RESUMEN
The epigenetic features of defined chromosomal domains condition their biochemical and functional properties. Therefore, there is considerable interest in studying the epigenetic marks present at relevant chromosomal loci. Telomeric regions, which include telomeres and subtelomeres, have been traditionally considered heterochromatic. However, whereas the heterochromatic nature of subtelomeres has been widely accepted, the epigenetic status of telomeres remains controversial. Here, we studied the epigenetic features of Arabidopsis (Arabidopsis thaliana) telomeres by analyzing multiple genome-wide ChIP-seq experiments. Our analyses revealed that Arabidopsis telomeres are not significantly enriched either in euchromatic marks like H3K4me2, H3K9ac, and H3K27me3 or in heterochromatic marks such as H3K27me1 and H3K9me2. Thus, telomeric regions in Arabidopsis have a bimodal chromatin organization with telomeres lacking significant levels of canonical euchromatic and heterochromatic marks followed by heterochromatic subtelomeres. Since heterochromatin is known to influence telomere function, the heterochromatic modifications present at Arabidopsis subtelomeres could play a relevant role in telomere biology.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Epigénesis Genética , Telómero/genética , Heterocromatina/genéticaRESUMEN
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.
Asunto(s)
Arabidopsis , Metilación de ADN , Arabidopsis/genética , Epigénesis Genética , Metiltransferasas/genética , Telómero/genéticaRESUMEN
Telomeres protect the ends of linear eukaryotic chromosomes from being recognized as DNA double-strand breaks. Two major protein complexes are involved in the protection of telomeres: shelterin and CST. The dysfunction of these complexes can challenge the function of telomeres and lead to telomere fusions, breakage-fusion-bridge cycles, and cell death. Therefore, monitoring telomere fusions helps to understand telomeres biology. Telomere fusions are often analyzed by Fluorescent In Situ Hybridization (FISH) or PCR. Usually, both methods involve hybridization with a telomeric probe, which allows the detection of fusions containing telomeric sequences, but not of those lacking them. With the aim of detecting both types of fusion events, we have developed a nested PCR method to analyze telomere fusions in Arabidopsis thaliana. This method is simple, accurate, and does not require hybridization. We have used it to analyze telomere fusions in wild-type and mutant plants altered in CTC1, one of the three components of the Arabidopsis CST telomere capping complex. Our results show that null ctc1-2 mutant plants display fusions between all telomeric regions present in Arabidopsis chromosomes 1, 3 and 5, thus highlighting the widespread end-capping protection achieved by CTC1.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Unión a Telómeros , Telómero , Arabidopsis/genética , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Complejo Shelterina , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteínas de Arabidopsis/genéticaRESUMEN
Although subtelomeric regions in humans are heterochromatic, the epigenetic nature of human telomeres remains controversial. This controversy might have been influenced by the confounding effect of subtelomeric regions and interstitial telomeric sequences (ITSs) on telomeric chromatin structure analyses. In addition, different human cell lines might carry diverse epigenetic marks at telomeres. We have developed a reliable procedure to study the chromatin structure of human telomeres independently of subtelomeres and ITSs. This procedure is based on the statistical analysis of multiple ChIP-seq experiments. We have found that human telomeres are not enriched in the heterochromatic H3K9me3 mark in most of the common laboratory cell lines, including embryonic stem cells. Instead, they are labeled with H4K20me1 and H3K27ac, which might be established by p300. These results together with previously published data argue that subtelomeric heterochromatin might control human telomere functions. Interestingly, U2OS cells that exhibit alternative lengthening of telomeres have heterochromatic levels of H3K9me3 in their telomeres.
Asunto(s)
Epigénesis Genética , Telómero/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Heterocromatina/metabolismo , Código de Histonas , HumanosRESUMEN
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.
Asunto(s)
Arabidopsis/genética , Metilación de ADN/genética , Homeostasis del Telómero/genética , Telómero/genética , Arabidopsis/crecimiento & desarrollo , Citosina/metabolismo , ADN Mitocondrial/genética , ARN/genéticaRESUMEN
The chromatin structure of eukaryotic telomeres plays an essential role in telomere functions. However, their study might be impaired by the presence of interstitial telomeric sequences (ITSs), which have a widespread distribution in different model systems. We have developed a simple approach to study the chromatin structure of Arabidopsis telomeres independently of ITSs by analyzing ChIP-seq data. This approach could be used to study the chromatin structure of telomeres in some other eukaryotes. The analysis of ChIP-seq experiments revealed that Arabidopsis telomeres have higher density of histone H3 than centromeres, which might reflects their short nucleosomal organization. These experiments also revealed that Arabidopsis telomeres have lower levels of heterochromatic marks than centromeres (H3K9(Me2) and H3K27(Me)), higher levels of some euchromatic marks (H3K4(Me2) and H3K9Ac) and similar or lower levels of other euchromatic marks (H3K4(Me3), H3K36(Me2), H3K36(Me3) and H3K18Ac). Interestingly, the ChIP-seq experiments also revealed that Arabidopsis telomeres exhibit high levels of H3K27(Me3), a repressive mark that associates with many euchromatic genes. The epigenetic profile of Arabidopsis telomeres is closely related to the previously defined chromatin state 2. This chromatin state is found in 23% of Arabidopsis genes, many of which are repressed or lowly expressed. At least, in part, this scenario is similar in rice.
