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The Human TTAGGG Repeat Factors 1 and 2 Bind to a Subset of Interstitial Telomeric Sequences and Satellite Repeats

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The Human TTAGGG Repeat Factors 1 and 2 Bind to a Subset of Interstitial Telomeric Sequences and Satellite Repeats npg Extratelomeric binding of TRF1 and TRF2 Cell Research (2011) 21:1028-1038. 1028 © 2011 IBCB, SIBS, CAS All rights reserved 1001-0602/11 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats Thomas Simonet1, Laure-Emmanuelle Zaragosi2, Claude Philippe3, Kevin Lebrigand2, Clémentine Schouteden1, Adeline Augereau1, 3, Serge Bauwens1, Jing Ye1, 3, Marco Santagostino4, Elena Giulotto4, Frederique Magdinier1, Béatrice Horard1, Pascal Barbry2, Rainer Waldmann2, Eric Gilson1, 3, 5 1Laboratoire de Biologie Moléculaire de la Cellule-UMR 5239 CNRS/ENS Lyon/ Université Lyon, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, Lyon 69364, France; 2CNRS and University of Nice Sophia Antipolis, Institut de Pharmacologie Molé- culaire et Cellulaire, 06560 Sophia Antipolis, France; 3Laboratory of Biology and Pathology of Genomes of University of Nice Sophia-Antipolis, CNRS UMR6267/INSERM U998, Faculty of Medicine, Nice, France; 4Dipartimento di Genetica e Microbiologia Adriano Buzzati-Traverso, Università di Pavia, Pavia, Italy; 5Department of Medical Genetics, CHU of Nice, Nice, France The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mech- anisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian te- lomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these ex- tratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences. Keywords: telomere; TRF1; TRF2; interstitial telomeric sequence; satellite DNA Cell Research (2011) 21:1028-1038. doi:10.1038/cr.2011.40; published online 22 March 2011 Introduction repetitions of a small G-rich motif (TTAGGG in mam- mals) and the presence of a single-stranded tail on the The paramount importance of telomeres to cell fate 3′-oriented strand (G tail). Telomeric DNA is transcribed likely stems from the great diversity in the functions into a UUAGGG repeat-containing RNA called TERRA, they perform [1, 2]. They control the replication of chro- which is believed to play fundamental roles in telomere mosomal DNA termini, protect chromosome ends from biology [4, 5]. DNA repair and checkpoint activation, control the mei- A key component of the mammalian telomere is the otic spindle, localize the chromosome ends within the shelterin complex, which is composed of six polypep- nuclear space and regulate long-range chromatin changes tides: TRF1, TRF2, Rap1, Tin2, TPP1 and Pot1 [5]. Of as well as gene expression. Telomeres consist of specific these, three bind specifically to TTAGGG repeats: TRF1 nucleoprotein complexes [3]. Telomeric DNA has sev- and TRF2, which recognize the duplex DNA, and Pot1, eral distinctive features, including a sequence formed by which binds to the single-stranded 3′ overhangs [3, 6]. TRF1 and TRF2 do not exist in budding yeast. Instead, yeast Rap1 acts as an essential capping factor that binds Correspondence: Eric Gilson to telomeric DNA, while yeast Cdc13 binds to the 3′ Tel: +33-472728453; Fax: +33-472728080 E-mail: [email protected] overhang and seems to perform functions that are similar Received 3 November 2010; revised 9 January 2011; accepted 11 January to those of Pot1 and TPP1 [7]. 2011; published online 22 March 2011 Telomeres in yeast and mammals can silence neigh- Cell Research | Vol 21 No 7 | July 2011 Thomas Simonet et al. npg 1029 boring genes by exerting telomeric position effect (or neuronal functions [21]. TPE) [8-10]. TPE is influenced by telomere length and In this study, we mapped the human chromosomal structure as well as by chromatin-remodeling machiner- sites to which TRF1 and TRF2 bind by combining chro- ies [11]. Telomeric and subtelomeric chromatin differ matin immunoprecipitation with high-throughput DNA from constitutive heterochromatin in terms of structure sequencing (ChIP-Seq). and dynamics, specificity of DNA sequences, and bind- ing of specific factors [12]. The mechanisms that initiate Results the formation of heterochromatin at telomeres are un- known but likely involve the binding of specific factors Identification of TRF binding sites by ChIP-Seq analysis to telomeric DNA. For instance, the N-terminal part of To establish global binding profiles of TRF1 and TRF2 TRF2 may facilitate heterochromatin formation by bind- (collectively named the TRF proteins), we performed a ing to ORC1 and TERRA [13]. ChIP-Seq analysis with one antibody specific for TRF1 Repetitions of the TTAGGG telomeric unit, called and two antibodies specific for