Identification and characterization of a novel enhancer for the human MCT-1 oncogene promoter.
Cloning and characterization of the promoter region for the MCT-1 oncogene is described. We used luciferase assays to identify cis-acting elements responsible for human MCT-1 promoter function. The MCT-1 promoter is TATA-less with a consensus initiator element located at the transcription start site and facilitated by two Sp1 sites that directs basal transcription. Deletion of a region of the MCT-1 promoter (-133 to -122) resulted in significant decrease in luciferase activity, suggesting that this region contains a positive cis-acting element. Using mobility shift assays with a 26-mer oligonucleotide, which contains this fragment and its flanking regions, we demonstrated the presence of sequence-specific DNA-binding protein in both Jurkat and Hela nuclear extracts that we designated as LMBF (for lymphoid MCT-1 binding factor). This 26-mer oligonucleotide containing the LMBF binding site is required for maximum transcriptional activity of the MCT-1 promoter. Although the 26-mer oligonucleotide contains a sequence with strong homology to a heat-shock factor consensus, competitive electrophoretic mobility shift assay (EMSA) analysis demonstrated that the binding protein is not a known member of heat shock family. Furthermore, this sequence when placed in reverse orientation downstream of the luciferase gene was able to enhance luciferase activity driven by a minimal promoter. These data are consistent with this sequence behaving as an enhancer. Finally, Southwestern blot analysis revealed a 96-kDa protein capable of binding a probe containing the LMBF binding site.
Shi
Bo
B
Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
Levenson
Victor
V
Gartenhaus
Ronald B
RB
D024506:5' Flanking Region; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; C115081:MCTS1 protein, human; D009841:Oligonucleotides; D015513:Oncogene Proteins; D010446:Peptide Fragments; D014157:Transcription Factors