telomeres: the silence is broken
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Telomeres: The silence is brokenClaus M. Azzalin & Joachim LingnerPublished online: 23 May 2008.
To cite this article: Claus M. Azzalin & Joachim Lingner (2008) Telomeres: The silence is broken, Cell Cycle, 7:9, 1161-1165,DOI: 10.4161/cc.7.9.5836
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The ends of linear eukaryotic chromosomes, telomeres, distin-guish natural chromosome ends from DNA double stranded breaks and thus promote genome stability. Telomeres comprise a repetitive DNA skeleton, which is wrapped in specific protein complexes. Recent data indicate that an additional building block of telomeres is RNA and that the longstanding idea that telomeres are silent genomic regions needs to be overturned. Mammalian telomeres are indeed transcribed into RNA molecules, which remain associated with telomeric chromatin, suggesting RNA-mediated mechanisms in organizing telomere architecture.
Telomeres fulfill essential functions for chromosome stability by protecting chromosome ends from inappropriate DNA repair and DNA degradation.1-4 The mammalian telomere core comprises tandem arrays of duplex 5'-TTAGGG-3' repeats (with the G-rich strand extending beyond its complement to form an overhang), bound to a multiprotein complex known as ‘shelterin’, which assures proper telomere length regulation and telomere protection.1-4 Human shelterin comprises the six factors TRF1, TRF2, TPP1, POT1, TIN2 and hRap1, while mouse shelterin is composed of seven factors due to a recent gene duplication event in the rodent lineage, which led to formation of two POT1 paralogs (POT1a and POT1b).2,5-7
In mammals, most adult somatic cells lack telomere lengthening mechanisms and their telomeres progressively shorten with each cell division cycle due to the inability of the replicative DNA polymerases to replicate the most 3' part of linear DNA molecules and due to nucleolytic processing of telomeres.1,3,8,9 Upon continuous short-ening, telomeres eventually reach a critical length (‘critically short telomeres’) and they become recognized by the cell as double stranded DNA breaks, triggering an irreversible cell cycle arrest known as cellular senescence.10-14 The immortal potential of cancer cells depends on the reactivation of telomere lengthening mechanisms, which counteract telomere sequence loss. In this light, telomere driven cellular senescence represents one of the most powerful cellular barriers against cancer development and progression.15,16
The majority (85–90%) of all human tumors maintain telomere length through reactivation of telomerase, a specialized reverse tran-scriptase, composed of a catalytic subunit and an RNA moiety, able to add telomeric repeats to the telomeric 3' end.1,3,4,17-20 In a small class of soft tissue sarcomas with complex karyotypes telomerase is not reactivated and telomeres are maintained by the so-called ALT (ALTernative lengthening of telomeres) mechanism, which involves homologous recombination between telomeres.21
Based on chromosome staining procedures, telomeres have been classified as constitutive heterochromatic domains for several decades.22 Indeed, we now know that mammalian telomeres are enriched in heterochromatin marks including histone H3 trimethyl-ated at lysine 9 (H3K9m3), histone H4 trimethylated at lysine 20 (H4K20m3) and Heterochromatin Protein 1 (HP1).23-25 Similarly, mammalian subtelomeric regions (the chromosomal regions adja-cent to telomeres) display high levels of DNA methylation, another heterochromatic signature.23,26 The heterochromatic state of telo-meres and their gene-less nature suggested the idea that telomeres are transcriptionally silent genomic regions. Supporting this notion, reporter genes experimentally inserted in subtelomeric regions undergo gene silencing, in a process known as telomere position effect.27-30
TERRA: A Regulated Nuclear RNA Transcribed from Telomeres
Contrary to the idea of telomeres being silent genomic regions, we have discovered that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules, which constitute a novel class of mammalian RNAs.31 More recently, Schoeftner and Blasco also reported on the existence of TERRA.32 Mammalian TERRA molecules contain UUAGGG repeats (while CCCUAA-contaning RNA seems not to be present in mammalian cells at detectable levels), range in size from about 100 bases up to >9 kilobases and are detected exclusively in nuclear fractions.31,32 TERRA is present in different human cell lines as well as in mouse, hamster and, outside mammals, in zebrafish cells indicating that TERRA is evolutionarily conserved in vertebrates.31,32 Using Northern blot and RT-PCR analysis we were also able to demonstrate that not only human telomeres but also subtelomeres are transcribed and that at least a fraction of TERRA molecules comprise subtelomeric-derived RNA and UUAGGG repeats.31 Thus, TERRA transcription starts from different subtelomeres towards chromosome ends. Further
