Assuntos
Regulação da Expressão Gênica , Glucose/metabolismo , Proteínas de Homeodomínio , Proteínas Quinases Ativadas por AMP , Animais , Humanos , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
L-type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP-dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site-dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA-dependent phosphorylation sites have been mutated still conferred cAMP-sensitive transactivation of a L3-dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4-dependent transactivation but this effect remained sensitive to cAMP inhibition.
Assuntos
AMP Cíclico/fisiologia , Proteínas de Ligação a DNA , Regiões Promotoras Genéticas , Piruvato Quinase/genética , Animais , Sítios de Ligação , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Fator 4 Nuclear de Hepatócito , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs). From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose. A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e. the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts. Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets. In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected. Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture. In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition. We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glucose/metabolismo , Piruvato Quinase/genética , Receptores de Esteroides , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Células COS , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Núcleo Celular/metabolismo , Galinhas , Clonagem Molecular , Primers do DNA , Proteínas de Ligação a DNA/genética , Dieta , Glucose/antagonistas & inibidores , Fígado/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Gênica , Fatores Estimuladores UpstreamRESUMO
Upon differentiation of 3T3-L1 preadipocytes into adipocytes transcription of many adipose-specific genes is coordinately activated. A differentiation-induced factor, later identified as C/EBP alpha, binds to and transactivates the promoters of these genes. Vector-directed expression of antisense C/EBP alpha RNA in preadipocytes blocked expression of C/EBP alpha, as well as adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the 'adipocyte phenotype' was accomplished by transfection of the antisense cells with a complementary sense C/EBP alpha RNA expression vector. Using an IPTG-inducible double-vector LacSwitch C/EBP alpha expression system, it was found that differentiation can be induced without exogenous hormone inducers. These findings indicate that C/EBP alpha is not only required, but is sufficient, to trigger differentiation of 3T3-L1 preadipocytes. The C/EBP alpha gene promoter possesses a C/EBP binding site through which C/EBP alpha autoactivates its own expression. A nuclear protein referred to as CUP (C/EBP undifferentiated protein) that binds to a bipartite element in the C/EBP alpha promoter just 5' to the C/EBP binding site has been purified and characterized. During differentiation of preadipocytes, expression of CUP activity decreases as expression of C/EBP alpha increases. Evidence suggests that a CUP-containing protein complex bridges between the CUP (repression) and C/EBP (autoactivation) elements in the promoter and may maintains the C/EBP alpha gene in the repressed state prior to differentiation.
Assuntos
Adipócitos/citologia , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Células 3T3 , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/genética , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , TransfecçãoRESUMO
During differentiation of 3T3-L1 preadipocytes into adipocytes, transcription of the C/EBP alpha (CCA-AT/enhancer binding protein alpha) gene is activated. The promoter of the C/EBP alpha gene contains a bipartite cis element with binding sites for C/EBP alpha undifferentiated protein (CUP) and an Sp1-like GT box binding protein. Binding of CUP to this element is markedly enhanced by its interaction with the Sp1-like protein. CUP, purified approximately 100,000-fold from HeLa cell nuclear extracts, appears to be composed of at least two types of subunit. Evidence is presented that a CUP-containing protein complex bridges between the CUP/Sp1-like GT box element and a downstream cis element, which contains a C/EBP binding site. During differentiation of 3T3-L1 preadipocytes into adipocytes, CUP activity or expression decreases as expression of C/EBP alpha increases. It is suggested that bridging by the CUP-containing protein complex may play a role in transcriptional regulation of the C/EBP alpha gene.
Assuntos
Tecido Adiposo/citologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular , Proteínas de Ligação a DNA/isolamento & purificação , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Oligodesoxirribonucleotídeos/química , Fator de Transcrição AP-2RESUMO
The differentiation of preadipocytes into adipocytes in culture is accompanied by the coordinate transcriptional activation of adipose-specific genes. Recent studies have identified cis-acting elements that are involved in activating (or derepressing) the transcription of many of these genes during differentiation. The identification of key trans-acting nuclear factors that interact with certain of these elements makes it possible to formulate models for the regulatory network governing the adipogenic differentiation program.