Regulation of human telomerase activity: repression by normal chromosome 3 abolishes nuclear telomerase reverse transcriptase transcripts but does not affect c-Myc activity

Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)-PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules was below the sensitivity of the assay (

A chromosome 3-encoded repressor of the human telomerase reverse transcriptase (hTERT) gene controls the state of hTERT chromatin

Joachim Lingner

Telomerase is crucial for human carcinogenesis. The limiting component of telomerase activity is telomerase reverse transcriptase (hTERT), undetectable in differentiated somatic cells but present in most tumor cells. There is evidence that hTERT transcription is shut down by a repressor in normal cells, but the mechanisms that turn on or maintain expression in tumor cells are not understood. To identify cis-acting regulatory elements, we scanned the hTERT gene for nuclease sensitive sites. In tumor cells and in in vitro transformed fibroblasts that contain hTERT mRNA, we detected a pattern of nuclease-sensitive sites in the second intron different from that in normal fibroblasts. To test whether the chromatin state characterized by the increased nuclease sensitivity plays a role in tumor-specific hTERT expression, we used a telomerase-positive breast carcinoma line, 21NT. Introduction of a normal chromosome 3 into these cells is known to down-regulate hTERT expression, probably through transcriptional silencing. 21NT cells displayed a similar pattern of micrococcal nuclease (MNase) sensitivity to other telomerase-positive lines, whereas the hTERT chromatin of the chromosome 3-hybrids resembled that of normal fibroblasts. In segregants that had lost the normal chromosome 3, the MNase sensitivity pattern characteristic of telomerase-positive cells was restored, and some (but not all) re-expressed the hTERT gene. The simplest model compatible with these results, and with data on the mapping of an hTERT repressor on chromosome 3, is that hTERT expression in tumor cells depends on an open state of intron 2-chromatin. We propose that, during the development of the breast carcinoma from which the 21NT cell line was derived, loss of function of this repressor led to chromatin remodeling necessary (but probably not sufficient) for expression of the hTERT gene. An improved understanding of the precise mechanism of hTERT dysregulation in human cancer may well find applications in the development of antitelomerase cancer therapy.

2003

Telomere length homeostasis

Joachim Lingner

The physical ends of chromosomes, known as telomeres, protect chromosome ends from nucleolytic degradation and DNA repair activities. Conventional DNA replication enzymes lack the ability to fully replicate telomere ends. In addition, nucleolytic activities contribute to telomere erosion. Short telomeres trigger DNA damage checkpoints, which mediate cellular senescence. Telomere length homeostasis requires telomerase, a cellular reverse transcriptase, which uses an internal RNA moiety as a template for the synthesis of telomere repeats. Telomerase elongates the 3' ends of chromosomes, whereas the complementary strand is filled in by conventional DNA polymerases. In humans, telomerase is ubiquitously expressed only during the first weeks of embryogenesis, and is subsequently downregulated in most cell types. Correct telomere length setting is crucial for long-term survival. The telomere length reserve must be sufficient to avoid premature cellular senescence and the acceleration of age-related disease. On the other side, telomere shortening suppresses tumor formation through limiting the replicative potential of cells. In recent years, novel insight into the regulation of telomerase at chromosome ends has increased our understanding on how telomere length homeostasis in telomerase-positive cells is achieved. Factors that recruit telomerase to telomeres in a cell cycle-dependent manner have been identified in Saccharomyces cerevisiae. In humans, telomerase assembles with telomeres during S phase of the cell cycle. Presumably through mediating formation of alternative telomere structures, telomere-binding proteins regulate telomerase activity in cis to favor preferential elongation of the shortest telomeres. Phosphoinositide 3-kinase related kinases are also required for telomerase activation at chromosome ends, at least in budding and fission yeast. In vivo analysis of telomere elongation kinetics shows that telomerase does not act on every telomere in each cell cycle but that it exhibits an increasing preference for telomeres as their lengths decline. This suggests a model in which telomeres switch between extendible and nonextendible states in a length-dependent manner. In this review we expand this model to incorporate the finding that telomerase levels also limit telomere length and we propose a second switch between a non-telomerase-associated "extendible" and a telomerase-associated "extending" state.

Springer-Verlag2006

Direct activation of TERT transcription by c-MYC

Joachim Lingner

The MYC proto-oncogene encodes a ubiquitous transcription factor (c- MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. Here we show that c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and can both induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, we find here that the TERT promoter contains numerous c-MYC-binding sites that mediate TERT transcriptional activation. c-MYC-induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. Our results indicate that TERT is a target of c-MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells.

1999

A chromosome 3-encoded repressor of the human telomerase reverse transcriptase (hTERT) gene controls the state of hTERT chromatin

Joachim Lingner

2003

Telomere length homeostasis

Joachim Lingner

Springer-Verlag2006