p53-dependent Down-regulation of Telomerase Is Mediated by p21

Abstract

Inactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominantnegative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F.Telomerase, a specialized RNA-directed DNA polymerase that extends telomeres at the end of eukaryotic chromosomes, has been implicated in aging, immortalization, and transformation. The human telomerase complex is composed of a catalytic subunit (hTERT) 1 with a reverse transcriptase activity(1) and an RNA-containing subunit (human telomerase RNA) (2) that is used as a template for extending telomere length.Telomerase activity is repressed in most normal human somatic tissues, whereas the enzyme is active in ϳ90% of human cancers (3). However, the mechanism through which telomerase is reactivated in the process of carcinogenesis remains unclear. Telomerase enzymatic activity can be regulated at multiple levels, including hTERT transcription, alternative splicing, chaperone-mediated folding, phosphorylation, and nuclear translocation; however, the major control mechanism of telomerase regulation seems to be at the level of hTERT transcription (for a review of telomerase regulation, see Ref. 4 and references therein). The tumor suppressor gene p53 is a sequence-specific transcription factor that can mediate many downstream effects such as growth arrest and apoptosis through activation or repression of its target genes (5). p53 is the most frequently altered gene in human cancer...

Regulation of telomerase activity by the p53 family member p73

“…In contrast to its family member p53, which only serves to repress hTERT transcription, regulation of hTERT by p73 is different for the various p73 isoforms that have been described so far (Kusumoto et al, 1999;Kanaya et al, 2000;Xu et al, 2000;Shats et al, 2004). Both N-and C-terminal variability influence the way how p73 affects hTERT transcription.…”

“…The second regulatory mechanism that was proposed is based on the observation that the core promoter contains an E2F binding site allowing p53 to regulate hTERT expression via modulation of the p16-RB pathway (Shats et al, 2004;Won et al, 2004). This E2F binding site was shown to recruit repressive E2F-RB-HDAC complexes to the hTERT core promoter in primary human fibroblasts rendering hTERT transcription susceptible to the cell cycle and to common, tumorassociated alterations in the p16-RB pathway (Won et al, 2004).…”

“…This E2F binding site was shown to recruit repressive E2F-RB-HDAC complexes to the hTERT core promoter in primary human fibroblasts rendering hTERT transcription susceptible to the cell cycle and to common, tumorassociated alterations in the p16-RB pathway (Won et al, 2004). Shats et al (2004) recently showed that p53-mediated repression of hTERT requires induction of the p53 target gene p21, which acts to direct HDACdependent repressor complexes to the hTERT promoter. As p21 is a common target gene of p53 and TAp73, it could be assumed that induction of p21 by TAp73 can contribute to hTERT repression.…”

“…Independent from induction of cell cycle arrest and apoptosis in response to various types of cellular stress, p53 has been reported to repress hTERT expression (Kusumoto et al, 1999;Kanaya et al, 2000;Xu et al, 2000;Shats et al, 2004). Mapping studies on the hTERT core promoter suggested a mechanism of hTERT repression in which p53 binds to Sp1 and renders it inaccessible to hTERT promoter activation Xu et al, 2000).…”

“…Mapping studies on the hTERT core promoter suggested a mechanism of hTERT repression in which p53 binds to Sp1 and renders it inaccessible to hTERT promoter activation Xu et al, 2000). A recent study using siRNA knockdown of p53 indicated that endogenous p53 induces the cyclin-dependent kinase inhibitor p21 to repress hTERT transcription via the assembly of repressive E2F-pocket protein complexes on an atypical E2F binding site in the proximal core promoter, which has been previously shown to be occupied by transcriptionally repressive E2F-pocket protein-HDAC complexes in primary human fibroblasts (Shats et al, 2004;Won et al, 2004).…”