On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.Key words: telomeres, subtelomeres, euchromatin, heterochromatin, ChIP, immunolocalizationTelomeric DNA usually contains tandem repeats of a short GC rich motif. The number of repeats and, therefore, the length of telomeres is subject to regulation and influences relevant biological processes like aging and cancer.^1–3 In situ hybridization studies have revealed that telomeric repeats are also present at interstitial chromosomal loci.^4,5 An analysis of the genome sequence from different eukaryotes indicates that ITSs have a widespread distribution in different model systems including zebrafish, chicken, opossum, mouse, dog, cattle, horse, human, rice, poplar or Arabidopsis (see Fig. 1 for an example; www.ncbi.nlm.nih.gov/mapview). These ITSs have been related to chromosomal aberrations, fragile sites, hot spots for recombination and diseases caused by genomic instability, although their functions remain unknown.⁶Open in a separate windowFigure 1Distribution of the main telomeric repeat arrays in the genome of several model organisms. These representations have been performed by using the megaBLAST program and the all assemblies genomic databases at NCBI (www.ncbi.nlm.nih.gov/mapview). Searches for homology with 100 tandem telomeric repeats were done using the default parameters except that the expected threshold was set to 10 and the filters were turned off. Chromosomes are represented as vertical bars and numbered at the bottom. The horizontal bars represent the telomeric repeat arrays. Colors indicate the BLAST scores (red ≥200; pink 80–200; green 50–80).Telomeres and ITSs have probably cross talk through evolution. In some instances, ITSs could have been generated by telomeric fusions. Pioneering studies performed by Hermann J. Muller in Drosophila and Barbara McClintock in maize showed that newly formed chromosome ends tend to fuse giving rise to the so-called breakage-fusion-bridge cycle.^7,8 This cycle can lead to stable chromosomal reorganizations after healing of the broken ends. In addition, Muller and McClintock found that, unlike these newly formed broken chromosome ends, natural chromosomal ends are quite stable and do not tend to fuse.⁹ It is currently known that telomere dysfunction due to mutations that cause telomeric shortening or abolish the expression of certain telomeric proteins can lead to telomeric fusions, anaphase bridges and genome reorganizations.^1–3,10,11 Therefore, telomeric shortening or alterations of telomeric chromatin structure might be expected to generate ITSs through evolution by promoting telomeric fusions.¹² ITSs might also originate through the activity of telomerase during the repair process of double strand breaks or by recombination.^13–16 In addition, telomerase activity might lead to the formation of new telomeres by healing of chromosome breaks within internal telomeric repeats and even within other sequences.^17–19 This process of healing involves the acquisition of telomeric chromatin structure.DNA folds into two major chromatin organizations inside the cell nucleus: heterochromatin and euchromatin. Heterochromatin is highly condensed in interphase nuclei and is usually associated with repetitive and silent DNA. By contrast, euchromatin has an open conformation and is often related to the capacity to be transcribed. Both kinds of chromatin exhibit defined epigenetic modifications that influence their biochemical behavior. Thus, the study of these epigenetic marks is an issue of major interest.The chromatin structures of telomeres and ITSs might be different. Therefore, they should be studied independently. Chromatin structure analyses are usually performed by immunocytolocalization or by chromatin immunoprecipitation (ChIP).^20–23 Special care should be taken when the epigenetic status of telomeres is analyzed by immunocytolocalization. This technique does not allow differentiating between telomeres and subtelomeric regions. Since subtelomeric regions are known to be heterochromatic in many eukaryotic organisms, heterochromatic marks should be immunolocalized at the chromosome ends of these organisms. However, these marks could correspond to subtelomeric regions and not to telomeres.The ChIP technique implies the immunoprecipitation of chromatin with specific antibodies and the further analysis of the immunoprecipitated DNA. DNA sequences immunoprecipitated by a specific antibody are thought to associate in vivo with the feature recognized by this antibody. Whereas the enrichment of single copy sequences in the immunoprecipitated DNA has been usually analyzed by quantitative PCR, the analyses of repetitive DNA sequences have been often performed by hybridization. Thus, multiple telomeric chromatin structure analyses have been performed by hybridizing immunoprecipitated DNA with a telomeric probe. However, these analyses displayed simultaneously the chromatin structures of telomeres and ITSs. High throughput sequencing analyses of the immunoprecipitated DNA might help overcome this problem. Nevertheless, since the reads obtained with these techniques at present are short, it is still difficult to ascertain whether the enrichment of immunoprecipitated telomeric sequences corresponds to telomeres or to ITSs. Third-generation long-read accurate technologies and new algorithms that discriminate between telomeres and ITSs should solve the problem.In principle, the combination of immunocytolocalization and ChIP experiments should help to differentiate between telomeres and ITSs. However, since subtelomeric regions are known to influence telomere function and contain degenerated ITSs, at least in some organisms like humans or Arabidopsis, this may not be necessarily true.