Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.
We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.
Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Shigeru
S
Takahashi
Naomi
N
Matsuura
Takako
T
Kurokawa
Yuji
Y
Takahashi
Takashi
T
Miura
D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D006419:Heme Oxygenase (Decyclizing)