Telomere epigenetics: a higher-order control of telomere length in mammalian cells

María A. Blasco1

Molecular Oncology Program, Spanish National Cancer Centre (CNIO), E-28029 Madrid, Spain

1 Email: mblasco{at}cnio.es

Abstract

Telomeres are capping structures at the ends of eukaryotic chromosomes composed of TTAGGG repeats bound to an array of specialized proteins. Telomeres, together with centromeres, have been classically considered heterochromatic regions. Constitutive heterochromatin domains typically consist of repetitive DNA and have a very low gene content. In addition, constitutive heterochromatin is characterized by a number of hallmark histone modifications, as well as DNA modifications. In the case of pericentric heterochromatin, several activities responsible for these epigenetic modifications have been recently identified and characterized. In contrast, very little is still known on the architecture of telomeric chromatin, as well as on the activities that may regulate its structure and function. Here, we will discuss recent findings suggesting that telomeric chromatin shares many features with pericentric chromatin, and that disruption of telomeric heterochromatin results in changes in telomere length.

Abbreviations: DSB, double-strand breaks; HMTases, histone methyltransferases; HP1, Heterochromatin Protein 1; NHEJ, non-homologous end-joining

Telomeric chromatin

Vertebrate telomeres are composed of tandem repeats of the TTAGGG DNA sequence bound to an array of specialized proteins (13). Telomeres are characterized by having a 3'-overhang of the G-rich strand, the G-strand overhang (3). The current model is that telomeres form a structure that physically hides the 3'-overhang, thus protecting chromosome ends from cellular activities. This structure is provided by the ability of the 3'-overhang to fold back and invade the double stranded region of the telomere forming the so-called T-loop and generating a displacement loop, or D-loop (4). Evidence for this type of structure has been obtained in different species using electron microscopy (4). In support of this model, telomere-binding proteins have been shown to influence the formation of T-loops (4). Indeed, T-loops have been recently proposed to represent a primordial mechanism for chromosome end protection (5).

When telomere function is lost, the ends of chromosomes become susceptible to fusing to other unprotected chromosome ends and/or to double strand DNA breaks (DSB), leading to end-to-end fusions and other types of chromosomal aberrations (6,7). The non-homologous end-joining DNA repair machinery seems to be essential for this process, since abrogation of this pathway leads to prevention of end-to-end fusions produced by dysfunctional telomeres (810). In particular, both Ku86 and the protein kinase complex DNA–PK catalytic subunit (DNA-PKcs), which are central to the non-homologous end-joining (NHEJ) pathway for DSB repair, are necessary for both end-to-end chromosome fusion and apoptosis triggered by critically short telomeres in the context of telomerase-deficient mice (6,8,9). These findings suggest that the NHEJ machinery detects and signals the presence of dysfunctional telomeres as damaged DNA and ‘repairs’ them leading to end-to-end chromosomal fusions and other chromosomal rearrangements (6,8,9). Mutation of telomere-binding proteins in the absence of significant telomere shortening also results in dysfunctional telomeres and end-to-end telomere fusions, which are also mediated by the NHEJ machinery (10).

The telomere-binding proteins

Proteins that bind to the double stranded TTAGGG repeats at telomeres, such as TRF1 and TRF2, or that bind to the single stranded G-strand overhang, such as Pot 1, have been shown to influence both telomere capping and telomere length (1115). TRF1 and TRF2 are located at telomeric T-loops and are negative regulators of telomere length (4,16). TRF1 function is regulated by TIN2 (17), and by the poly(ADP-ribose) polymerases TANK1 (also known as tankyrase) and TANK 2 (18,19). TANK1 has been recently demonstrated to be essential for separation of sister chromatid telomeres during mitosis, suggesting the existence of a new telomere-specific cohesion, which is regulated by poly(ADP-ribosylation) (20). In addition, TRF1 interacts with Pot 1, and this interaction has been proposed to convey information from the double stranded telomere region to the single stranded 3'-overhang (15).

TRF2 has been shown to stabilize the G-strand overhang, and prevent telomeric fusions (21). TRF2 also recruits hRAP1 to human telomeres. hRAP1 is the homologue of yeast RAP1 protein and its over-expression causes telomere elongation (22,23). In addition, TRF2 recruits the MRE11 complex to human telomeres (24). The MRE11 complex is composed of RAD50, MRE11 and NBS1 and is a key component of the homologous recombination and NHEJ involved in DSB repair. TRF2 has been also shown to interact with the nucleotide excision repair complex XPF/ERCC1, which in turn is the exonuclease involved in 3'-overhang resection in the absence of TRF2-mediated telomere capping (25).

Another NHEJ DNA repair complex found at mammalian telomeres is the DNA-dependent DNA–PK, involved in NHEJ DSB repair. DNA–PK is composed of Ku70 and Ku86 proteins, and of the DNA–PKcs (26). The study of Ku86 and DNA–PKcs-deficient mice indicated that these proteins have a role in telomere capping (6). In particular, abrogation of either Ku86 or DNA–PKcs results in telomeric fusions characterized by showing long TTAGGG regions at the fusion point (8,2731). Therefore, these end-to-end chromosome fusions are not the result of telomere shortening below a minimum length, but rather of the loss of telomere capping. In addition, the fact that these fusions preferentially involve telomeres produced by leading strand synthesis suggested a role for these activities in the post-replicative processing of the leading strand telomere, most likely to generate the 3' G strand overhang (32).