*Correspondence to: Claus M. Azzalin; ETH Zürich; Institute of Biochemistry (IBC); Schafmattstrasse 18; Zürich CH-8093 Switzerland; Tel.: +41.44.633.4410; Fax: +41.44.632.12.98; Email: [email protected]
Submitted: 03/03/08; Accepted: 03/04/08
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5836
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TelomeresThe silence is broken
Claus M. Azzalin1,* and Joachim Lingner2,3
1ETHZ-Eidgenössische Technische Hochschule Zürich; Institute of Biochemistry (IBC); Zürich, Switzerland; 2EPFL-Ecole Polytechnique Fédérale de Lausanne; ISREC-Swiss institute for experimental cancer research; Epalinges, Switzerland; 3“Frontiers in Genetics” National Center for Competence in Research (NCCR); Geneva, Switzerland
Key words: telomeres, transcription, TERRA, heterochromatin, SMG, RNAi
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Transcription of telomeric heterochromatin
analysis will be necessary to map TERRA transcrip-tion start sites and promoter regions.
While the length distribution of TERRA molecules seems not to vary substantially among the different mammalian cell lines, TERRA abundance is variable, suggesting cell type- and species-specific TERRA regu-latory mechanisms.31,32 Several lines of evidence seem to indicate that TERRA abundance might positively correlate with telomere length; for example HeLa cell lines bearing very long telomeres (HeLa-E1 and HeLaI.2.11) show higher TERRA levels as compared to HeLa cell lines with much shorter telomeres.31,32 Nevertheless, mouse RenCa cells, which have 4–6 kb long telomeres, display TERRA amounts similar to the ones detected in mouse embryonic fibroblasts (MEFs) with telomeres longer than 23 kb; similarly, mouse NS1 cells and mouse ES cells display similar TERRA levels while their telomere lengths are substantially different.31,32 However, it may be difficult to establish a connection between TERRA levels and telomere length when using non-isogenic cell lines because of possible cell line specific variations in TERRA regulatory mechanisms. Interestingly, Schoeftner and Blasco show that TERRA levels are lower in MEFs deficient for telomerase as compared to wild type cells, suggesting that telomere length might correlate with TERRA expression and/or telomerase might be directly involved in regulating TERRA expression. However, TERRA is present both in telomerase-positive and negative cells.31,32
TERRA is detectable in most adult mouse tissues (Fig. 1A and ref. 32) and in skeletal muscle at different developmental stages from E17 embryonic stage (Fig. 1B). As for different cell lines and species, TERRA abundance varies among the different tissues and different developmental stages. Interestingly, low TERRA levels are detected in some cancerous tissues as compared to healthy tissues, suggesting that TERRA might be downregulated during cancer development in vivo.32
Experiments performed using the RNA polymerase II (RNA polII) specific inhibitor alpha-amanitin demonstrated that TERRA molecules are at least in part produced by RNA polII-mediated transcription of mammalian telomeres. Consistently, RNA polII could be localized to telomeres by chromatin immuno-precipitation experiments and was reported to physically interact with the shelterin component TRF1.32 Schoeftner and Blasco also showed that TERRA molecules can be recovered in polyA+ enriched RNA fractions from mouse and human cell lines, suggesting that TERRA is 3' polyadenylated.32 However, we only find 7% of TERRA molecules in polyA+ nuclear RNA fractions from human HeLa cells (Fig. 2), while 62–64% of c-myc and actin mRNA molecules could be detected in polyA+ fractions in our experimental conditions (Fig. 2). As expected, only a negligible fraction (1%) of non-polyadenylated U1 RNA was detected in polyA+ fractions. Notably, polyA+ TERRA molecules were almost exclusively larger
than 2 kb in length (Fig. 2) indicating that short TERRA molecules are not polyadenylated. Nonetheless, it is important to point out that the information about the sequences of the subtelomeric tract of TERRA is very limited and that one cannot formally exclude that internal polyA rich sequences also contributed to the retention of TERRA on the oligo dT column.
