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ABSTRACT We have read with interest thearticle “Telomere length regulates TERRA levels through increasedtrimethylation of telomeric H3K9 and HP1α” by Arnoult and colleagues [1]. Thisstudy focuses on human telomeric chromatin structure using different techniqueslike Chromatin Immunoprecipitation (ChIP), cytolocalization or RT-qPCR.However, it has been performed without taking into consideration the presenceof Interstitial Telomeric Sequences (ITSs) in the human genome. Some of the conclusionsof the article are undoubtedly clear but there are others that might beexplained in alternative ways, considering the existence of ITSs. Following, wemention some comments that arise from this interesting article. Keywords:Telomeres; TERRA; Epigenetics; Human 1. Introduction First, we would like topoint out that there is a misunderstanding in the introduction section, whenthe authors comment on Telomere Position Effect (TPE) in Saccharomycescerevisiae. Native TPE in Saccharomyces cerevisiae was first discovered in 1997[2] and not in 1999 [3,4], as Arnoult and colleagues suggested. The 1997publication showed that the yeast retrotransposon Ty5-1 was repressed by thetelomeric SIR silencing complex, which constitutes one of the earliest demonstrationof mobile elements heterochromatic regulation in eukaryotes based on theanalysis of heterochromatin mutants [2,5]. Secondly, referring to thewell-performed experiments presented by Arnoult and colleagues, we would liketo mention that humans, as many other organisms, contain ITSs. In Arabidopisthaliana, ITSs, as an average, exhibit heterochromatic features and have beenshown to be transcribed [6,7]. Similarly, human ITSs are most probablytranscribed, at least at certain levels, and are bound by TRF2 [8-11].Therefore, human TERRA transcripts might be generated from telomeres and fromITSs. Arnoult and colleagues demonstrated that the increase of telomere length,by means of ectopic expression of either the hTERT catalytic subunit oftelomerase or both, hTERT and hTR subunits, caused a reduction of telomericTERRA levels. In turn, the telomeric TERRA transcripts contained more telomericsequences. They showed that the reduction of telomeric TERRA transcripts wascoincident with an increase of TRF2 loading at the longer telomeres. Inaddition, they ascribed the lower levels of telomeric TERRA transcripts to anincrease of heterochromatic marks at telomeres (H3K9me3 and HP1α) anddemonstrated that the levels of telomeric TERRA, of the association of H3K9me3with telomeric sequences and of the association of HP1α with TRF2 correlatethrough the cell cycle (Figure 7 [1]). All these conclusions were essentiallybased on 1) the study of telomeric TERRA levels by RT-qPCR of subtelomericregions in different cell types, including cells containing siRNAs for SUV39H1or HP1α, 2) ChIP experiments using antibodies against H3K9me3 followed bydot-blot hybridization with a telomeric probe or by subtelomeric qPCR, 3) thecytolocalization of TERRA transcripts, TRF2 and HP1α and 4) the study of thecell cycle dependent cellular distribution of these marks. Following, wecomment some of the experiments shown by Arnoult and colleagues: 1) Arnoult andcolleagues demonstrated by RT-qPCR that the levels of subtelomeric transcriptsare lower in cells with elongated telomeres than in cells with normaltelomeres, which indicated that the levels of telomeric TERRA transcripts arelower in cells with elongated telomeres (Figures 1 and 2 [1]). 2) Arnoult andcolleagues showed by ChIP and qPCR that changes of telomeric TERRA levels incells with Copyright © 2013 SciRes. CellBio M. I. VAQUERO-SEDAS, M. A.VEGA-PALAS 71 longer telomeres cannot be attributed to changes at the level ofTERRA promoter chromatin (Supplementary Figure 3 [1]). 