RESUMO
Five Dnase 1 footprints, termed boxes L1 to L5, have been characterized in the 280 bp upstream from the cap site of the L-type pyruvate kinase (L-PK) gene. They bind from 3' to 5', the factors HNF1, HNF4, MLTF, and a non-identified protein referred to as L5 binding factor (L5-BF). These elements, individually or variably combined, were tested by cell-free transcription. The L1 box stimulates both L-PK and TK promoters, but in the context of L-PK promoter, it cooperates with the L3 element to reach a high level of transcriptional activation. The L3 and L4 elements exhibit weaker influences, increased by homologous or heterologous interactions. The L5 box behaves as a promoter-dependent negative regulatory element.
Assuntos
Encéfalo/enzimologia , Proteínas de Ligação a DNA/metabolismo , Isoenzimas/genética , Fígado/enzimologia , Proteínas Nucleares/metabolismo , Fosfoproteínas , Regiões Promotoras Genéticas , Piruvato Quinase/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Desoxirribonuclease I , Fator 1 Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Fator 4 Nuclear de Hepatócito , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Deleção de Sequência , Transcrição GênicaRESUMO
The glucose/insulin response element of the L-pyruvate kinase gene is a perfect palindrome located from nt -168 to -144 with respect to the cap site. This element (L4) is partially homologous to MLTF binding sites. Its full efficiency requires cooperation with a contiguous binding site for HNF4, termed L3 and located from nt -145 to -125. In the presence of the L4 element contiguous to L3, cyclic AMP inhibits activity of the L-PK promoter while in its absence, or when the normal L4-L3 contiguity is modified, cyclic AMP behaves as a transcriptional activator that does not seem to be sequence-specific. Therefore, we propose that the mechanism of inhibition of the L-PK gene by cyclic AMP requires precise interactions between the nucleoprotein complex built up at sites L4 and L3 and other components of the L-PK transcription initiation complex.
Assuntos
AMP Cíclico/farmacologia , Glucose/farmacologia , Insulina/farmacologia , Regiões Promotoras Genéticas/genética , Piruvato Quinase/genética , Animais , Sequência de Bases , Células Cultivadas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
L-type pyruvate kinase gene regulation is an excellent model of gene control by hormones and diet. In vivo and ex vivo experiments allowed us to established that thyroid hormones and glucocorticoids act on pyruvate kinase gene expression at the post-transcriptional level. In contrast, glucose and insulin together stimulate transcription of this gene while glucagon inhibits it. Insulin or glucose are individually inefficient and glucagon-dependent transcriptional inhibition seems to be dominant in insulin + glucose-dependent activation. A 14-kbp fragment encompassing the entire pyruvate kinase gene and 3.2-kbp of 5' flanking sequences is expressed in transgenic mice exactly like the endogenous gene; the 3.2-kbp upstream region is sufficient to confer this tissue-specific and hormone/diet-regulated expression to reporter genes. In vivo, DNAse I hypersensitivity analysis revealed the presence of 3 liver-specific groups of hypersensitive sites (HSS). The proximal sites, between + 1 and -183 bp with respect to the start site of transcription, were, in addition, transcription-dependent. The nature and functional role of proteins binding to this proximal upstream sequence were analyzed by in vitro binding and cell free transcription experiments. The existence of more upstream cis-acting elements was investigated by transient transfection assays using differentiated hepatoma cell lines and hepatocytes in primary culture. These experiments permitted the detection of an extinguisher active in hepatoma Hep G2 cells but not in hepatocytes, and of an activating element which could correspond to a distal HSS. Unfortunately, this investigation has not yet allowed us to determine with accuracy the DNA elements responsible for response to diet and hormones.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucagon/fisiologia , Insulina/fisiologia , Piruvato Quinase/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Ingestão de Alimentos/fisiologia , Jejum/fisiologia , Humanos , Sequências Reguladoras de Ácido NucleicoRESUMO
A protein with a molecular mass of 19 kDa has been purified to homogeneity from 11-day-old chick embryos using a procedure involving chromatography on heparin - Sepharose, immunoaffinity resin and C4 reversed-phase. Indirect immunofluorescence studies, using polyclonal and monoclonal antibodies raised against this protein, indicate that it is essentially localized within the basement membranes in early embryonic tissues. After the 18th day of embryonic life and in post-hatched chicken, this protein could only be detected in some eye basement membranes. It appears to be bound to heparan sulfate chains of the proteoglycan present in these structures. Thus, the protein exhibits similar properties to those previously described for fibroblast growth factors (FGF), such as heparin affinity, molecular mass and localization in the basement membranes. In contrast, this protein is present in much larger amounts than FGFs, at least in 11-day-old embryos. Furthermore, the first 17 amino acid residues of the N-terminal sequence show that it does not strictly correspond to any previously described protein.
Assuntos
Heparina/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Anticorpos , Anticorpos Monoclonais , Membrana Basal/metabolismo , Embrião de Galinha , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Olho/embriologia , Olho/metabolismo , Imunofluorescência , Cinética , Dados de Sequência Molecular , Peso Molecular , Receptores de Superfície Celular/metabolismoRESUMO
A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor A1 (LF-A1), nuclear factor 1 (NF1), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNF1-binding site. In contrast, deletion of the HNF1-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.
Assuntos
Fígado/enzimologia , Regiões Promotoras Genéticas/fisiologia , Piruvato Quinase/genética , Animais , Sequência de Bases , Ligação Competitiva , Núcleo Celular/análise , Sistema Livre de Células , Glucagon/fisiologia , Masculino , Dados de Sequência Molecular , Fenômenos Fisiológicos da Nutrição/fisiologia , Oligonucleotídeos/metabolismo , Especificidade de Órgãos , Plasmídeos , Ratos , Ratos Endogâmicos , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
A 183 base-pair fragment of the liver-specific promoter of the L-type puruvate kinase (L-PK) gene has been shown by transfection assay to be sufficient to confer a tissue-specific expression to a reporter gene. The proteins binding in vitro to this fragment have been investigated by a combination of DNase I footprinting, gel retardation of synthetic oligonucleotides and ultraviolet cross-linking. Four proteins from liver nuclear extracts bind to the investigated fragment. They were called, from 3' to 5', L1 to L4 binding factors. The L1 site (nucleotides -95 to -66 with respect to the cap site) binds hepatocyte nuclear factor 1 (HNF1), a liver-specific protein. The L2 site (nucleotides -114 to -97) binds the ubiquitous nuclear factor 1 (NF1), or a related factor. The L3 site (nucleotides -144 to -126) binds liver factor A1 (LF-A1), another liver-specific protein. Finally, the L4 site (nucleotides -168 to -145) binds major late transcription factor (MLTF/USF/UEF), an ubiquitous protein. Each of these proteins has been detected in other liver-specific promoters, but their combination is unique to the liver-specific promoter of the L-PK gene.