Requirement for transcription factor NFAT in interleukin-2 expression.
(17/75781)
The nuclear factor of activated T cells (NFAT) transcription factor is implicated in expression of the cytokine interleukin-2 (IL-2). Binding sites for NFAT are located in the IL-2 promoter. Furthermore, pharmacological studies demonstrate that the drug cyclosporin A inhibits both NFAT activation and IL-2 expression. However, targeted disruption of the NFAT1 and NFAT2 genes in mice does not cause decreased IL-2 secretion. The role of NFAT in IL-2 gene expression is therefore unclear. Here we report the construction of a dominant-negative NFAT mutant (dnNFAT) that selectively inhibits NFAT-mediated gene expression. The inhibitory effect of dnNFAT is mediated by suppression of activation-induced nuclear translocation of NFAT. Expression of dnNFAT in cultured T cells caused inhibition of IL-2 promoter activity and decreased expression of IL-2 protein. Similarly, expression of dnNFAT in transgenic mice also caused decreased IL-2 gene expression. These data demonstrate that NFAT is a critical component of the signaling pathway that regulates IL-2 expression. (+info)
p38 mitogen-activated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis.
(18/75781)
p38 mitogen-activated protein kinase (p38) has been extensively studied as a stress-responsive kinase, but its role in development remains unknown. The fruit fly, Drosophila melanogaster, has two p38 genes, D-p38a and D-p38b. To elucidate the developmental function of the Drosophila p38's, we used various genetic and pharmacological manipulations to interfere with their functions: expression of a dominant-negative form of D-p38b, expression of antisense D-p38b RNA, reduction of the D-p38 gene dosage, and treatment with the p38 inhibitor SB203580. Expression of a dominant-negative D-p38b in the wing imaginal disc caused a decapentaplegic (dpp)-like phenotype and enhanced the phenotype of a dpp mutant. Dpp is a secretory ligand belonging to the transforming growth factor beta superfamily which triggers various morphogenetic processes through interaction with the receptor Thick veins (Tkv). Inhibition of D-p38b function also caused the suppression of the wing phenotype induced by constitutively active Tkv (TkvCA). Mosaic analysis revealed that D-p38b regulates the Tkv-dependent transcription of the optomotor-blind (omb) gene in non-Dpp-producing cells, indicating that the site of D-p38b action is downstream of Tkv. Furthermore, forced expression of TkvCA induced an increase in the phosphorylated active form(s) of D-p38(s). These results demonstrate that p38, in addition to its role as a transducer of emergency stress signaling, may function to modulate Dpp signaling. (+info)
Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element.
(19/75781)
Typical metazoan core promoter elements, such as TATA boxes and Inr motifs, have yet to be identified in early-evolving eukaryotes, underscoring the extensive divergence of these organisms. Towards the identification of core promoters in protists, we have studied transcription of protein-encoding genes in one of the earliest-diverging lineages of Eukaryota, that represented by the parasitic protist Trichomonas vaginalis. A highly conserved element, comprised of a motif similar to a metazoan initiator (Inr) element, surrounds the start site of transcription in all examined T. vaginalis genes. In contrast, a metazoan-like TATA element appears to be absent in trichomonad promoters. We demonstrate that the conserved motif found in T. vaginalis protein-encoding genes is an Inr promoter element. This trichomonad Inr is essential for transcription, responsible for accurate start site selection, and interchangeable between genes, demonstrating its role as a core promoter element. The sequence requirements of the trichomonad Inr are similar to metazoan Inrs and can be replaced by a mammalian Inr. These studies show that the Inr is a ubiquitous, core promoter element for protein-encoding genes in an early-evolving eukaryote. Functional and structural similarities between this protist Inr and the metazoan Inr strongly indicate that the Inr promoter element evolved early in eukaryotic evolution. (+info)
Isolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis.
(20/75781)
Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells. The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C). The driver cDNAs were generated from a control hamster cell line that did not contain the YAC that expressed NPC1. Among the gene fragments obtained by RDA, NPC1 was the most abundant product. In addition, two non-NPC1 fragments were isolated that were mapped to and expressed from 911D5. One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone reported previously as an expressed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) that had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs 911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two-part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval. (+info)
Functional studies by site-directed mutagenesis on the role of Sp1 in the expression of the pyruvate kinase M and aldolase A genes.
