Functional characterization of the TERRA transcriptome at damaged telomeres

Telomere deprotection occurs during tumorigenesis and aging upon telomere shortening or loss of the telomeric shelterin component TRF2. Deprotected telomeres undergo changes in chromatin structure and elicit a DNA damage response (DDR) that leads to cellular senescence. The telomeric long noncoding RNA TERRA has been implicated in modulating the structure and processing of deprotected telomeres. Here, we characterize the human TERRA transcriptome at normal and TRF2-depleted telomeres and demonstrate that TERRA upregulation is occurring upon depletion of TRF2 at all transcribed telomeres. TRF2 represses TERRA transcription through its homodimerization domain, which was previously shown to induce chromatin compaction and to prevent the early steps of DDR activation. We show that TERRA associates with SUV39H1 H3K9 histone methyltransferase, which promotes accumulation of H3K9me3 at damaged telomeres and end-to-end fusions. Altogether our data elucidate the TERRA landscape and defines critical roles for this RNA in the telomeric DNA damage response.

Telomeric Repeat Containing RNAs and the Response to Telomere Deprotection in Saccharomyces Cerevisiae

Andrea Bernardo Panza

Telomeres are the DNA/RNA/protein structures at the end of linear eukaryotic chromosomes, which protect them against the DNA damage repair machinery, preventing chromosome end-to-end fusions and aberrant recombination. Moreover, telomeres compensate for the erosion that occurs at every cycle of DNA replication maintaining telomere length through the action of the telomerase reverse transcriptase. Even if telomeres have many features in common with heterochromatic DNA, they have been shown to be transcribed into telomeric repeat-containing RNA (TERRA) both in vertebrates and plants. In the first part of this work we demonstrate that TERRA expression is conserved in the yeast Saccharomyces cerevisiae. Moreover, TERRA is transcribed by RNA polymerase II, it is polyadenylated and its levels in wild type cells are maintained low through the action of the 5’ to 3’ RNA exonuclease Rati. Indeed cells harboring the thermosensitive mutant allele ratl-1 accumulate high levels of TERRA even at semipermissive temperature. Moreover, ratl-1 mutant cells display a telomere shortening phenotype that is rescued by RNase H overexpression, indicating that DNA/RNA hybrids, which can accumulate in this mutant, inhibit the normal telomere maintenance. In the second part of this work we tried to define TERRA function(s) in response to telomere alterations in yeast cells. We observed that TERRA increases during senescence and survivor formation in est2A mutant cells, which undergo through telomere shortening due to telomerase deletion. The observed increase in TERRA does not require the recombination machinery. Moreover, though TERRA levels are not altered by global DNA damage, they are raised in cdcl3-l, stnl-13 and ku70A mutants, which display telomere resection and DNA damage checkpoint activation at restrictive temperature, suggesting a specific role for TERRA in response to telomere alterations. Since the TERRA increase in telomere deprotection-mutants is suppressed by the deletion of either the 5’ to 3’ exonuclease EXOl or one of the checkpoint factors MEC1, TEL1 and RAD9, we suggest that TERRA is part of a specific checkpoint response, which is induced upon telomere resection. Finally, since est2A cells harboring the ratl-1 mutation display a prolonged senescence phenotype, we suspect a role for TERRA in telomere protection and in particular in preventing telomeric recombination.

EPFL2012

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

Telomeric Repeat Containing RNAs and the Response to Telomere Deprotection in Saccharomyces Cerevisiae

Andrea Bernardo Panza

EPFL2012

Telomere length homeostasis

Joachim Lingner

Springer-Verlag2006

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

Joachim Lingner

2003