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

Abstract

We have previously described a simple method to analyze the chromatin structure of Arabidopsis telomeres independently of that of Interstitial Telomeric Sequences (ITSs). By using this method, we found that, whereas ITSs are heterochromatic, Arabidopsis telomeres exhibit euchromatic features [1]. Some concerns have been recently raised about the accuracy of this procedure [2]. Here, we summarize these concerns and justify our experimental approaches and interpretation of results. Keywords: Arabidopsis thaliana; Telomeres; ITSs; Euchromatin; HeterochromatinIt is important to differentiate between telomeres and ITSs when the chromatin structure of telomeres is analyzed by ChIP and hybridization with a telomeric probe [1,3]. We have previously addressed this problem in Arabidopsis by using the frequently cutting restriction enzyme Tru9I [1,4]. Since Arabidopsis telomeres are composed of perfect telomeric repeat arrays, they remain uncut after digestion with Tru9I. In contrast, ITSs are frequently cut because they are composed of very short arrays of perfect telomeric repeats interspersed with degenerated repeats [4][5][6][7]. When Arabidopsis genomic DNA is digested with Tru9I, resolved on an agarose gel and hybridized with a telomeric probe, most of the signals corresponding to ITSs disappear. Only three ITSs bands smaller than 500 bp remain. Therefore, the signals detected above 500 bp after Tru9I digestion correspond only to telomeres. In turn, the signals detected above 500 bp when the DNA is undigested correspond to both, telomeres and ITSs [4]. This observation led us to study the chromatin structure of Arabidopsis telomeres and ITSs independently. After performing ChIP experiments, we amplified the input and the immunoprecipitated DNA samples following a whole genome amplification proto- Our experimental analyses undoubtedly showed that most of the signals obtained after hybridizing Arabidopsis genomic DNA with a telomeric probe corresponded to ITSs [4]. This conclusion was supported by a micrococcal nuclease digestion analysis of the Arabidopsis genome [4]: after digesting Arabidopsis chromatin with micrococcal nuclease, resolving the resulting DNA samples in an agarose gel and hybridizing them with a telomeric probe, the nucleosome ladder displayed revealed a nucleosomal spacing similar to that of bulk chromatin [4]. If the assumption made by Majerová and colleagues were true, the nucleosomal spacing revealed by the telomeric probe should have been shorter because, in all eukaryotic systems analyzed, telomeres are known to fold into shorter nucleosomes than bulk chromatin [8].2) We have previously reported that ITSs show heterochromatic structure while telomeres exhibit euchromatic features [1]. We analyzed histone marks and DNA methylation of ITSs and telomeres independently, using a technique based on a protocol described by Lippman et al. [9]. This protocol starts with random fragmentation of cross-linked chromatin and is followed by immunoprecipitation using an appropriate antibody. Then, the ...

Determination of Arabidopsis thaliana telomere length by PCR

“…These discrepancies should arise from the different technologies that we have used to analyze telomeric chromatin structure. We have discussed these technologies elsewhere 34 36 37 38 . Currently, we believe that DNA methylation is essentially absent from Arabidopsis telomeres (our unpublished results).…”

Frequently cutting restriction enzymes: Clearing the fog to see the ends

“…Although some concerns have been raised about this experimental procedure by Majerová and colleagues [21], these concerns are unjustified [22]. The results obtained using the Tru9I approach have been confirmed with the second technique mentioned above, which does not use frequently cutting restriction enzymes.…”