RESUMO
The chum salmon insulin-like growth factor II (IGF-II) gene is highly expressed in liver tissue. In this study we demonstrate that two transcription factors, Sp1 and C/EBPbeta, are involved in the enhanced expression of the salmon IGF-II gene. The presence of the fish homolog for C/EBPbeta in salmon liver RNA was confirmed by Northern blotting. The sIGF-II promoter was activated up to 20-fold by co-transfection with C/EBPbeta. The functional importance of four out of the five putative C/EBPbeta binding sites was demonstrated with mutational analysis in transient transfection assays. The transcription factor Sp1 binds to two sites within the salmon IGF-II promoter. Interestingly, mutation of the Sp1 binding sites decreases not only the basal IGF-II promoter activity but also the C/EBPbeta-induced transactivation. These results demonstrate that liver-enriched C/EBPbeta and ubiquitously expressed Sp1 each activate the sIGF-II promoter and that Sp1 is required for full transactivation of the sIGF-II gene by C/EBPbeta. This suggests that C/EBPbeta and Sp1 act in synergy.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/farmacologia , Fator de Crescimento Insulin-Like I/genética , Oncorhynchus keta/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator de Transcrição Sp1/farmacologia , Animais , Sequência de Bases , Sítios de Ligação/genética , Northern Blotting , Sinergismo Farmacológico , Fígado/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Mensageiro/análise , Ativação Transcricional/efeitos dos fármacosRESUMO
IGF-II plays an important role in growth and development of vertebrates and is highly expressed in adult salmon liver. In the present study, we demonstrate that a liver-enriched transcription factor, hepatocyte nuclear factor 3beta (HNF-3beta), is an activator of the chum salmon IGF-II gene. Multiple binding sites for HNF-3beta were identified within the 5'-UTR using electrophoretic mobility shift assays and mutation of these sites prevents binding of HNF-3beta. In transient transfection assays it was shown that mutation of the HNF-3beta binding sites results in a substantial decrease of HNF-3beta-activated salmon IGF-II gene expression. This is the first identified transcription factor that is functionally involved in the regulation of fish IGF-II expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Fator 3-beta Nuclear de Hepatócito , Proteínas Nucleares/genética , Oncorhynchus keta , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
IGF-II plays an important role in growth and development of vertebrates. In the present study, the characterization of the first fish IGF-II gene, chum salmon IGF-II, is described. The sIGF-II gene consists of four exons, spanning a region of 9 kbp, that encode the 214 aa IGF-II precursor. While the amino acid sequences of fully processed IGF-II of salmon and mammalian species are very similar, the prepro-peptide sequence deviates extensively in the signal- and E-peptide domains. The transcription initiation site of the sIGF-II gene was localized within a 30 nt region employing RT-PCR. Using sIGF-II promoter-luciferase constructs it was demonstrated that the sIGF-II gene has a relatively strong promoter that contains tissue-specific regulatory elements.