[Cell Cycle 8:7, 1099-1100; 1 April 2009]; ©2009 Landes Bioscience

à L’Energie Atomique; DSV-LRO; Fontenay aux Roses, France; de Physique Théorique de la Matière Condensée; Université Pierre et Marie Curie; Paris, France 2Laboratoire

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Abbreviations: CHO, chinese hamster ovary; ITS, interstitial telomeric sequence; NRL, nucleosome repeat length; MNase, micrococcal nuclease; (k)bp, (kilo) base pair

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Key words: chromatin structure, nucleosome spacing, telomere, interstitial telomeric sequence, radiosensitivity

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Déborah Revaud,1 Julien Mozziconacci,2 Laure Sabatier,1 Chantal Desmaze1,† and Christophe Lavelle1,†,*

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Sequence-driven telomeric chromatin structure

NRL in CHO cells has been measured by nuclease digestion followed by mobility assay on agarose gel (Fig. 1B and C, and Suppl. Fig. 1A and B). Mean NRL was estimated from the length/number ratio of the bands. We found ~187 bp (+/-5 bp) for bulk chromatin. Note that this value is slightly above reported values from older studies (see for instance Rill et al.10), the difference probably being due to the various digestion conditions (times and amount of nuclease per DNA). Indeed, it was shown that NRL measurement in CHO cells can vary from ~185 bp to ~175 bp during the course of the digestion (whose intensity was assessed by the final amount of acid soluble DNA).11 Remarkably, a striking difference was seen upon measurement on the same gel after blotting and hybridization by a telomeric (TTAGGG) probe: ITS from CHO cells have a ~20 bp shorter NRL than bulk chromatin, giving a ~167 bp (+/-5 bp) repeat length, slightly above the ~157 bp value already reported for rat telomere.6 Note here that, due to the overwhelming amount of interstitial telomeric DNA (usually several hundreds of kbp per chromosome) compared to true telomeric DNA (a few kbp at the ends of each chromosome), the signal observed after hybridization can reasonably be entirely attributed to ITS. (Note also that, for the same reason, ITS but no telomeres are visible in Figure 1A, confirming the dominating signal of ITS). Interestingly, in the study already discussed above,11 d’Anna and Tobey made the same measurements by using as a probe what was merely qualified in their paper as an hexameric repeated sequence known to hybridize both with telomeric and centromeric regions of some CHO chromosomes;12 this probe has since been recognized as the consensual telomeric repeat in higher eukaryotes. The result they obtained (NRL ~165 bp) is in very good agreement with our result, confirming the robustness of chromatin conformation with respect to the sequence of wrapped DNA. The same results were obtained with chinese hamster primary cells and lung fibroblasts derivative cells (data not shown), confirming the true difference in nucleosomal occupancy, independently of the cell type, between telomeric sequences and the rest of the chromosome. Another remarkable feature of ITS chromatin is its high regularity, as seen from the number of bands visible in Figure 1B and C (10 bands can be clearly visualized after Southern blotting whereas only 5 are seen in gel); this has also formerly been observed for telomeres.6 Sedimentation analysis of DNA extracted from various sucrose gradient fractions was then used to assess the physical properties of ITS chromatin (Suppl. Fig. 2). The same kind of analysis was formerly carried out to compare centromeric versus bulk chromatin structure of mouse fibroblasts: in this study, the difference in sedimentation coefficient of centromeric versus bulk chromatin suggested that the higher regularity of centromeric chromatin fibers allowed a better compaction.13 Here we show that bulk chromatin sediments faster than ITS chromatin. Indeed, bands corresponding to various sucrose fractions show no shift between BET (bulk) and blot (ITS), which means bulk and ITS DNA fragments of the same length migrate together despite their difference in nucleosome occupancy. In other words: a ~20 nucleosome long fiber, for instance, would sediment within the 9th fraction for bulk chromatin whereas it would be found in the 8th fraction for ITS chromatin. Hence, densely nucleosome-covered ITS fibers must be less compact, thus migrating with the less heavy but more compact bulk fibers. These results are compatible with chromatin fiber structures obtained in vitro using regular positioning nucleosomal arrays and linker histones.14,15 167 bp NRL oligomers were indeed shown to

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Letter to the Editor

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Interstitial (also called internal or intrachromosomal) telomeric sequences (ITS) are found in many organisms.1 In hamsters, CHO cells show long (up to several Mbp) ITS2 (Fig. 1A) which are over involved in spontaneous or radiation induced chromosome aberrations.3 ITS are also found in human, but they are much shorter (several hundreds of bp maximum) and show no radiosensitivity.4 We report here the striking observation that interstitial telomeric chromatin, although located near centromeres in hamster cells, shares common structural features with truly telomeric chromatin (located at the end of chromosomes), and notably a higher nucleosome density than bulk chromatin, which correlates with a highly regular chromatin structure. This study emphasizes the critical role of DNA in establishing particular chromatin folding, confirming that the DNA sequence encodes important information concerning local chromatin properties.

