DH site to exist upstream of the IL-3 gene during hemopoietic development before IL-3 expression.     

Analysis of basal regulatory elements in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene promoter

5'-Deletion analysis of the 1.7-kb mouse 1alpha-hydroxylase gene promoter reveals that the minimal promoter region for basal activity is -85/+22 and requires a functional CCAAT element. Mutational analysis also demonstrates that deletion of the internal promoter region from nucleotides -1125 to -86 leads to a 25- to 30-fold increase in basal promoter activity. The increased activity is not the result of positional effects, but is caused in part by the removal of an AC repeat. Further analysis of the promoter revealed an enhancer element localizes to an upstream region -1385 to -1125, which contains three consensus AP-1 sites. Deletion of the most proximal AP-1 site causes a 60% loss of enhancer activity. The data suggest the presence of the AC repeat prevents the full potential activation of the 1alpha-hydroxylase promoter by factors binding to AP-1 sites.     

Characterization of constitutive and inducible transcription factors binding to the P2 NF-AT site in the human interleukin-4 promoter

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites—the P2 site—located between –173 and –150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer proteins were suggested to bind to this region and to cooperatively activate the promoter activity (Chuvpilo et al., 1993). To precisely analyze the P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, Jun, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T-cells form two P2-binding complexes—a PMA/ionomycin-inducible and a constitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c, Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of the two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU domains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at –173 to –150 of the IL-4 promoter is a binding site for NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with NF-ATp/c at this site.© 1997 Elsevier Science B.V. All rights reserved.     

Sequence analysis of the promoter region of the glioblastoma derived T cell suppressor factor/transforming growth factor (TGF)-beta 2 gene reveals striking differences to the TGF-beta 1 and -beta 3 genes

Human glioblastoma cells secrete a factor termed glioblastoma derived T cell suppressor factor (G-TsF) or transforming growth factor beta 2 (TGF-beta 2) which inhibits the response of T cells to mitogenic or antigenic stimulation. In the present study we isolated the promoter region of the G-TsF/TGF-beta 2 gene. The promoter region shares no homology to the promoter of the TGF-beta 1 or the 5' region of the TGF-beta 3 gene and harbours several familiar DNA motifs, including the cytokine-1 region, an octamer-like sequence, Sp1- and AP-2-like elements and a putative NF-kappa B site. In contrast to the TGF-beta 1 gene, the G-TsF/TGF-beta 2 gene contains three TATA-like sequences but lacks an AP-1 site. To understand the cell type specificity of expression of G-TsF/TGF-beta 2, the individual contribution of the DNA elements detected in the promoter has to be analysed in further studies.     

T cell-specific negative regulation of transcription of the human cytokine IL-4.

IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells, B cells, and mast cells. We investigated the upstream regulatory elements of the human IL-4 promoter. A novel T cell-specific negative regulatory element (NRE) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene: -311CTCCCTTCT-303 (NRE-I) and -288CTTTTTGCTT-TGC-300 (NRE-II). A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II, respectively, were identified. Furthermore, a positive regulatory element was found 45 bp downstream of the NRE. The enhancer activity of the PRE was completely suppressed when the NRE was present. These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element. These data may have implications for the stringent control of IL-4 expression in T cells.