Deficiency in either Ku86 or DNA–PKcs also influences telomere length, in accordance with their proposed role in generating or maintaining a proper telomere capping structure. In particular, Ku86 acts as a negative regulator of telomerase, since Ku86-deficient mice show a telomerase-mediated telomere elongation compared with the wild-type controls (8). In contrast, DNA–PKcs cooperates with telomerase in telomere length maintenance, and mice doubly deficient in both activities show an accelerated rate of telomere loss (9). In addition, single mutant DNA–PKcs mice show decreased telomere length with age, as well as with increasing mouse generations compared with the wild-type controls (33), again suggesting a role for DNA–PKcs in telomere length maintenance. Besides their roles in telomere capping and telomere length regulation, Ku86 and DNA–PKcs have been also shown to be essential in signalling and processing critically short telomeres as damaged DNA, as discussed above (8,9).

Finally, recent works suggest that proteins involved in homologous recombination DNA repair, such as Rad54 and Rad51D, are important for telomere capping and telomere length regulation, suggesting that homologous recombination has an important role at mammalian telomeres (34,35).

Nucleosomes and histone modifications at telomeres

It has been long described that human and mouse telomeres display a canonical chromatin structure with extensive arrays of tightly packed nucleosomes that had a shorter repeat size than bulk nucleosomes, suggesting a special spacing of nucleosomes at telomeres (36). The N-terminal tails of histones can be subjected to post-translational modifications including acetylation, methylation and phosphorylation generating an extensive repertoire of chromatin structures, which in turn can regulate a plethora of cellular responses (37,38). However, very little was known on histone and DNA modifications at mammalian telomeric chromatin, which has been classically considered part of the constitutive heterochromatin. In particular, constitutive heterochromatin, such as that of pericentric chromatin, is characterized by hypermethylation of DNA, hypoacetylation of histones, and hypermethylation of histone H3 at different lysines, most notably at lysine 9 (H3K9). In particular, H3-K9 tri-methylation by the Suv39 h histone methyltransferases (HMTases) is one of the hallmarks of pericentric heterochromatin (39,40). This enrichment for H3-K9 tri-methylation at pericentric chromatin creates a binding site for the Heterochromatin Protein 1 (HP1) family of proteins (38) that mediate heterochromatin formation. In mammals, however, the putative role of these chromatin-modifying activities on regulating telomere length and function is largely unknown. Interestingly, yeast and flies defective for activities that modify the state of chromatin, also show abnormal telomere function and telomere length regulation (41,42). In particular, flies with HP1 mutations show defective telomere capping, as well as increased recombination at telomeres by the In HeT-A and TART retrotransposons, suggesting a role for chromatin modifications in regulating telomeric function (42,43,44).

Do mammalian telomeres have the hallmarks of constitutive heterochromatin?

Histone modifications at telomeres have been recently investigated by using mice doubly null for the Suv39h1 and Suv39h2 HMTases, SUV39DN mice. The Suv39h1 and Suv39h2 HMTases govern histone H3 lysine 9 (H3-K9) methylation at pericentric heterochromatin (39). In particular, cells derived from SUV39DN mice show a decreased tri-methylation of H3-K9 at pericentric chromatin both using immunofluorescence and chromatin Immunoprecipitation (ChIP) techniques (39,40). In addition, cells derived from SUV39DN mice show a marked genetic instability, which has been proposed to be the consequence of a defective architecture at pericentric chromatin (39).

Interestingly, telomeres showed similar epigenetic changes to those present at pericentric chromatin in SUV39DN cells. In particular, Garcia-Cao et al. recently described that telomeres are enriched for di- and tri-methylated H3-K9, and that these histone modifications are decreased at SUV39DN telomeres, similar to that described previously for pericentric chromatin (40,45). Moreover, both telomeric and pericentric chromatin accumulate H3-K9 mono-methylation in the absence of the Suv39h1 and Suv39h2 HMTases, highlighting the similarities between telomeric and centromeric chromatin (45). However, while pericentric heterochromatin shows increased H3-K27 tri-methylation in the absence of Suv39h1 and Suv39h2 HMTases, this modification was not detected at telomeric heterochromatin, revealing possible differences between these two heterochromatic domains with distinct functions in the cell (45).

In addition, SUV39DN telomeres showed a decreased binding of the HP1 family of proteins HP1{alpha}, HP1ß and HP1{gamma}, concomitant with the decrease in H3-K9 tri- and di-methylation (45). Therefore, telomeric chromatin shares similar hallmark epigenetic modifications to those present at pericentric heterochromatin and those modifications are maintained by the Suv39h1 and Suv39h2 HMTases.