Asunto(s)
Cromatina/metabolismo , Epigénesis Genética , Telómero/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina/química , Inmunoprecipitación de Cromatina , Histonas/metabolismo , Oryza/genética , Oryza/metabolismo , Análisis de Secuencia de ADN , Telómero/químicaRESUMEN
Telomere function is influenced by chromatin structure and organization, which usually involves epigenetic modifications. We describe here the chromatin structure of Arabidopsis thaliana telomeres. Based on the study of six different epigenetic marks we show that Arabidopsis telomeres exhibit euchromatic features. In contrast, subtelomeric regions and telomeric sequences present at interstitial chromosomal loci are heterochromatic. Histone methyltransferases and the chromatin remodeling protein DDM1 control subtelomeric heterochromatin formation. Whereas histone methyltransferases are required for histone H3K9(2Me) and non-CpG DNA methylation, DDM1 directs CpG methylation but not H3K9(2Me) or non-CpG methylation. These results argue that both kinds of proteins participate in different pathways to reinforce subtelomeric heterochromatin formation.
Asunto(s)
Arabidopsis/genética , Eucromatina/química , Telómero/química , Arabidopsis/enzimología , Proteínas de Arabidopsis/fisiología , Metilación de ADN , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/fisiología , Epigénesis Genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The functions of telomeres and, probably, of interstitial telomeric sequences (ITSs) are influenced by their chromatin structure and organization. Telomeres in higher eukaryotes fold into nucleosomes that are about 20-40 bp shorter than the nucleosomes associated with bulk chromatin. Although the functional relevance of this short nucleosomal organization remains unknown, it is believed that short nucleosomes should contribute to telomere function. Whereas telomeric nucleosomes have been widely studied in different organisms, very little is known about the nucleosomal organization of ITSs. Chinese hamster ITSs have been found to associate with short nucleosomes. However, we have found that Arabidopsis thaliana ITSs fold into nucleosomes that have a repeat length similar to bulk chromatin. We discuss how the primary sequence of telomeres and ITSs could influence their nucleosomal organization.
Asunto(s)
Nucleosomas/metabolismo , Telómero/metabolismo , Animales , Arabidopsis/genética , Secuencia de Bases , Ensamble y Desensamble de Cromatina , Cromosomas de las Plantas/química , Cromosomas de las Plantas/metabolismo , Cricetinae , Cricetulus , ADN Intergénico/genética , Nucleosomas/química , Secuencias Repetitivas de Ácidos Nucleicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Telómero/química , Telómero/genéticaRESUMEN
Majerová et al. (Plant Mol Biol, 2011) have recently reported that a considerable fraction of cytosines at tobacco telomeres is methylated. Although the data presented in this report indicate that tobacco telomeric sequences undergo certain levels of DNA methylation, it is not clear whether the methylated sequences are at telomeres, at internal chromosomal loci or at both.
Asunto(s)
Adenina/análogos & derivados , Citidina/análogos & derivados , ADN de Plantas/efectos de los fármacos , Nicotiana/efectos de los fármacos , Telomerasa/metabolismo , Telómero/efectos de los fármacosRESUMEN
The epigenetic modifications of human telomeres play a relevant role in telomere functions and cell proliferation. Therefore, their study is becoming an issue of major interest. These epigenetic modifications are usually analyzed by microscopy or by chromatin immunoprecipitation (ChIP). However, these analyses could be challenged by subtelomeres and/or interstitial telomeric sequences (ITSs). Whereas telomeres and subtelomeres cannot be differentiated by microscopy techniques, telomeres and ITSs might not be differentiated in ChIP analyses. In addition, ChIP analyses of telomeres should be properly controlled. Hence, studies focusing on the epigenetic features of human telomeres have to be carefully designed and interpreted. Here, we present a comprehensive discussion on how subtelomeres and ITSs might influence studies of human telomere epigenetics. We specially focus on the influence of ITSs and some experimental aspects of the ChIP technique on ChIP analyses. In addition, we propose a specific pipeline to accurately perform these studies. This pipeline is very simple and can be applied to a wide variety of cells, including cancer cells. Since the epigenetic status of telomeres could influence cancer cells proliferation, this pipeline might help design precise epigenetic treatments for specific cancer types.
Asunto(s)
Epigénesis Genética/genética , Epigenómica/métodos , Telómero/genética , Cromatina/genética , Inmunoprecipitación de Cromatina/métodos , Epigénesis Genética/fisiología , Histonas/genética , Humanos , Microscopía/métodos , Telómero/metabolismo , Telómero/fisiologíaRESUMEN
In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length.
Asunto(s)
Arabidopsis/genética , Cromosomas de las Plantas/genética , Reacción en Cadena de la Polimerasa/métodos , Homeostasis del Telómero/genética , Telómero/genética , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Two different groups, using ChIP-seq data, have recently published the genome-wide distribution of histones H3.1 and H3.3 in Arabidopsis thaliana. In one report, Stroud and colleagues determined that, whereas H3.1 was enriched in repetitive pericentromeric and silent chromatin, H3.3 was enriched in transcriptionally active regions. This work was performed using seedlings, which contained dividing and non-dividing cells. In a second report, Wollmann and colleagues found similar results analyzing dividing or non-dividing tissue. None of these reports addressed the analysis of telomeres or centromeres. Our group has recently described an experimental approach that allows the study of the epigenetic status of some Arabidopsis repetitive sequences by analyzing ChIP-seq data. By using this approach and the data generated by Stroud, Wollmann and colleagues, we found that telomeres are enriched in H3.3 with regard to the centromeric 178â bp repeats, whereas the centromeric repeats are enriched in H3.1 with regard to telomeres.
Asunto(s)
Arabidopsis/genética , Centrómero/metabolismo , Histonas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Telómero/metabolismo , Arabidopsis/metabolismo , Secuencia de Bases , Cromatina/metabolismo , Variación Genética , Histonas/genética , Transcripción GenéticaRESUMEN
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.