DNA methylation at tobacco telomeric sequences

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

Human Long Telomeres and Epigenetic Marks

We have read with interest the article “Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and HP1α” by Arnoult and colleagues [1]. This study focuses on human telomeric chromatin structure using different techniques like Chromatin Immunoprecipitation (ChIP), cytolocalization or RT-qPCR. However, it has been performed without taking into consideration the presence of Interstitial Telomeric Sequences (ITSs) in the human genome. Some of the conclusions of the article are undoubtedly clear but there are others that might be explained in alternative ways, considering the existence of ITSs. Following, we mention some comments that arise from this interesting article

Assessing the Epigenetic Status of Human Telomeres

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.Spanish Agency of ResearchEuropean Fund for Regional Development European Union BIO2016-78955-

The restriction endonuclease Tru9I is a useful tool to analyze telomere sequences separately from interstitial telomeric sequences in Arabidopsis thaliana

We have previously described a simple method to analyze the chromatin structure of Arabidopsis te- lomeres independently of that of Interstitial Telome- ric Sequences (ITSs). By using this method, we found that, whereas ITSs are heterochromatic, Arabidopsis telomeres exhibit euchromatic features [1]. Some con- cerns have been recently raised about the accuracy of this procedure [2]. Here, we summarize these concerns and justify our experimental approaches and inter- pretation of results.Ministerio de Ciencia e Innovación BIO2011-2479

Determination of Arabidopsis thaliana telomere length by PCR

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.España Ministerio de Economía y Competitividad Grant BIO2011-2479

Analysis of the epigenetic status of telomeres by using ChIP-seq data

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 (H3K9Me2 and H3K27 Me), higher levels of some euchromatic marks (H3K4Me2 and H3K9Ac) and similar or lower levels of other euchromatic marks (H3K4 Me3, H3K36Me2, H3K36Me3 and H3K18Ac). Interestingly, the ChIP-seq experiments also revealed that Arabidopsis telomeres exhibit high levels of H3K27Me3, 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.Ministerio de Educación y Ciencia BFU2008-02497/BM

Conditional Expression of RNase P in the CyanobacteriumSynechocystis sp. PCC6803 Allows Detection of Precursor RNAs

We have constructed a strain (CT1) that expresses RNase P conditionally with the aim to analyze the in vivotRNA processing pathway and the biological role that RNase P plays inSynechocystis 6803. In this strain, the rnpBgene, coding for the RNA subunit of RNase P, has been placed under the control of the petJ gene promoter (PpetJ), which is repressed by copper, cell growth, and accumulation of RNase P RNA is inhibited in CT1 after the addition of copper, indicating that the regulation by copper is maintained in the chimerical PpetJ -rnpB gene and that RNase P is essential for growth in Synechocystis. We have analyzed several RNAs by Northern blot and primer extension in CT1. Upon addition of copper to the culture medium, precursors of the mature tRNAs are detected. Furthermore, our results indicate that there is a preferred order in the action of RNase P when it processes a dimeric tRNA precursor. The precursors detected are 3′-processed, indicating that 3′ processing can occur before 5′ processing by RNase P. The size of the precursors suggests that the terminal CCA sequence is already present before RNase P processing. We have also analyzed other potential RNase P substrates, such as the precursors of tmRNA and 4.5 S RNA. In both cases, accumulation of larger than mature size RNAs is observed after transferring the cells to a copper-containing medium

Differential association of Arabidopsis telomeres and centromeres with histone H3 variants

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 178 bp repeats, whereas the centromeric repeats are enriched in H3.1 with regard to telomeres