TERRA is a Constitutive Component of Telomeric Heterochromatin
RNA fluorescence in situ hybridization experiments revealed that TERRA molecules form discrete nuclear foci overlapping with telomeric heterochromatin in interphase cells as well as in tran-
Figure 1. (A) RNA from different adult mouse tissues immobilized on nylon filters (Zyagen Laboratories, San Diego, USA) was hybridized to radiolabelled (CCCTAA)n probes.31 Actin was used as a loading control. (B) RNA from skeletal muscles from mice at different develop-mental stages (Zyagen Laboratories, San Diego, USA) was hybridized as in (A). E, embryo; d, day; w, week; m, month. (C) HeLa cells were treated with 100 nM Trichostatin A (TCA) for 24 h or left untreated (untr). Total RNA was prepared and subjected to Northern blot analysis as in (A). Actin was used as a loading control.
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scriptionally inactive metaphase chromosomes.31,32 The ability of TERRA molecules to interact with telomeres independently on their transcription indicates that TERRA is an integral component of telomeric heterochromatin and suggests that TERRA might partici-pate in maintaining telomere architecture. It is possible that TERRA regulates the epigenetic state of telomeres, perhaps by mechanisms similar to the inactivation of the mammalian X chromosome in females, which is mediated by the long noncoding RNA Xist.33,34 Intriguingly, some TERRA foci at least partially overlap with inactive X chromosome domains.32
TERRA Regulatory Pathways
The recent discovery of TERRA was also accompanied by the discovery of different pathways involved in the regulation of either TERRA abundance or cellular localization. A careful dissection of such pathways will help us to decipher TERRA associated functions as well as to clarify the general role of non-coding RNAs in modula-tion of transcription and in heterochromatin formation.
SMG proteins. Supressors with Morphogenetic defects in Genitalia (SMG) proteins are essential to sustain Nonsense Mediated mRNA Decay (NMD), a highly conserved eukaryotic RNA quality control pathway, which targets aberrant mRNA molecules for degradation.35 Intriguingly, three human SMG proteins including the DNA/RNA helicase UPF1 (or SMG2), the putative RNA
endonuclease hEST1A (or SMG6) and the PI3-kinase-like kinase SMG1 promote genome stability by mechanisms separated from their function in NMD.36-39 We have shown that a fraction of all seven human SMG proteins associate with telomeres in vivo and that short hairpin RNA (shRNA) mediated depletion of different SMG proteins induces accumulation of telomere associated TERRA molecules, without affecting TERRA degradation rate or total cellular levels.31 Thus, SMG proteins promote TERRA displace-ment from telomeres. Interestingly, while depletion of UPF1, hEST1A or SMG1 drastically impaired with TERRA displacement from telomeres, only a mild effect was obtained when cells were depleted for the other SMG factor UPF2.31 Because similar impair-ment of NMD was observed when UPF1, hEST1A, SMG1 or UPF2 were depleted, it seems conceivable that different SMG proteins play differently important roles in modulating TERRA association with chromatin.31 In this respect, the reported enzymatic activi-ties of UPF1 and hEST1A may be largely responsible for TERRA regulation. Human UPF1 is a 5'–3' DNA and RNA helicase with nucleic acid-dependent ATPase and ATP-dependent activities.40,41 The C-terminal PIN domain of hEST1A exhibits an overall struc-ture similar to ribonucleases of the RNase H family and displays single stranded RNA endonuclease activity under certain experi-mental conditions.42 UPF1 and hEST1A mutants lacking specific enzymatic activities will be used to understand if UPF1-mediated unwinding and hEST1A-mediated cleavage of TERRA molecules play a role in TERRA displacement from chromatin.
