Determination of Arabidopsis thaliana telomere length by PCR

Vaquero-Sedas, María I.; Vega-Palas, Miguel A.

doi:10.1038/srep05540

Cited by 23 publications

(26 citation statements)

References 45 publications

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“…Consistent with the delayed onset of aberrant phenotypes in ddm1-2 mutants, we found that telomere length is maintained at the wild-type level throughout the first five generations of the mutants, but then it is abruptly disrupted in G6 when telomeres dramatically shorten. Thus, in addition to its role in maintaining heterochromatin and DNA methylation across the A. thaliana genome, our results support the conclusion of two previous studies (Ogrocka et al 2014; Vaquero-Sedas and Vega-Palas 2014), indicating that DDM1 promotes telomere length homeostasis. Our data further argue that dysregulation of telomere length is correlated with a metastable genome caused by transposon mobilization.…”

Section: Discussionsupporting

confidence: 90%

“…Two recent studies indicate that DNA methylation is required for telomere length homeostasis in Arabidopsis (Ogrocka et al 2014; Vaquero-Sedas and Vega-Palas 2014). Plants bearing the ddm1-2 allele of Deficient in DNA Methylation 1 (DDM1) exhibit telomeres shorter than wild-type (Vaquero-Sedas and Vega-Palas 2014).…”

Section: Introductionmentioning

confidence: 99%

“…Plants bearing the ddm1-2 allele of Deficient in DNA Methylation 1 (DDM1) exhibit telomeres shorter than wild-type (Vaquero-Sedas and Vega-Palas 2014). Telomere shortening is also reported with a second DDM1 allele, ddm1-8 (Ogrocka et al 2014).…”

Section: Introductionmentioning

confidence: 99%

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DDM1 guards against telomere truncation in Arabidopsis

Xie

Shippen

2018

Plant Cell Rep

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Epigenetic pathways, including DNA methylation, are crucial for telomere maintenance. Deficient in DNA Methylation 1 (DDM1) encodes a nucleosome remodeling protein, required to maintain DNA methylation in Arabidopsis thaliana. Plants lacking DDM1 can be self-propagated, but in the sixth generation (G6) hypomethylation leads to rampant transposon activation and infertility. Here we examine the role of DDM1 in telomere length homeostasis through a longitudinal study of successive generations of ddm1-2 mutants. We report that bulk telomere length remains within the wild-type range for the first five generations (G1–G5), and then precipitously drops in G6. While telomerase activity becomes more variable in later generation ddm1-2 mutants, there is no correlation between enzyme activity and telomere length. Plants lacking DDM1 also exhibit no dysregulation of several known telomere-associated transcripts, including TERRA. Instead, telomere shortening coincides with increased G-overhangs and extra-chromosomal circles, consistent with deletional recombination. Telomere shortening also correlates with transcriptional activation of retrotransposons, and a hypersensitive DNA damage response in root apical meristems. Since abiotic stresses, including DNA damage, stimulate homologous recombination, we hypothesize that telomere deletion in G6 ddm1-2 mutants is a by-product of elevated genome-wide recombination in response to trans-poson mobilization. Further, we speculate that telomere truncation may be beneficial in adverse environmental conditions by accelerating the elimination of stem cells with aberrant genomes.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

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DDM1 guards against telomere truncation in Arabidopsis

Xie

Shippen

2018

Plant Cell Rep

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“…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).…”

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confidence: 99%

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

et al. 2016

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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...

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“…K (20 mg/mL) at 55°C for 1 h. After overnight de-cross-linking at 65°C, phenol-chloroform extraction, isolated DNA was dissolved in 50 mL of 5 mM Tris-Cl, pH 8.0. Quantification of telomeric DNA was performed by qPCR as described (Vaquero-Sedas and Vega-Palas, 2014). DNA purified from ChIP (1 mL) was used in 20 mL of qPCR reaction containing 13 LightCycler 480 High Resolution Melting Master mix, 3 mM MgCl 2 , and 0.25 mM each TelA and TelB primers (Supplemental Table 1).…”

Section: Chipmentioning

confidence: 99%

Protection of Arabidopsis Blunt-Ended Telomeres Is Mediated by a Physical Association with the Ku Heterodimer

et al. 2017

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Telomeres form specialized chromatin that protects natural chromosome termini from being recognized as DNA doublestrand breaks. Plants possess unusual blunt-ended telomeres that are unable to form t-loops or complex with single-strand DNA binding proteins, raising the question of the mechanism behind their protection. We have previously suggested that blunt-ended telomeres in Arabidopsis thaliana are protected by Ku, a DNA repair factor with a high affinity for DNA ends. In nonhomologous end joining, Ku loads onto broken DNA via a channel consisting of positively charged amino acids. Here, we demonstrate that while association of Ku with plant telomeres also depends on this channel, Ku's requirements for DNA binding differ between DNA repair and telomere protection. We show that a Ku complex proficient in DNA loading but impaired in translocation along DNA is able to protect blunt-ended telomeres but is deficient in DNA repair. This suggests that Ku physically sequesters blunt-ended telomeres within its DNA binding channel, shielding them from other DNA repair machineries.

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Determination of Arabidopsis thaliana telomere length by PCR

Cited by 23 publications

References 45 publications

DDM1 guards against telomere truncation in Arabidopsis

DDM1 guards against telomere truncation in Arabidopsis

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

Protection of Arabidopsis Blunt-Ended Telomeres Is Mediated by a Physical Association with the Ku Heterodimer

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