RESUMO
SHP (small heterodimer partner; NR0B2) is an atypical orphan NR (nuclear receptor) that functions as a transcriptional co-repressor by interacting with a diverse set of NRs and transcriptional factors. HNF-6 (hepatocyte nuclear factor-6) is a key regulatory factor in pancreatic development, endocrine differentiation and the formation of the biliary tract, as well as glucose metabolism. In this study, we have investigated the function of SHP as a putative repressor of HNF-6. Using transient transfection assays, we have shown that SHP represses the transcriptional activity of HNF-6. Confocal microscopy revealed that both SHP and HNF-6 co-localize in the nuclei of cells. SHP physically interacted with HNF-6 in protein-protein association assays in vitro. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated that SHP inhibits the DNA-binding activity of HNF-6 to an HNF-6-response element consensus sequence, and the HNF-6 target region of the endogenous G6Pase (glucose 6-phosphatase) promoter respectively. Northern blot analysis of HNF-6 target genes in cells infected with adenoviral vectors for SHP and SHP siRNAs (small inhibitory RNAs) indicated that SHP represses the expression of endogenous G6Pase and PEPCK (phosphoenolpyruvate carboxykinase). Our results suggest that HNF-6 is a novel target of SHP in the regulation of gluconeogenesis.
Assuntos
Fator 6 Nuclear de Hepatócito/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Fator 6 Nuclear de Hepatócito/química , Fator 6 Nuclear de Hepatócito/genética , Humanos , Microscopia Confocal , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hepatocyte nuclear factor-6 (HNF-6), a liver-enriched transcription factor, controls the development of various tissues, such as the pancreas and liver, and regulates the expression of several hepatic genes. This protein belongs to the ONECUT class of homeodomain proteins and contains a bipartite DNA-binding domain composed of a single cut domain and a characteristic homeodomain. This transcription factor has two distinct modes of DNA binding and transcriptional activation that use different coactivators depending on the target gene. The crystal structure of the bipartite DNA-binding domain of HNF-6alpha complexed with the HNF-6-binding site of the TTR promoter revealed the DNA recognition mechanism of this protein. Comparing our structure with the DNA-free structure of HNF-6 or the structure of Oct-1, we discuss characteristic features associated with DNA binding and the structural basis for the dual mode of action of this protein, and we suggest a strategy for variability of transcriptional activation of the target gene.
Assuntos
DNA/química , Fator 6 Nuclear de Hepatócito/química , Proteínas de Homeodomínio/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cristalografia por Raios X , Dados de Sequência Molecular , Fator 1 de Transcrição de Octâmero/química , Regiões Promotoras Genéticas , Conformação Proteica , Estrutura Terciária de Proteína , RatosRESUMO
Hepatocyte nuclear factor 6 (HNF-6)/OC-1, a part of liver-enriched transcription factor, controls pancreas and liver development and regulates expression of several hepatic genes. DNA-binding region of HNF-6alpha bound to a 14-mer DNA fragment has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P2 with unit cell parameters of a = 73.0 A, b = 39.0 A, c = 106.5 A, beta = 107.6 degrees. X-ray diffraction data were collected to 2.0 A resolution.
