Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT).
Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. The human telomerase catalytic subunit (hTERT) is a rate limiting determinant of the enzymatic activity of human telomerase. In the previous study, we identified the proximal 181 bp core promoter responsible for transcriptional activity of the hTERT gene. To identify the regulatory factors of transcription, transient expression assays were performed using hTERT promoter reporter plasmids. Serial deletion assays of the core promoter revealed that the 5'-region containing the E-box, which binds Myc/Max, as well as the 3'-region containing the GC-box, which binds Sp1, are essential for transactivation. The mutations introduced in the E-box or GC-box significantly decreased transcriptional activity of the promoter. Overexpression of Myc/Max or Sp1 led to significant activation of transcription in a cell type-specific manner, while Mad/Max introduction repressed it. However, the effects of Myc/Max on transactivation were marginal when Sp1 sites were mutated. Western blot analysis using various cell lines revealed a positive correlation between c-Myc and Sp1 expression and transcriptional activity of hTERT. Using fibroblast lineages in different stages of transformation, we found that c-Myc and Sp1 were induced to a dramatic extent when cells overcame replicative senescence and obtained immortal characteristics, in association with telomerase activation. These findings suggest that c-Myc and Sp1 cooperatively function as the major determinants of hTERT expression, and that the switching functions of Myc/Max and Mad/Max might also play roles in telomerase regulation.
Kyo
S
S
Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, 13-1 Takaramachi, Kanazawa, Ishikawa 920-0934, Japan, satoruky@med.kanazawa-u.ac.jp
Takakura
M
M
Taira
T
T
Kanaya
T
T
Itoh
H
H
Yutsudo
M
M
Ariga
H
H
Inoue
M
M
D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001667:Binding, Competitive; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C079439:MXD1 protein, human; C067850:Myc associated factor X; D009838:Oligodeoxyribonucleotides; D016271:Proto-Oncogene Proteins c-myc; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D019098:Telomerase