RESUMO
XB-S is an amino-terminal truncated protein of tenascin-X (TNX) in humans. The levels of the XB-S transcript, but not those of TNX transcripts, were increased upon hypoxia. We identified a critical hypoxia-responsive element (HRE) localized to a GT-rich element positioned from -1410 to -1368 in the XB-S promoter. Using an electrophoretic mobility shift assay (EMSA), we found that the HRE forms a DNA-protein complex with Sp1 and that GG positioned in -1379 and -1378 is essential for the binding of the nuclear complex. Transfection experiments in SL2 cells, an Sp1-deficient model system, with an Sp1 expression vector demonstrated that the region from -1380 to -1371, an HRE, is sufficient for efficient activation of the XB-S promoter upon hypoxia. The EMSA and a chromatin immunoprecipitation (ChIP) assay showed that Sp1 together with the transcriptional repressor histone deacetylase 1 (HDAC1) binds to the HRE of the XB-S promoter under normoxia and that hypoxia causes dissociation of HDAC1 from the Sp1/HDAC1 complex. The HRE promoter activity was induced in the presence of a histone deacetylase inhibitor, trichostatin A, even under normoxia. Our results indicate that the hypoxia-induced activation of the XB-S promoter is regulated through dissociation of HDAC1 from an Sp1-binding HRE site.
Assuntos
Histona Desacetilases/metabolismo , Fator de Transcrição Sp1/metabolismo , Tenascina/genética , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Drosophila , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1 , Humanos , Ácidos Hidroxâmicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
Matrix metalloproteinase 2 (MMP-2) is a metalloproteinase belonging to a family of structurally related zinc-dependent endopeptidases capable of degrading extracellular matrix components. To elucidate the functional promoter of the mouse MMP-2 gene, systematic transient expression analysis of the 5'-flanking region of the MMP-2 gene was performed using serially nested deletions. The deletion analysis indicated that the proximal 327-bp sequence from nucleotide positions -313 to +14 relative to the transcription start site is essential for minimal promoter activity and that a 10-bp sequence of the promoter at positions -939 to -930 is required for high expression level of the MMP-2 gene. The 10-bp fragment functioned as a potent stimulator of heterologous SV40 promoter activity. This element is identical to the YB-1 binding motif (Y-box) present within the responsive element-1 (RE-1), which has been shown to act as a potent cis-activator of transcription of the rat MMP-2 gene. The binding of a nuclear factor(s) to the 10-bp fragment was also revealed by electrophoretic mobility shift assays (EMSAs). Antibody-supershift EMSAs of nuclear extracts from NIH 3T3 cells demonstrated YB-1 binding to the RE-1 sequence. It was concluded that the RE-1 is the conserved element for potent expression of MMP-2 gene among rodents.