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

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.

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

Joachim Lingner

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 (

2001

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 Quantitative Telomeric Chromatin Isolation Protocol (Q-TIP) to Characterize Telomere Composition Changes

Larissa Grolimund

Telomeres protect linear ends of eukaryotic chromosomes by preventing chromosomal end-to-end fusions and telomere attrition. They consist of repetitive DNA sequences, telomeric repeat containing RNAs (TERRAs) and proteins. Telomeres play a crucial role in chromosome stability, cancer biology, and ageing. With each cell division, telomeres shorten, as the replication machinery of the cell is not capable to fully copy the very terminal part of chromosomes. Therefore, after a certain number of cell divisions, telomeres get critically short and elicit signals in order to stop the proliferation. A cell can circumvent the signal for critically short telomeres by activating mechanisms that elongate telomeres. In most of the cases, telomere elongation is achieved by the expression of the telomerase enzyme. In this case, cells gain the ability to divide indefinitely, which can, in combination with other events, eventually lead to cancer. The telomeric protein composition changes during tumorigenesis, aging and in telomere syndromes are poorly defined. Here, we develop a quantitative telomeric chromatin isolation protocol (q-TIP) for human cells in which chromatin is crosslinked, immunopurified and analyzed by mass spectrometry. Q-TIP involves stable isotope labeling by amino acids in cell culture (SILAC) in order to compare and identify quantitative differences in telomere protein composition of cells from various states. With q-TIP, we specifically enrich telomeric DNA and all shelterin components. We identify and validate known and novel telomere associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply q-TIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterin components TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate q-TIP to study telomeric states during normal development and in disease.

EPFL2013

A Quantitative Telomeric Chromatin Isolation Protocol (Q-TIP) to Characterize Telomere Composition Changes

Larissa Grolimund

EPFL2013