(21/75781)
During the cell cycle of mitogen stimulated rat thymocytes, an 8-10-fold induction of glycolytic enzymes and a corresponding increase in the mRNA levels has been observed. This prompted us to study the transcriptional regulation of the rat aldolase A and pyruvate kinase M genes. cis-Regulatory elements of both promoters were evaluated by site-directed mutagenesis of promoter/luciferase constructs and transient transfections of rat hepatoma FTO2B cells. Furthermore, the binding proteins were identified by mobility shift assays in the presence of specific antibodies. In the aldolase AH1 promoter, five binding sites for Sp1 and Sp3 and a TPA responsive element were identified as essential for transcriptional regulation. Most of the promoter activity can be attributed to these regulatory elements. In the pyruvate kinase M promoter three out of five binding sites of Sp1 and Sp3 (B box and GC boxes 1 and 3) turned out to be functional in the transfection assays whereas the disruption of GC box 2 had no effect, and the disruption of the GC box 4 had only a minor effect on the promoter activity. Both promoters are stimulated by Sp1 as well as Sp3, as judged by cotransfection experiments of Drosophila SL2 cells. Therefore, the Sp1- and Sp3-directed transcription provides a means for common regulatory mechanism of the aldolase A and the pyruvate kinase M genes. (+info)
Thyroid hormone effects on Krox-24 transcription in the post-natal mouse brain are developmentally regulated but are not correlated with mitosis.
(22/75781)
Krox-24 (NGFI-A, Egr-1) is an immediate-early gene encoding a zinc finger transcription factor. As Krox-24 is expressed in brain areas showing post-natal neurogenesis during a thyroid hormone (T3)-sensitive period, we followed T3 effects on Krox-24 expression in newborn mice. We analysed whether regulation was associated with changes in mitotic activity in the subventricular zone and the cerebellum. In vivo T3-dependent Krox-24 transcription was studied by polyethylenimine-based gene transfer. T3 increased transcription from the Krox-24 promoter in both areas studied at post-natal day 2, but was without effect at day 6. An intact thyroid hormone response element (TRE) in the Krox-24 promoter was necessary for these inductions. These stage-dependent effects were also seen in endogenous Krox-24 mRNA levels: activation at day 2 and no effect at day 6. Moreover, similar results were obtained by examining beta-galactosidase expression in heterozygous mice in which one allele of the Krox-24 gene was disrupted with an inframe Lac-Z insertion. However, bromodeoxyuridine incorporation showed mitosis to continue through to day 6. We conclude first, that T3 activates Krox-24 transcription during early post-natal mitosis but that this effect is extinguished as development proceeds and second, loss of T3-dependent Krox-24 expression is not correlated with loss of mitotic activity. (+info)
Expression and differential regulation of connective tissue growth factor in pancreatic cancer cells.
(23/75781)
CTGF is an immediate early growth responsive gene that has been shown to be a downstream mediator of TGFbeta actions in fibroblasts and vascular endothelial cells. In the present study hCTGF was isolated as immediate early target gene of EGF/TGFalpha in human pancreatic cancer cells by suppression hybridization. CTGF transcripts were found in 13/15 pancreatic cancer cell lines incubated with 10% serum. In 3/7 pancreatic cancer cell lines EGF/TGFalpha induced a significant rise of CTGF transcript levels peaking 1-2 h after the start of treatment. TGFbeta increased CTGF transcript levels in 2/7 pancreatic cancer cell lines after 4 h of treatment and this elevation was sustained after 24 h. Only treatment with TGFbeta was accompanied by a parallel induction of collagen type I transcription. 15/19 human pancreatic cancer tissues were shown to overexpress high levels of CTGF transcripts. CTGF transcript levels in pancreatic cancer tissues and nude mouse xenograft tumors showed a good correlation to the degree of fibrosis. In situ hybridization and the nude mouse experiments revealed that in pancreatic cancer tissues, fibroblasts are the predominant site of CTGF transcription, whereas the tumor cells appear to contribute to a lesser extent. We conclude that CTGF may be of paramount importance for the development of the characteristic desmoplastic reaction in pancreatic cancer tissues. (+info)
Induction of the tumor suppressor protein p53 restricts cellular proliferation. Since actively growing cells require the ongoing synthesis of ribosomal RNA to sustain cellular biosynthesis, we studied the effect of p53 on ribosomal gene transcription by RNA polymerase I (Pol I). We have measured rDNA transcriptional activity in different cell lines which either lack or overexpress p53 and demonstrate that wild-type but not mutant p53 inhibits cellular pre-rRNA synthesis. Conversely, pre-rRNA levels are elevated both in cells which express mutant p53 and in fibroblasts from p53 knock-out mice. Transient transfection assays with a set of rDNA deletion mutants demonstrate that intergenic spacer sequences are dispensable and the minimal rDNA promoter is sufficient for p53-mediated repression of Pol I transcription. However, in a cell-free transcription system, recombinant p53 does not inhibit rDNA transcription, indicating that p53 does not directly interfere with the basal Pol I transcriptional machinery. Thus, repression of Pol I transcription by p53 may be a consequence of p53-induced growth arrest. (+info)