Downstream E-Box–mediated Regulation of the Human Telomerase Reverse Transcriptase (hTERT) Gene Transcription: Evidence for an Endogenous Mechanism of Transcriptional Repression

Abstract

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/hTERTnegative counterpart containing a transferred, normal chromosome 3 (RCC23ϩ3). This E-box element mediated repression of hTERT transcription in RCC23ϩ3 but not in RCC23. A copy number-dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23ϩ3. 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 RCC23ϩ3 and, importantly, detected an RCC23ϩ3-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.

TAK1 represses transcription of the human telomerase reverse transcriptase gene

“…Such transcription factors, for example Mad1 and MT-box-binding factor, have been reported to function as repressors of the hTERT promoter that act when cancer cell differentiation is induced by antagonizing the transcriptional activation ability of c-Myc (Tzukerman et al, 2000;Xu et al, 2001;Horikawa et al, 2002). Although our results showed that TAK1 represses the hTERT promoter activity in an E-box-independent manner, we cannot exclude the possibility that TAK1 represses the hTERT promoter even in an E-box-dependent manner.…”

The human telomerase gene: complete genomic sequence and analysis of tandem repeat polymorphisms in intronic regions

“…These studies show that the human and mouse TERT genes are regulated by inducible transcription factors including c-Myc, Sp1 and NFkB (Horikawa et al, 1999;Wick et al, 1999;Wu et al, 1999;Kyo et al, 2000;Yin et al, 2000;Oh et al, 2000;Tzukerman et al, 2000;Poole et al, 2001). However, these studies were not carried out with a complete hTERT gene including all putative regulatory elements and introns.…”

TGF-β and cancer: Is Smad3 a repressor of hTERT gene?

“…Transcriptional activation of the hTERT gene is a critical, initial rate-limiting step in telomerase activation in human cancers. The hTERT gene promoter provides a platform with a number of binding sites for transcriptional regulation, including two typical E-boxes and several GCboxes for the transcription factors c-myc/Max and Sp1, respectively [29][30][31]. Expression of c-myc rapidly induces hTERT gene transcription [32], with the binding of c-myc to the E-box being coupled to cell proliferation and that is disengaged by Mad1 during differentiation [33].…”