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Nucleosomal density in chromosomes is indicated by the nucleosome repeat length (NRL), which represents the mean periodicity (in bp) at which nucleosomes are found. The most common NRL value is ~200 bp, but remarkable variations have been reported.5 Differences occur between different organisms, different cell types in the same organism, or at different loci in the same cell type. With respect to this latter, the clearest example concerns telomeres, in which the NRL is systematically reduced by ~20 bp compared to the mean NRL from bulk DNA in the same cell type.6,7 This has led several authors to propose a specific chromatin structures at telomeres.8,9

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*Correspondence to: Christophe Lavelle; Interdisciplinary Research Institute; CNRS—USR 3078; 50 Av Halley; Parc Scientifique de la Haute Borne; Villeneuve d’Ascq 59655 France; Tel.: 33.6.24.71.44.03; Email: christophe.lavelle@iri. univ-lille1.fr Submitted: 02/04/09; Accepted: 02/04/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8081

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fold in ~2 times longer fibers than 187 bp NRL oligomers (6 nuc/11 nm versus 11 nuc/11 nm). Even though the diameter of these less compact fibers is smaller, the increase in the fiber length when switching from 167 to 187 bp NRL could explain the slower sedimentation rate observed here. Chromatin folding and compaction generally depends on linker histone incorporation, competing with the binding of various non-histone proteins. These orchestrated interactions, directed by the relative affinity of the various proteins for the local DNA sequence, could define a particular NRL which would in turn allow for a unique chromatin folding around this specific region.16 Interestingly, in a study following the NRL measurement on synchronized CHO cells mentioned above, d’Anna and Tobey further showed that this bulk NRL was reduced upon H1 destabilization.17 In a less “dynamic” fashion, one can thus speculate that specific chromatin folding may be stabilized in ITS by the interaction of telomerespecific binding factors (TRF1/2) that would compete with linker histone, hence establishing this striking short-NRL chromatin structure in Figure 1. Structural analysis of chromatin from chinese hamster ovary cells. (A) Telomere PNA FISH all telomeric sequence. The fixation of TRF1 in on CHO cells. (a) Overview of metaphase spreads and interphasic nuclei; ITS are in red. (b) Zoom CHO ITS has indeed been reported18 as has on chromosome X (see white box in a). (B) Gel analysis of chromatin digestion by MNase. (a) The the case for TRF2 in human ITS.19 gel is first revealed by BET (left) and then transferred to a membrane and hybridized (Southern From a functional point of view, the fact blotting) with 32P (TTAGGG)10 oligo (right); (i and i’) DNA ladder (1 kbp ladder from Promega; that short ITS show no radiosensitivity4 would see Suppl. Fig. 3), (ii and ii’) MNase digestions. (C) Density profiles of lanes (i/i’, ii and ii’); note imply that long stretches of repeat sequences how much clearer the signal is after hybridization, showing that ITS form a regular chromatin are required to allow the formation of a given structure (compared to the smeared BET signal due to structural polymorphism in bulk chromatin). chromatin folding in a locus. This structure, (D) Proposed models of chromatin fiber taking into account the ~167 bp (ITS) and ~187 bp (bulk) NRL estimated from gel measurements (see Suppl. Fig. 1). less compact (due to short NRL) would make ITS loci more sensitive to radiation, as was formerly measured for X-rays20 or γ-rays.21 In this sense, CHO ITS References have been suggested to exhibit a particular chromatin structure 1. Lin KW, et al. Mutat Res 2008; 658:95-110. enriched in short unpaired DNA segments, potentially affecting DNA 2. Faravelli M, et al. Gene 2002; 283:11-6. repair process in these regions.22 3. Alvarez L, et al. Genes Chromosomes Cancer 1993; 8:8-14. Despite their common feature regarding reduced nucleosome 4. Desmaze C, et al. Cytogenet Genome Res 2004; 104:123-30. spacing, “true” telomeric and interstitial telomeric chromatin may still 5. Kornberg RD. Annu Rev Biochem 1977; 46:931-54. retain specific organizational properties,23 in particular due to the 6. Makarov VL, et al. Cell 1993; 73:775-87. 7. Tommerup H, et al. Mol Cell Biol 1994; 14:5777-85. simple fact that true telomeres must deal with a free DNA end, poten- 8. Besker N, et al. FEBS Lett 2003; 554:369-72. tially by T-loop formation.24 It is also possible that the chromosomal 9. Fajkus J, et al. Biochem Biophys Res Commun 2001; 280:961-3. environment of ITS and telomeres influence the binding of various 10. Rill RL, et al. Nucleic Acids Res 1977; 4:771-89. protein known to interact with telomeric sequences, hence modulating 11. D’Anna JA, et al. Biochemistry 1989; 28:2895-902. telomeric chromatin structure.25 Chromatin function would thus be 12. Moyzis RK, et al. Proc Natl Acad Sci USA 1988; 85:6622-6. 13. Gilbert N, et al. Proc Natl Acad Sci USA 2001; 98:11949-54. regulated by a balance between DNA sequence (“cis” influence) and 14. Robinson PJF. et al. Proc Natl Acad Sci USA 2006; 103:6506-11. nuclear environment (“trans” influence). 15. Routh A, et al. Proc Natl Acad Sci USA 2008; 105:8872-7.

