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...

DNA methylation modulates telomere function. In Arabidopsis thaliana, telomeric regions have a bimodal chromatin organization with unmethylated telomeres and methylated subtelomeres. To gain insight into this organization we have generated TAIR10-Tel, a modified version of the Arabidopsis reference genome with additional sequences at most chromosome ends. TAIR10-Tel has allowed us to analyse DNA methylation at nucleotide resolution level in telomeric regions. We have analysed the wild-type strain and mutants that encode inactive versions of all currently known relevant methyltransferases involved in cytosine methylation. These analyses have revealed that subtelomeric DNA methylation extends 1 to 2 kbp from Interstitial Telomeric Sequences (ITSs) that abut or are very near to telomeres. However, DNA methylation drops at the telomeric side of the telomere-subtelomere boundaries and disappears at the inner part of telomeres. We present a comprehensive and integrative model for subtelomeric DNA methylation that should help to decipher the mechanisms that govern the epigenetic regulation of telomeres. This model involves a complex network of interactions between methyltransferases and subtelomeric DNA sequences.