RESUMO
The chipmunk hibernation-specific protein HP-20 is a component of the 140 kDa complex that drastically decreases in the blood during hibernation, and its gene is expressed specifically in the liver. To reveal molecular mechanisms underlying the liver-specific transcription of the HP-20 gene, we isolated chipmunk HP-20 genomic clones. The HP-20 gene spans approximately 6 kb, and consists of three exons. The transcription start site, as determined by 5' RACE-PCR analysis, was found to be 160 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 57 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. A database search revealed that this region contains a potential binding site for hepatocyte nuclear factor-1 (HNF-1). In a gel retardation assay, in vitro-synthesized HNF-1 bound to the 5' flanking sequence from -52 to -26. A similar shifted band was also observed with HepG2 nuclear extracts, and this complex was super-shifted by an anti-(HNF-1) Ig. When transfected into COS-7 cells, HNF-1 transactivated transcription from the HP-20 gene promoter, and this activity was abolished by a mutation of the HNF-1 binding site, indicating that HNF-1 plays an important role in HP-20 gene expression.
Assuntos
Proteínas Sanguíneas/genética , Proteínas de Ligação a DNA , Fígado/metabolismo , Proteínas Nucleares , Sciuridae/genética , Fatores de Transcrição/fisiologia , Região 5'-Flanqueadora/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Células COS , DNA/química , DNA/genética , Regulação da Expressão Gênica , Genes/genética , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
A-type lamins are not present in either early embryos or the embryonal carcinoma (EC) cell line. P19 cells, which are EC cell line, are able to express A-type lamins upon retinoic acid (RA) treatment. Here we report that a novel RA-responsive element, termed lamin A/C-RA-responsive element (L-RARE), is located within the lamin A/C promoter. RA activated the luciferase activity of the reporter which had four tandem repeats of the wild-type L-RARE, while a loss of function mutant, which altered CACCCCC to CACtatC within L-RARE, did not respond. Four specific binding complexes of L-RARE, Complexes-A, -B, -C, and -D, were detected in protein extracts obtained from P19 cells treated with and without RA. Specific antibodies revealed that Sp1 and Sp3 were included in Complex-A and Complexes-B and -C, respectively. Thus, L-RARE was important in the RA-mediated activation of the lamin A/C promoter and was recognized by DNA binding proteins.