Acknowledgements

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We are grateful to Hua Wong for help with modeling, Eric le Cam for electron microscopy experiments, and Malcolm Buckle for the reading of the manuscript. D. R. and C. L. benefited respectively from a doctoral and a post-doctoral training grant funded by CEA.

Note Supplementary materials can be found at: www.landesbioscience.com/supplement/RevaudCC8-7-Sup.pdf 1100

16. Wong H, et al. PLoS ONE 2007; 2:877. 17. D’Anna JA, et al. Biochemistry 1983; 22:5631-40. 18. Krutilina RI, et al. Oncogene 2003; 22:6690-8. 19. Mignon-Ravix C, et al. Eur J Hum Genet 2002; 10:107-12. 20. Warters RL, et al. Radiat Res 1992; 130:309-18. 21. Elia MC, et al. Cancer Res 1992; 52:1580-6. 22. Rivero MT, et al. Exp Cell Res 2004; 295:161-72. 23. Fernandez JL, et al. Cytogenet Cell Genet 1998; 82:195-8. 24. Nikitina T, et al. J Cell Biol 2004; 166:161-5. 25. Pisano S, et al. Cell Mol Life Sci 2008; 65:3553-63.

Cell Cycle

2009; Vol. 8 Issue 7

Supplementary material for

Sequence-driven Telomeric Chromatin Structure Déborah Revaud, Julien Mozziconacci, Laure Sabatier, Chantal Desmaze and Christophe Lavelle

Materials and methods PNA-FISH CHO-K1 cells were cultured in DMEMF12+Glutamax supplemented with 10% fetal calf serum (FCS) in 5% CO2 at 37°C in a humidified incubator. Cell cultures were harvested in PBS containing 0.25mg/ml trypsin and washed twice in PBS before being divided in two flasks. Cells returned to incubator for 14 hours to harvest metaphases. 0.06µg/ml colchicin was added 2 hours before harvesting metaphases according to standard protocol: cells were trypsinized, incubated in hypotonic solution (KCl 0.075M:1/6 human serum in H2O; vol. 0,5:0,5) 15 minutes at 37°C and fixed in acetic acid:absolute ethanol (vol. 1:3). Cells were then spread on wet and cold slides and conserved at –20°C before experiments. Interstitial telomere sequences were detected by hybridization of 0.3 µg.ml-1 (C3TA2)3 PNA oligonucleotide coupled with the Cyanin 3 fluorochrome (Perceptive Biosystems) according to previously described FISH protocol (Lansdorp et al., 1996). Hybridized metaphases were captured and analyzed with a Zeiss Axioplan epifluorescence microscope equipped with a CCD camera (Cohu) coupled with filters for observation of the different fluorochromes. Images were processed with ISIS software (Metasystems S.A.).

Nuclei preparation Cell pellets (as prepared above) were resuspended in 5 volumes of ice-cold hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) until lysis was checked on microscope. Nuclei were further washed twice in 2 volumes of ice cold hypotonic buffer and resuspended in sucrose buffer (15 mM Tris-HCl pH 7.5, 60mM KCl, 15 mM NaCl, 1 mM EDTA, 0.15 mM βME, 0.5 mM spermidine, 0.15 mM spermine, 0.345 M sucrose) at ~2 A260 units of DNA per ml and conserved at 80°C for further use.

Nuclei digestion For NRL measurements, nuclei were digested with micrococcal nuclease (MNase): 1 ml of nuclei solution was supplemented with 5 mM MgCl2 and 2.5 CaCl2 and digested with 15 units MNase (Roche) for 3 min at 37°C (figure 1B). The digestion was stopped by adding EDTA to 25 mM and immediately followed by phenol-chloroforme DNA extraction.