We have also demonstrated that human cells depleted for UPF1, hEST1A and SMG1 lose entire telomeric tracts at a high rate, suggesting a causal link between proper TERRA displace-ment from telomeres and telomere integrity.31 Interestingly, human UPF1 physically interacts with DNA polymerase δ, accumulates on chromatin during S-phase and is essential to complete DNA replica-tion.36,41 We speculate that UPF1 (as well as other SMG factors) might ensure proper telomere replication by displacing TERRA from telomeres during progression of the replication fork, thereby avoiding unwanted annealing of TERRA to single stranded telomeric DNA. Lack of such a displacement mechanism could lead to arrest of the replication fork and ultimately generate double stranded breaks in the telomeric tract. Moreover, hEST1A, which shares partial sequence homology with the yeast telomerase subunit Est1p, inter-acts with active telomerase.38,39,43 Thus, UPF1 and hEST1A (and eventually other SMG factors) appear to coordinate higher order molecular processes including TERRA localization to telomeres, telomere replication and telomerase action. Finally, female mouse embryonic stem cell lines depleted for RENT1 (the murine ortholog of UPF1) or for UPF2 fail to form Xist RNA domains upon differ-entiation and to undergo X-inactivation, suggesting possible broad roles for nuclear SMG proteins in chromatin organization pathways that involve RNA.44
Chromatin state. As already mentioned, mammalian telomeres are enriched in heterochromatic marks including H3K9me3, H4K20me3 and HP1. Interestingly, mouse cells deficient for the histone methyl transferases SUV39H1/SUV39H2 and SUV4-20H1/SUV4-20H2 show decreased levels of histone methylation at telo-meres and aberrant telomere elongation.24,25 Moreover, progressive telomere shortening induces a decrease in the density of H3K9me3, H4K20me3 and HP1 at telomeres as well as diminished methyla-
Figure 2. Nuclear RNA was prepared from HeLa cells and polyA+ RNA was prepared using a polyT resin (Qiagen). Elutions were performed in volumes corresponding to the volumes of starting input RNA. Increasing volumes (2.5, 5 and 10 ml) of input RNA as well as 1.25, 2.5, 5 and 10 ml of polyA+ RNA were subjected to Northern blot analysis using radiolabelled (CCCTAA)n probes.31 Actin and c-myc mRNAs were detected on the same blot to verify the efficiency of purification of polyA+ RNA, while U1 non polyadenylated RNA was detected as a negative control. The percentage (%) of each RNA retrieved in the polyA+ fraction is indicated.
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tion of subtelomeric DNA.26 Collectively, these results indicate that the structure of mammalian telomeres is regulated epigenetically and this might contribute to TERRA regulation. Consistently, TERRA accumulates in mouse cells deficient for SUV39H1/2 and SUV4-20H1/232 as well as in human cells treated with the histone deacetylase inhibitor Trichostatin A (Fig. 1C), consistent with a model where hetero-chromatinization of genomic loci promotes downregulation of transcription of the same loci. On the contrary, TERRA abundance is decreased in mouse cells deficient for the DNA methyltransferases DNMT1 and DNMT3a/b, even though the global methylation levels of subtelomeric regions are diminished in this background.32,45
Mutations in genes encoding for enzymatic activities involved in chromatin remodeling result in a number of genetic disorders characterized by a widespread pattern of developmental defects. For example, the a-Thalassaemia/mental retardation syndrome, X-linked is due to mutations in the human ATRX gene, which codes for a nuclear ATP-dependent translocase essential for proper methylation of rDNA arrays and other repetitive subtelomeric DNA loci.46 The Rett syndrome and the Immunodeficiency-centromeric instability-facial anomalies syndrome arise from mutations in genes involved in the regulation of CpG dinucleotide methylation.46 The Rubinstein-Taybi syndrome derives from mutations in a histone acetyl transferase gene.46 It will be interesting to analyze the tran-scriptional state of telomeres in cells from patients affected by such syndromes and eventually determine if TERRA might contribute to the disease phenotype.