The consequences of losing heterochromatic features at telomeric chromatin

The fact that telomeric chromatin is characterized by complex epigenetic modifications, predicts that epigenetic errors, such as abrogation of the Suv39 h HMTases or defective HP1 binding, which disrupt the normal architecture of telomeric chromatin, may also affect telomere length regulation and telomere function. In fact, an unprecedented abnormal elongation of telomeres was detected in SUV39DN cells coincidental with changes in telomeric heterochromatin, suggesting that loss of heterochromatic features at telomeres results in a more ‘open’ chromatin state, which in turn could facilitate the access of telomerase or other telomere-elongating activities to the chromosome end (45). Interestingly, the abnormal telomere length elongation in SUV39DN cells was observed indistinguishably at p- and q-chromosome arms, suggesting that it was unlikely to be the consequence of changes at the neighbouring pericentric chromatin domains (i.e. mouse chromosomes are acrocentric).

It is likely that the elongated telomeres present in SUV39DN cells are produced by telomerase activity. Given the fact that SUV39DN cells show similar levels of telomerase activity to those of the wild-type controls (45) it is possible that accessibility of telomerase to telomeres is altered in SUV39DN cells due to changes in telomeric chromatin. Indeed, the elongated telomeres present in SUV39DN cells were preserved at both sister chromatids within a given chromosome arm (45), indicating that they were generated during DNA replication.

Finally, it is possible that epigenetic modifications at telomeric chromatin could also affect the binding of specific TTAGGG-binding proteins, such as TRF1 and TRF2. Indeed, a reproducible increase in TRF1 binding per amount of TTAGGG repeats was detected in SUV39DN telomeres, suggesting a change in the compaction of telomeric chromatin (45). The increased TRF1 binding at SUV39DN telomeres could represent a feedback mechanism to regulate telomere length in these cells, since TRF1 is a known negative regulator of telomerase-mediated elongation at telomeres (16).

Normal telomere capping despite loss of heterochromatin features

Changes in the architecture of telomeric chromatin could also affect telomeric capping. Loss of telomeric capping primarily gives rise to end-to-end chromosomal fusions (3,6). However, the abnormally elongated telomeres present in the SUV39DN cells seem to retain their end-capping function as indicated by the fact that no increased telomere fusions were observed in these cells (45). These findings indicate that the abnormally elongated telomeres in SUV39DN cells retain a normal capping function. These results are in agreement with the fact that ChIP analysis showed that the abundance of TRF2 at telomeric repeats was normal in SUV39DN cells, indicating that TRF2 binding to telomeres is independent of their degree of H3-K9 methylation (45).

Future directions

These recent findings have demonstrated that histone H3 Lys-9 methylation by the Suv39h1 and Suv39h2 HMTases occurs at mammalian telomeres and that is important for telomere length regulation (Figure 1). It is probable that the loss of heterochromatic features at SUV39DN telomeres could explain the abnormal telomere elongation in these cells, for instance by increasing the access of telomerase to the telomere. These findings imply that telomere length can be regulated by epigenetic factors, such as H3-K9 methylation, linking for the first time histone modifications with telomere length regulation in mammalian cells. Since the Suv39h1 and Suv39h2 HMTases are not the only HMTases responsible for H3-K9 methylation in mammalian heterochromatic regions, it is possible that other HMTases of the same family (SET-domain containing enzymes) (46) may also contribute to the epigenetic regulation of telomere length in mammalian cells.



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Fig. 1. Model for epigenetic regulation of telomere length. Mammalian telomeres contain features of the constitutive heterochromatin such as enrichment for H3-K9 di-, and tri-methylation, as well as binding of the HP1 family of proteins (HP1{alpha}, HP1ß and HP1{gamma}), similar to that described previously for pericentric hetochromatin. In the absence of the Suv39h1 and Suv39h2 HMTases, di- and tri-methylation of H3-K9 is decreased at telomeres, while they are enriched for H3-K9 mono-methylation, similar to that observed at pericentric chromatin. Telomeric chromatin in SUV39DN cells also shows decreased binding of the HP1 proteins. These changes in the composition of the telomeric chromatin in SUV39DN cells suggest a loss of heterochromatic features, which in turn could explain the telomere elongation phenotype shown by these cells. The binding of a specific telomere-binding protein, TRF2, which is essential for telomere protection, was normal in SUV39DN cells, in agreement with a normal telomere capping function in these cells. TRF1, another telomere-specific binding protein, showed a slight increase at SUV39DN telomeres, which may reflect the fact that these telomeres have an altered chromatin structure.

 
Finally, the fact that epigenetic errors can alter telomere length in mammals, may explain the abnormal re-setting of telomere length in different cloned animals (47,48). In addition, the fact that telomere length can be regulated by epigenetic modifications provides a connection between the transcriptional silencing of genes near the telomeres (the so-called Telomere Position Effect or TPE) (49,50,51), and the architecture of telomeric chromatin.

Acknowledgments

Research at the laboratory of M.A.B. is funded by grants from the Spanish Ministry of Science and Technology, the Regional Government of Madrid, the European Union, and by the Spanish National Cancer Centre (CNIO).

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accepted May 5, 2004.