Novel features of telomere biology revealed by the absence of telomeric DNA methylation

Abstract

Cytosine methylation regulates the length and stability of telomeres, which can affect a wide variety of biological features, including cell differentiation, development, or illness. Although it is well established that subtelomeric regions are methylated, the presence of methylated cytosines at telomeres has remained controversial. Here, we have analyzed multiple bisulfite sequencing studies to address the methylation status of Arabidopsis thaliana telomeres. We found that the levels of estimated telomeric DNA methylation varied among studies. Interestingly, we estimated higher levels of telomeric DNA methylation in studies that produced C-rich telomeric strands with lower efficiency. However, these high methylation estimates arose due to experimental limitations of the bisulfite technique. We found a similar phenomenon for mitochondrial DNA: The levels of mitochondrial DNA methylation detected were higher in experiments with lower mitochondrial read production efficiencies. Based on experiments with high telomeric C-rich strand production efficiencies, we concluded that Arabidopsis telomeres are not methylated, which was confirmed by methylation-dependent restriction enzyme analyses. Thus, our studies indicate that telomeres are refractory to de novo DNA methylation by the RNA-directed DNA methylation machinery. This result, together with previously reported data, reveals that subtelomeric DNA methylation controls the homeostasis of telomere length.[Supplemental material is available for this article.]Telomeres guarantee the complete replication of chromosomal termini, prevent genome instability, and influence relevant systemic processes like aging, cancer, or illness (Blackburn 2010). The length of telomeres and the chromatin organization of telomeric regions influence telomere functions. Hence, the epigenetic marks that label telomeric regions, which include telomeres and subtelomeres, play important roles in telomere biology (Blasco 2007;Galati et al. 2013;Giraud-Panis et al. 2013).One of the major epigenetic signatures found in eukaryotes is cytosine methylation. This DNA modification regulates multiple processes in plants and animals, including the homeostasis of telomere length (Blasco 2007;Suzuki and Bird 2008;Ooi et al. 2009;Law and Jacobsen 2010;Castel and Martienssen 2013;Ogrocká et al. 2014;Vaquero-Sedas and Vega-Palas 2014). Mammalian DNA methylation is primarily found in the CG context (Ramsahoye et al. 2000;Lister et al. 2009). In contrast, plants have significant levels of DNA methylation in all sequence contexts (CG, CHG, and CHH, where H can be A, C, or T) (Law and Jacobsen 2010).Although subtelomeric DNA methylation has been reported in animals and plants, the presence of DNA methylation at telomeres remains an open question in both kingdoms (Blasco 2007;Vrbsky et al. 2010;Vaquero-Sedas et al. 2011;Ogrocká et al. 2014). The methylation status of mammalian telomeres has not been investigated because, as mentioned above, mammals have low levels of non-CG methylation, which is the type of DNA me...

Epigenetic features of human telomeres

“…In summary, we have seen that telomeres in most of the commonly studied human cell lines are not heterochromatic but exhibit euchromatic marks. However, heterochromatin is known to play a major role in telomere biology, which suggests that the integrity of subtelomeric heterochromatin might be important for the proper functioning of telomeres, as previously proposed for A. thaliana ( 10 , 14 ). Interestingly, we have also seen that certain cancer cells that maintain their telomeres through ALT have heterochromatic levels of H3K9me3 in their telomeres.…”

DDM1 guards against telomere truncation in Arabidopsis

“…There has been considerable debate concerning the presence of DNA methylation at A. thaliana telomeres and its impact on telomere length (Cokus et al 2008; Vrbsky et al 2010; Vega-Vaquero et al 2016). One group reported that plants lacking RDR2, a core component of the DNA methylation pathway at asymmetric cytosines (such as telomeric repeats), do not display any telomere length dysregulation (Vrbsky et al 2010), while other studies, including this one, find telomere shortening in plants lacking DDM1 (Ogrocka et al 2014; Vaquero-Sedas and Vega-Palas 2014).…”

“…One group reported that plants lacking RDR2, a core component of the DNA methylation pathway at asymmetric cytosines (such as telomeric repeats), do not display any telomere length dysregulation (Vrbsky et al 2010), while other studies, including this one, find telomere shortening in plants lacking DDM1 (Ogrocka et al 2014; Vaquero-Sedas and Vega-Palas 2014). Recent data indicate that subtelomeric sequences flanking telomere repeats, not telomeric DNA itself, are subject to methylation (Vega-Vaquero et al 2016). Thus, it has been proposed that changes in telomeric chromatin are responsible for telomere shortening in ddm1 mutants (Vaquero-Sedas et al 2011).…”

“…Two groups reported that A. thaliana telomeres are methylated by asymmetrical DNA methylation pathways directed by RNA-dependent DNA methylation (RdDM) (Cokus et al 2008; Vrbsky et al 2010). More recent studies suggest that the extent of DNA methylation at telomeres may be quite low or confined to subtelomeric regions (Vaquero-Sedas et al 2011; Ogrocka et al 2014; Vega-Vaquero et al 2016). While both telomeric and subtelomeric regions of yeast and mammal chromosomes are exclusively heterochromatic (Blasco 2007; Ottaviani et al 2008), subtelomeric regions in A. thaliana are reported to contain euchromatic (H3K4 Me2 and H3K9 Ac ) as well as heterochromatic (H3K9 Me2 and H3K27 Me ) features (Vaquero-Sedas et al 2011, 2012; Vrbsky et al 2010).…”

The distribution pattern of 5-methylcytosine in rye (Secale L.) chromosomes

“…Methylation studies of telomeric sequences, as with studies of the post-translational modifications of histones, have yielded ambiguous results. In Arabidopsis thaliana, one study showed the presence of methylation in telomeric sequences [47], whereas another study indicated the lack of methylation in this region [48]. In turn, DNA methylation of the telomeric and subtelomeric regions in three cotton species (G. hirsutum, G. herbaceum and G. arboreum) showed interspecific variation [49].…”