Enhanced activity of cloned hamster TERT gene promoter in transformed cells

In 7,12-dimethylbenz[a]anthracene-treated hamster pouch epithelial cells, t elomerase activity increased within I week of treatment and reached a 6-7-f old increase within 3 weeks. To investigate this phenomenon, we have cloned and sequenced the hamster telomerase catalytic subunit (hamTERT) promoter. Transient transfection with different genomic segments upstream of the ATG translation initiation codon linked to the luciferase reporter gene mapped the core promoter within a 250 bp region. Three major transcription initia tion sites and several minor sites were found between -42 and -140 bp relat ive to the ATG site. Like the human and murine TERT promoters, the hamTERT promoter lacks TATA and CAT boxes and all three promoters share similar reg ulatory factor binding sites. DNase I footprint analysis revealed six prote cted regions which contain sequences homologous with known transcription fa ctor binding sites. Three protein binding regions (I, II, and III) were ess ential for the promoter activity. Regions I and III bound to Spl and Sp3 tr anscriptional factors, whereas region II bound to an unknown factor. Transi ent transfection of a promoter-luciferase plasmid into Drosophila SL2 cells showed that Spl and Sp3 regulated the hamster TERT promoter in a concentra tion-dependent and synergistic manner. Telomerase activity showed a 2-4-fol d and 8-10-fold increase in immortalized cells and tumor cells, respectivel y, but hamTERT expression was only increased 1.7-fold and 2.4-fold, respect ively, in the same cells. (C) 2001 Elsevier Science B.V. All rights reserve d.