Identification and characterization of a novel enhancer for the human MCT-1 oncogene promoter

Bo Shi, Victor Levenson, Ronald B. Gartenhaus*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)68-79
Number of pages12
JournalJournal of Cellular Biochemistry
Volume90
Issue number1
DOIs
StatePublished - Sep 1 2003

Keywords

  • DNA-binding protein
  • EMSA
  • Enhancer
  • Heat shock factors
  • Jurkat
  • MCT-1
  • Oncogene
  • Promoter
  • Southwestern
  • Transcription factor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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