3) Arnoult andcolleagues also showed by Northernblot and hybridization with a telomeric probethat the levels of TERRA transcripts of similar size, including short RNAmolecules, are higher in cells with elongated telomeres than in normal cells,which, in principle, is in contradiction with the results obtained by RT-qPCRmentioned above (Figure 6a [1]). They explained this contradiction arguing thatthe Northern-blot should not be considered quantitative because a concentrationof 2.2 M of formaldehyde was used in the experiment (Figure 6a [1]). However,if the Northern-blot were considered quantitative, we should conclude thatTERRA molecules originnated from ITSs are more abundant in cells with elongatedtelomeres. 4) Arnoult and colleagues found by cytolocalization that the numberof TERRA and TRF2 foci in normal cells were very high, which indicates that aportion of them localize at internal loci (Figure 5b [1]). In addition, not allthe TERRA foci were found coincident with TRF2 in normal cells. In contrast,the major foci detected in cells with elongated telomeres were more intensethan in normal cells and contained both, TERRA and TRF2 (Figure 5b [1]). Theseresults are in agreement with their proposal that elongated telomeres containlonger telomeric TERRA transcripts with more telomeric sequences and more TRF2proteins than normal cells. In principle, TRF2 proteins should be sequesteredto the elongated telomeres from other genomic loci. Therefore, TRF2 moleculescould migrate from ITSs to telomeres in cells with elongated telomeres causinga change in the chromatin structure of ITSs. 5) Arnoult and colleaguesdemonstrated by cytolocalization that HP1α associates with some, but not all,TRF2 foci with very low intensity in cells with longer telomeres (Figure 7e[1]). They concluded from this result that longer telomeres were loaded withHP1α. However, the TRF2 foci associated with HP1α might also correspond toITSs. 6) Arnoult and colleagues also showed by ChIP and dot-blot hybridizationwith a telomeric probe that telomeric sequences in cells with elongatedtelomeres are enriched in H3K9me3 with regard to normal cells (Figure 3 [1]).They concluded from this result that longer telomeres are loaded with H3K9me3.However, this increase of H3K9me3 at telomeric sequences might also be due tothe loading of H3K9me3 at ITSs. 7) Arnoult and colleagues studied, by RT-qPCRof subtelomeric sequences, the levels of telomeric TERRA transcripts in normalcells and in cells with elongated telomeres after the introduction of SUV39H1or of HP1α siRNAs. The introduction of these siRNAs leaded to decreased levelsof H3K9me3 or HP1α in both kinds of cells. Arnoult and colleagues showed thatthe levels of telomeric TERRA transcripts in cells with elongated telomeres containingthese siRNAs were similar to those found in their parental normal cellscontaining the same siRNAs (Figure 4 [1]). They also showed that in some normalcells containing the siRNAs, the levels of TERRA transcripts at certaintelomeres were higher than in the same cells without the siRNAs (Figure 4b[1]). Therefore, H3K9me3 and HP1α are involved in the regulation of telomericTERRA levels in cells with elongated telomeres and might also regulate TERRAlevels in normal cells. In our opinion, further experiments should establishthat this regulation is exerted directly by binding of H3K9me3 and HP1α attelomeres and not indirectly by their binding at ITSs or even at other genomicloci. In summary, Arnoult and colleagues have elegantly demonstrated that elongatedtelomeres lead to lower levels of telomeric TERRA transcripts containing longertracts of telomeric sequences, and higher levels of telomeric TRF2. Theseconclusions are very robust. However, some of the experiments presented byArnoult and colleagues might be explained in alternative ways taking intoconsideration the presence of ITSs. For example, the increased number oftelomeric TRF2 molecules present at longer telomeres could be sequestered fromITSs, which, in turn, would lead to higher levels of TERRA transcripts