RNAi. RNA interference (RNAi) is a highly conserved gene silencing mechanism triggered by small interfering RNA molecules (siRNAs). SiRNAs are about 22 nucleotide long double stranded (ds) RNA molecules generated by the degradation of long ds RNA precursor by the ribonuclease III enzyme Dicer.47 Recent data in fission yeast suggest that the nuclear RNAi machinery, comprising the Argonaute 1 protein, could directly promote heterochromatiniza-tion of a genomic locus by associating with nascent RNA molecules transcribed from the same locus.48,49 Moreover, the RNAi machinery has been involved in telomeric heterochromatin formation in fission yeast.50 It is possible that transcription of a telomere could trigger heterochromatin formation by RNAi mediated mechanisms and that TERRA could be a precursor RNA to generate telomeric siRNAs. Two lines of evidence suggest a possible function for RNAi in maintaining the mammalian telomeric structure and in TERRA regulation. First is the recently observed increased association of Argonaute 1 protein to telomeres and the concomitant over-produc-tion of TERRA-like molecules upon transfection of mouse cells with synthetic RNAi, irrespective of the RNAi sequence used.51 Second, Dicer deficient mouse ES cells display lower TERRA levels as compared to wild type counterparts.31 While the molecular basis of the connection between telomeres, TERRA and RNAi are still unclear, these observations pose a technical problem which needs to be taken into consideration during the design of experimental settings: when analyzing the effects of depletion of one specific factor on TERRA levels one should consider the possible TERRA upregula-tion deriving from transfecting siRNAs into mammalian cells.51 One possible alternative is to use shRNA-expressing vectors, which have been reported not to affect TERRA total levels.31,51
Concluding Remarks
Herman J. Muller, who first coined the term “telomere”, predicted in 1938 that: “the terminal gene must have a special function, that of sealing the end of the chromosome, so to speak, and that for some reason a chromosome cannot persist indefinitely without having its ends thus sealed”.52 Structural and functional studies during the last decade demonstrated that telomeres, rather than corresponding to a gene, fulfill their functions through their specialized architecture. We now know that mammalian telomeres have indeed gene-like prop-erties in that they are actively transcribed into TERRA molecules. Moreover, at least a fraction of TERRA molecules remain associated to telomeres defining a new specific component of mammalian telomeric heterochromatin, together with repetitive DNA and shelterin (Fig. 3). The main future challenge will be to understand what function TERRA plays at telomeres. As already mentioned, TERRA could participate in RNA-based mechanisms of epigenetic regulation of telomeres, similar to the Xist-mediated inactivation of mammalian X chromosomes or to the RNAi mediated heterochoma-tinization of telomeric regions in fission yeast.33,34,50 An alternative and not exclusive hypothesis is suggested by the observation that short TERRA-like RNA oligonuclotides inhibit telomerase activity in PCR-based in vitro assays.32 Telomere-bound TERRA could negatively regulate telomerase in vivo by annealing to the template region of the telomerase RNA moiety, thereby preventing telomerase action at telomeres.32 Additional efforts are now required to test these and alternative hypotheses as well as to understand the level of conservation of putative TERRA associated functions among different eukaryotes.
AcknowledgementsWork in JL’s laboratory is supported by grants from the Swiss
Cancer League, the Swiss National Science Foundation, the HFSP Program and the EU 6th and 7th Framework Programme. CMA’s laboratory is supported by grants from the Swiss National Science Foundation and ETH-Zürich.
Figure 3. Mammalian telomeres comprise (TTAGGG)n DNA sequences, (UUAGGG)n-containing RNA molecules (TERRA) and shelterin. Many addi-tional telomere interacting proteins are not shown. See text for details.
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