RESUMO
Chromatin architecture plays a decisive role in many aspects of transcription regulation. We have tested the role of specific chromatin structures in c-fos gene regulation, using a gene transfer system based on episomes derived from the Epstein-Barr virus (EBV). This system reproduces in several respects the chromatin structure and regulation of the chromosomal c-fos gene. Using this approach, we first demonstrate that the pausing of RNA polymerase II downstream of the transcriptional start site does not require precisely positioned nucleosomes. Indeed, changing the pattern of MNase hypersensitive sites along the transcribed sequence does not perturb RNA polymerase II pausing or the regulation of the c-fos gene. Next, we show that a putative nucleosome positioned between the SIE/SRE elements (-300) and the CRE/TATA elements (-36) is not necessary for activation by a variety of inducers. Accordingly, total or partial deletion of the putative nucleosome sequence does not disturb c-fos regulation while the two regulatory sites flanking the nucleosome sequence remain hypersensitive to MNase. As described in this paper, EBV episomes are useful vectors to critically examine the role of the chromatin structure in gene transcription for human cells.
Assuntos
Nucleossomos/genética , Proteínas Proto-Oncogênicas c-fos/genética , Animais , Linhagem Celular , Cromatina/genética , Colforsina/farmacologia , DNA/genética , DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/genética , Humanos , Ionomicina/farmacologia , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Camundongos , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Transcription elongation regulates c-fos expression in mouse and human cells. In the inactive state of the gene, RNA polymerases are engaged only in the promoter-proximal region. Upon activation, RNA polymerases move efficiently along the complete gene. We have used Epstein-Barr virus (EBV) episomes as a gene transfer system to study the role of promoter-proximal pausing and transcript elongation in c-fos expression. We find that the sequence located immediately downstream of the transcriptional start site specifies pausing of RNA polymerases, dependent on both its orientation and position relative to the promoter. This sequence is, however, not necessary to maintain repression in the absence of a stimulus. As promoter-proximal pausing is therefore not a repression mechanism for the c-fos gene, the promoter and enhancer sequences are the main determinants of RNA polymerase elongation competence. Surprisingly, we find that promoter-proximal pausing further increases transcriptional levels from a variety of promoters. These observations lead us to hypothesize that promoter-proximal pausing of RNA polymerase II augments c-fos expression by allowing more efficient phosphorylation of the C-terminal domain of the large subunit.
Assuntos
Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Polimerase II/metabolismo , Animais , Linhagem Celular , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/genética , Humanos , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Camundongos , Plasmídeos/genética , RNA/efeitos dos fármacos , RNA/genética , RNA/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Hepatocyte growth factor/scatter factor (HGF/SF) is a recently characterised molecule with many remarkable functions. Its involvement in important processes such as cell proliferation, cell migration, morphogenesis and organ development implies that its activity should be tightly regulated. To understand the molecular mechanisms controlling HGF/SF transcription, we have analysed DNaseI hypersensitive sites (DHS) along rat and human HGF/SF genes in various tissues and cell types. We identified five DHS along the rat gene, two in the 5'-flanking region and three in the first intron. These sites are only found in rat tissues and rat cell lines, which express HGF/SF. The strongest hypersensitive site map to a region that corresponds to the promoter by start site analysis. A single tissue-specific DHS is present in human cell lines that express HGF/SF and corresponds to the promoter region. Our results suggest that chromatin accessibility plays a major role in the regulation of HGF/SF transcription regulation.
Assuntos
Cromatina/genética , Fator de Crescimento de Hepatócito/genética , Interleucina-3/química , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Nuclease do Micrococo/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transcrição Gênica/genéticaRESUMO
Genomic and cDNA clones derived from the gene (dodA) coding for DOPA dioxygenase, a key enzyme in the betalain pathway, were obtained from the basidiomycete Amanita muscaria. A cDNA library was established in the phage lambda ZapII and dodA clones were isolated using polyclonal antibodies raised against the purified enzyme. Their identity was confirmed by comparison of the deduced amino acid sequence with the sequence of several tryptic peptide fragments of DOPA dioxygenase. The gene coded for a 228-amino acid protein that showed no homology to published sequences. The coding region was interrupted by five short introns. Regulation was shown to occur at the transcriptional level; the mRNA accumulated to high levels only in the coloured cap tissue. dodA was found to be a single-copy gene in A. muscaria. To our knowledge, this is the first gene from the betalain pathway to be cloned. It encodes a type of aromatic ring-cleaving dioxygenase that has not been previously described.