Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression.
Regulation of the hTERT gene encoding the telomerase catalytic subunit plays an important role in human cell senescence, immortalization, and carcinogenesis. By examining the activity of various deleted or mutated hTERT promoter fragments, we show that an E-box element downstream of the transcription initiation site is critical to differential hTERT transcription between the telomerase/hTERT-positive renal cell carcinoma cell line (RCC23) and its telomerase/hTERT-negative counterpart containing a transferred, normal chromosome 3 (RCC23+3). This E-box element mediated repression of hTERT transcription in RCC23+3 but not in RCC23. A copy number-dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23+3. Endogenous expression levels of c-Myc or Mad1, which could activate or repress hTERT transcription when overexpressed, did not account for the differential hTERT transcription. Gel mobility shift assays identified the upstream stimulatory factors (USFs) as a major E-box-binding protein complex in both RCC23 and RCC23+3 and, importantly, detected an RCC23+3-specific, E-box-binding factor that was distinct from the USF and Myc/Mad families. The E-box-mediated repression was also active in normal human fibroblasts and epithelial cells and inactive in some, but not all, telomerase/hTERT-positive cancer cells. These findings provide evidence for an endogenous, repressive mechanism that actively functions in telomerase/hTERT-negative normal cells and becomes defective during carcinogenic processes, e.g., by an inactivation of the telomerase repressor gene on chromosome 3.
Horikawa
Izumi
I
Laboratory of Biosystems and Cancer, Cancer and Aging Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. horikawi@mail.nih.gov
Cable
P LouAnn
PL
Mazur
Sharlyn J
SJ
Appella
Ettore
E
Afshari
Cynthia A
CA
Barrett
J Carl
JC
D018797:Cell Cycle Proteins; D002893:Chromosomes, Human, Pair 3; D004268:DNA-Binding Proteins / Q000378:metabolism; D024721:E-Box Elements / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D016615:Telomere / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C501384:MAD1L1 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016271:Proto-Oncogene Proteins c-myc; D012097:Repressor Proteins; D019098:Telomerase