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. J. Cell. Biochem. 90: 68–79, 2003. © 2003 Wiley‐Liss, Inc.