TRF2 (one monoclonal interstitial telomeric sequences, or ITSs, are also present or TRF2m, one polyclonal or TRF2p). We used the BJ- within chromosomes [14]. In humans, three classes of HELTRasmc tumor cell line because TRF2 is required for ITSs were identified [15]: (i) subtelomeric ITSs, located tumorigenicity through a pathway that involves uncou- within subtelomeric domains and composed of extended pling of telomere protection and the DNA damage re- arrays (usually several hundreds of base pairs), including sponse mechanism, suggesting a role for extratelomeric many degenerate units; they probably arose from recom- TRF2 binding sites in oncogenesis (Biroccio et al, sub- bination events involving chromosome termini [16]; (ii) mitted). The specificity of the anti-TRF1 and anti-TRF2 short internal ITSs, located away from telomeres and antibodies was confirmed by slot blot analysis (Figure composed of relatively few TTAGGG units; these ITSs 1A). We found up to 50-fold enrichment of telomeric are likely to have been generated during the repair of sequences in the TRF antibody-immunoprecipitated sam- DNA double-strand breaks that occurred during evolu- ples when compared to Protein G-Sepharose-precipitated tion [17]; (iii) one fusion ITS, located in 2q14, derived control samples and total histone H3-immunoprecipi- from the end fusion between the two ancestral chromo- tation. This result was confirmed by the analysis of the somes that gave rise to human chromosome 2 [18]. No ChIP-Seq reads. In TRF antibody-immunoprecipitated clear indication of any particular function of ITSs has samples we detected 90 to 150 times more sequences that been provided so far. contain solely the (TTAGGG)n motif than in the control Emerging evidence indicates that the shelterin com- samples (Figure 1B). ponents have non-telomeric functions in DNA repair To identify extratelomeric binding sites for the TRF [19], Epstein-Barr virus replication [20], transcriptional proteins, we retained only reads that were uniquely regulation [21] and NF-κB activation [22]. These non- aligned on the 2006 Human genome assembly (NCBI36/ telomeric functions might be, at least partially, explained hg18), and we checked that pure (TTAGGG)n reads, by their binding to ITSs. Indeed, there is mounting indi- which likely originate from telomeric DNA, had been cation that shelterin components can bind to interstitial indeed completely discarded. Significantly read-enriched DNA sequences: (i) TRF1 and TRF2 bind to the peri- positions or peaks were identified using the SISSR centromeric regions of hamster chromosomes containing software [28] with a P value threshold of 0.001 using large blocks of ITSs [23, 24]; (ii) TRF2 and TIN2 bind protein G immunoprecipitation as background. We fur- to an ITS formed by a rare human chromosome rear- ther removed the seemingly artifactual (non-specific) rangement [25]; (iii) TRF2 binds to a stretch of telomeric peaks through a visual inspection of a density profile of sequence that is artificially inserted in the middle of the matched reads (see the example for chromosome 1, the long arm of chromosome 4 [26]; (iv) Rap1 binds to shown in Supplementary information, Figure S1). Fol- several ITSs of the mouse genome [27]. However, three lowing this filtering, we identified 68 peaks present in naturally occurring ITSs of human chromosomes do not all three TRF ChIP-Seq samples (TRF1, TRF2m and appear to be bound by TRF2 [26]. Therefore, it is still TRF2p) (Figure 2A). Results for chromosome 1 are unclear whether TRF1 and TRF2 really bind to the ITSs shown in Figure 2B and those for other chromosomes are normally found in human chromosomes or even to unre- shown in Supplementary information, Figure S2. lated sequences. Moreover, there is evidence that TRF2 Notably, 18 peaks from the TRF2m ChIP (among modulates gene expression outside from telomeres since n = 90, 20%) were not found using TRF2p antibody, it interacts with the repressor element 1-silencing tran- while 21 peaks identified by the TRF2p ChIP (n = 93, scription factor (REST), a repressor of genes devoted to 22.5%) were not present in TRF2m ChIP. For most of www.cell-research.com | Cell Research npg Extratelomeric binding of TRF1 and TRF2 1030 Figure 1 (A) Slot blot showing the telomeric enrichment of DNA immunoprecipitated with anti-TRF1 or TRF2 antibodies. DNA immunoprecipitated by a total H3 antibody and pulled down by protein G alone was used as a control. Half of the precipitated DNA was loaded, along with an input scale (2 500 ng to 10 ng, corresponding to 10% to 0.04% of the total input), and hybrid- ized sequentially to a telomeric probe and a genomic probe.
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