fromITSs and to the recruitment of heterochromatic marks (H3K9me3 and HP1α) atITSs, and not at telomeres, in a cell cycle regulated manner. In this context,the lower levels of telomeric TERRA transcripts found at cells with elongatedtelomeres might be caused by alternative features. We do not favor theheterochromatinization of long telomeres versus ITSs or vice versa. However, webelieve that the influence of ITSs should be ruled out in telomeric chromatinstructure studies to strongly support conclusions [6]. Two different approacheshave been designed to study the chromatin structure of telomeres independentlyof ITSs in another model system [12-14]. These approaches could be adapted toother organisms including humans [12,15]. REFERENCES [1] N. Arnoult, A. VanBeneden and A. Decottignies, “Telomere Length Regulates TERRA Levels throughIncreased Trimethylation of Telomeric H3K9 and HP1α,” Nature Structural &Molecular Biology, Vol. 19, 2012, pp. 948-957 [2] M. Vega-Palas, S. Vendittiand E. Di Mauro, “Telomeric Transcriptional Silencing in a Natural Context,”Nature Genetics, Vol. 15, 1997, pp. 232-233. doi:10.1038/ng0397-232 [3] G.Fourel, E. Revardel, C. Koering and E. Gilson, “CohaCopyright © 2013 SciRes.CellBio M. I. VAQUERO-SEDAS, M. A. VEGA-PALAS Copyright © 2013 SciRes. CellBio72 bitation of Insulators and Silencing Elements in Yeast SubtelomericRegions,” EMBO Journal, Vol. 18, 1999, pp. 2522-2537.doi:10.1093/emboj/18.9.2522 [4] F. Pryde and E. Louis, “Limitations of Silencingat Native Yeast Telomeres,” EMBO Journal, Vol. 18, 1999, pp. 2538-2550.doi:10.1093/emboj/18.9.2538 [5] M. Bryk, M. Banerjee, M. Murphy, K. Knudsen, K.Garfinkel and M. Curcio, “Transcriptional Silencing of Ty1 Elements in the RDN1Locus of Yeast,” Genes and Development, Vol. 11, 1997, pp. 255-269.doi:10.1101/gad.11.2.255 [6] M. Vaquero-Sedas and M. Vega-Palas, “On theChromatin Structure of Eukaryotic Telomeres,” Epigenetics, Vol. 6, No. 9, 2011,pp. 1055-1058. doi:10.4161/epi.6.9.16845 [7] J. Vrbsky, S. Akimcheva, J.Watson, T. Turner, L. Daxinger, B. Vyskot, W. Aufsatz and K. Riha,“siRNA-Mediated Methylation of Arabidopsis Telomeres,” PLoS Genetics, Vol. 6,No. 6, 2010, e1000986. doi:10.1371/journal.pgen.1000986 [8] C. Azzalin, P.Reichenbach, L. Khoriauli, E. Giulotto and J. Lingner, “Telomeric RepeatContaining RNA and RNA Surveillance Factors at Mammalian Chromosome Ends,”Science, Vol. 318, 2007, pp. 798-801. [9] S. Feuerhahn, N. Iglesias, A. Panza,A. Porro and J. Lingner, “TERRA Biogenesis, Turnover and Implications forFunction,” FEBS Letters, Vol. 584, No. 17, 2010, pp. 3812-3818.doi:10.1016/j.febslet.2010.07.032 [10] D. Yang, Y. Xiong, H. Kim, Q. He, Y. Li,R. Chen and Z. Songyang, “Human Telomeric Proteins Occupy Selective InterstitialSites,” Cell Research, Vol. 21, 2011, pp. 1013- 1027. doi:10.1038/cr.2011.39[11] T. Simonet, L. E. Zaragosi, C. Philippe, K. Lebrigand, C. Schouteden, A.Augereau, S. Bauwens, J. Ye, M. Santagostino, E. Giulotto, F. Magdinier, B.Horard, P. Barbry, R. Waldmann and E. Gilson, “The Human TTAGGG Repeat Factors1 and 2 Bind to a Subset of Interstitial Telomeric Sequences and SatelliteRepeats,” Cell Research, Vol. 21, 2011, pp. 1028-1038. doi:10.1038/cr.2011.40[12] M. Vaquero-Sedas, F. Gámez-Arjona and M. Vega-Palas, “Arabidopsis thalianaTelomeres Exhibit Euchromatic Features,” Nucleic Acids Research, Vol. 39, 2011,pp. 2007-2017. doi:10.1093/nar/gkq1119 [13] M. Vaquero-Sedas, C. Luo and M.Vega-Palas, “Analysis of the Epigenetic Status of Telomeres by Using ChIP-seqData,” Nucleic Acids Research, Vol. 40, 2012, e163. doi:10.1093/nar/gks730 [14]M. Vaquero-Sedas and M. Vega-Palas, “Differential Association of Arabidopsistelomeres and Centromeres with Histone H3 Variants,” Scientific Reports, Vol.3, 2013, 1202. doi:10.1038/srep01202 [15] M. Vega-Palas and M. Vaquero-Sedas,“Integrating Telomeres into Genome-Wide ChIP-seq Analyses,” Protocol Exchange,2013. doi:10.1038/protex.2013.039