For sucrose sedimentation analysis, nuclei were digested with a cocktail of restriction enzymes to favour long chromatin fragments (up to 10kbp) that tend otherwise to quickly disappear upon MNase treatment. Digestion was carried overnight with 10 units of EcoRI, BamHI, Rsa1 and HinfI restriction enzymes (New England Biolabs), followed by RNAse treatment before loading on gradient (see below).

Gradient sucrose separation of digested chromatin fibers Following enzyme digestion, nuclei were pelleted and resuspended in 150 µl TEEP20 (10 mM TrisHCL pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.25 mM PMSF, 20 mM NaCl) and incubated at 4°C overnight for nuclear lysis. Nuclear debris was removed by centrifugation (12000g, 5 min at 4°C) and the soluble chromatin was recovered in supernatant. This supernatant was fractionated on 6% to 40% (wt/vol) sucrose gradient in TEEP80 (10 mM Tris-HCL pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.25 mM PMSF, 80 mM NaCl) by centrifugation at 4°C for 2h at 38000 rpm in a Beckman SW41 rotor. 500 µl fractions were collected and phenol-chloroform extracted for gel analysis.

Gel electrophoresis For NRL: extracted DNA from MNase digested nuclei was resolved in 1% agarose gel; for sedimentation analysis, DNA extracted from fractions of sucrose gradient was resolved in 0.8% agarose gel. Migrations are in 1X Tris acetate EDTA buffer and revealed by 5 min incubation in 0.5µg/ml ethidium bromide solution. 1Kb DNA size marker (Promega; see suppl. Fig. 3) was added in the gels (first and/or last lane).

Blot For Southern blotting, DNA was transferred to Hybond XL membrane (Amersham). Membrane was UV cross-linked and prehybridized in Church buffer (0.25M Sodium Phosphate buffer, 1mM EDTA, 1% BSA and 7% SDS) 4h at 55°C. Telomeric signal and ladder signal were jointly revealed by hybridization 2h at 55°C with (TTAGGG)10 oligonucleotides and 1kb ladder (Promega) labelled with 32

P and pre-melted (5 min at 95°C). Membrane was then washed 15minutes in 1X SSC-0.1% SDS,

15min in 0.1X SSC-0.1% SDS, 5 min in 0.1 X SSC. Revelation and analysis were done with Typhoon scan 9400 system and Image Quant.

Chromatin fiber modeling The 3D models of the chromatin fiber were designed using the Blender software (www.blender.org) as described in (Wong et al., 2007).

References Lansdorp, P.M., Verwoerd, N.P., van de Rijke, F.M., Dragowska, V., Little, M.T., Dirks, R.W., Raap, A.K., and Tanke, H.J. (1996). Heterogeneity in telomere length of human chromosomes. Hum Mol Genet 5, 685-691. Wong, H., Victor, J.M., and Mozziconacci, J. (2007). An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat length. PLoS ONE 2, e877.

Figure legends Figure S1. CHO nuclei digestion by MNase. (A) The same kind of experiment as the one reported in fig. 1 B and C was carried out independently, however using a briefer MNase digestion. This limits the number of high molecular weight visible bands (hidden in the smear, with non-digested material) but eliminates any risk due to exonuclease activity of MNase that could artefactly led to underestimation of NRL (see how signal decrease in lower bands compare to its increasing in fig 1, showing that digestion was really stopped at its very beginning). A high resolution gel was then performed to obtain clear separation between each band. (B) Density profiles from gel A. See how band 4 in bulk signal (BET) perfectly matches the 750 bp marker size, giving a 750/4 ~ 187 bp NRL; at the same time, see how band 6 in ITS signal (blot) matches the 1000 bp marker size, giving a 1000/6 ~ 167 bp NRL.

Figure S2. (A) Sedimentation analysis of DNA extracted from various sucrose gradient fractions. No shift is seen, showing that the more densely nucleosome-covered ITS fibers must be less compact, hence migrating along the less heavy but more compact bulk fibers. Note incidentally the relative enrichment in long chromatin fractions in ITS (right) compare to bulk chromatin (left): this can easily be explained by the poor occurrence of restriction enzymes sites in ITS regions. (B) Integrity of chromatin fraction after sucrose sedimentation was systematically checked by transmission electron microscopy (images courtesy provided by Eric Le Cam, IGR, Villejuif). Resolution is however not sufficient to measure NRL, hence to distinguish between bulk (NRL ~187 bp) or ITS (NRL ~167 bp) chromatin on these images. Figure S3. 1 kbp DNA ladder from Promega, with size corresponding to each band (picture from Promega catalog). This marker is used in every gel (fig. 1B, S1A and S2A).

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