⁶ A specific epigenetic mark might be required for telomere function, found associated with telomeric repeats by ChIP and with the end of chromosomes by immunocytolocalization and still not associate with true telomeres but with subtelomeric regions and ITSs or just with subtelomeric ITSs.An alternative way to analyze the chromatin structure of telomeres by ChIP involves the use of frequently cutting restriction enzymes. The chromatin structures of Arabidopsis telomeres and ITSs have been independently studied by using Tru9I, a restriction enzyme that recognizes the sequence TTAA.²⁴ Since telomeres in Arabidopsis and in other model systems are composed of perfect telomeric repeat arrays, they remain uncut after digestion with Tru9I.²⁵ In contrast, Arabidopsis ITSs are frequently cut because they are composed of short arrays of perfect telomeric repeats interspersed with degenerated repeats.^25–28 Thus, when Arabidopsis genomic DNA is digested with Tru9I and hybridized with a telomeric probe, most of the signals corresponding to ITSs disappear.²⁵ The use of Tru9I has made possible to discover that Arabidopsis telomeres exhibit euchromatic features. In contrast, Arabidopsis ITSs and subtelomeric regions are heterochromatic.²⁴ In Arabidopsis, heterochromatin is characterized by cytosine methylation, which can be targeted at CpG, CpNpG or CpNpN residues (where N is any nucleotide), and by H3K9me1,2, H3K27me1,2 and H4K20me1. In turn, Arabidopsis euchromatin is characterized by H3K4me1,2,3, H3K36me1,2,3, H4K20me2,3 and by histones acetylation.²⁹ ChIP experiments processed with Tru9I have revealed that Arabidopsis telomeres have high levels of euchromatic marks (H3K4me2, H3K9 and H4K16 acetylation) and low levels of heterochromatic marks (H3K9me2, H3K27me1 and DNA methylation).²⁴ Therefore, Arabidopsis telomeres exhibit epigenetic modifications characteristic of euchromatin.Different studies in mice, humans or Arabidopsis have reported that telomeres are heterochromatic based on the existence of siRNAs containing telomeric sequences, on the association of telomeric sequences with telomeric and with heterochromatin proteins, on the methylation of telomeric sequences or on the histones modifications associated with telomeric sequences.^30–34 However, the experiments presented in those studies addressed simultaneously the chromatin organizations of telomeres and subtelomeric regions or of telomeres and ITSs. Telomeres have also been reported to be heterochromatic based on the existence of the so-called TElomeric Repeat containing RNAs (TERRA), which are present in different eukaryotes.³⁵ At telomeric regions, TERRA are transcribed from subtelomeric promoters towards chromosome ends. Since human subtelomeric TERRA are mostly composed of subtelomeric sequences, with only about 200 bp of telomeric sequences at their 3′ ends, they might be related to subtelomeric heterochromatin formation rather than to the formation of telomeric chromatin. Nevertheless, TERRA interact with human telomeric proteins and influence telomere function. In addition, TERRA might also be related to ITSs heterochromatinization.^34,35We believe that the scenario found in Arabidopsis could also be found in other model systems if the chromatin structures of telomeres, subtelomeric regions and ITSs are independently analyzed. Several reports have described the presence of histone H3.3 at mice telomeres.^36–39 Since this histone variant has been previously associated with active chromatin, these studies are compatible with a euchromatic organization of telomeres. However, again in these reports, the experiments shown addressed simultaneously the chromatin organization of telomeres and subtelomeric regions or of telomeres and ITSs. In general terms, we believe that a clear distinction between telomeres and ITSs should be established when future ChIP experiments are analyzed. The use of third generation high throughput sequencing technologies or of frequently cutting restriction enzymes might help in this task.As mentioned above, the epigenetic modifications associated with telomeric regions are known to be important for telomere function. These modifications are required to provide genome stability.³³ In this context, it will be relevant to ascertain how the function of Arabidopsis telomeres is influenced by their euchromatic marks and by the presence of heterochromatin at subtelomeric regions.