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Human telomerase reverse transcriptase (hTERT) is a novel target of the Wnt/?-catenin pathway in human cancer.

ABSTRACT: Telomerase activation plays a critical role in human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. The human telomerase reverse transcriptase (hTERT) promoter has been shown to promote hTERT gene expression selectively in tumor cells but not in normal cells. Deregulation of the Wnt/?-catenin signaling pathway is reported to be associated with human carcinogenesis. However, little is known about whether the Wnt/?-catenin pathway is involved in activating hTERT transcription and inducing telomerase activity (TA). In this study, we report that hTERT is a novel target of the Wnt/?-catenin pathway. Transient activation of the Wnt/?-catenin pathway either by transfection of a constitutively active form of ?-catenin or by LiCl or Wnt-3a conditioned medium treatment induced hTERT mRNA expression and elevated TA in different cell lines. Furthermore, we found that silencing endogenous ?-catenin expression by ?-catenin gene-specific shRNA effectively decreased hTERT expression, suppressed TA, and accelerated telomere shortening. Of the four members of the lymphoid-enhancing factor (LEF)/T-cell factor (TCF) family, only TCF4 showed more effective stimulation on the hTERT promoter. Ectopic expression of a dominant negative form of TCF4 inhibited hTERT expression in cancer cells. Through promoter mapping, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we found that hTERT is a direct target of ?-catenin·TCF4-mediated transcription and that the TCF4 binding site at the hTERT promoter is critical for ?-catenin·TCF4-dependent expression regulation. Given the pivotal role of telomerase in carcinogenesis, these results may offer insight into the regulation of telomerase in human cancer.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC3463325 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Human telomerase reverse transcriptase (hTERT) is a novel target of the Wnt/β-catenin pathway in human cancer.

Zhang Yong Y Toh LingLing L Lau Peishan P Wang Xueying X

The Journal of biological chemistry 20120731 39

Telomerase activation plays a critical role in human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. The human telomerase reverse transcriptase (hTERT) promoter has been shown to promote hTERT gene expression selectively in tumor cells but not in normal cells. Deregulation of the Wnt/β-catenin signaling pathway is reported to be associated with human carcinogenesis. However, little is known about whether the Wnt/β-catenin p ...[more]

PMID: 22854964

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Project description:BackgroundTelomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated.Methodology/principal findingsTo understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function.Conclusions/significanceWe provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.

Dataset Information

Human telomerase reverse transcriptase (hTERT) is a novel target of the Wnt/?-catenin pathway in human cancer.

Publications

Human telomerase reverse transcriptase (hTERT) is a novel target of the Wnt/β-catenin pathway in human cancer.

Similar Datasets

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