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Address correspondence to Christopher L. Woodcock, Biology Dept., University of Massachusetts, Amherst, MA 01003. Tel.: (413) 545-2602. Fax: (413) 545-3243. email: chris{at}bio.umass.edu
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Abstract |
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Key Words: telomere; chromatin; electron microscopy; TRF1
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Introduction |
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In addition to the terminal single-stranded DNA sequence, most telomeres contain several kilobases of double-stranded TTAGGG repeats. In mammals (Griffith et al., 1999), as well as Trypanosoma brucei (Munoz-Jordan et al., 2001), Oxytricha fallax (Murti and Prestcott, 1999), and Pisum sativum (Cesare et al., 2003), this repetitive DNA contributes to a unique "t-loop" sealed by an insertion of the single-stranded terminus into duplex DNA. Telomere chromatin contains nucleosomes but differs from bulk nuclear chromatin in having a nucleosome repeat length (NRL) that is 40 bp shorter (Tommerup et al., 1994; Lejnine et al., 1995; Makarov et al., 1997), and has been predicted to induce a unique pattern of higher order folding (Fajkus and Trifonov, 2001). Intriguingly, in nuclei, telomere DNA occurs in distinct circles or rings (Luderus et al., 1996, Pierron and Puvion-Dutilleul, 1999). Telomeres are strongly anchored in nuclei, and remain with the insoluble "matrix" fraction after nuclease digestion (de Lange, 1992; Luderus et al., 1996).
The inability to extract native telomeres from nuclei effectively prevents the full examination and characterization of the unique higher order chromatin structure of these critical chromosomal components. We surmised that telomere anchoring to the insoluble nuclear matrix fraction, as reported by de Lange (1992) for HeLa cells, might be reduced in differentiated cells in which nuclei have a reduced complement of nonhistone chromosomal proteins. Here, we report the successful isolation of telomere chromatin from chicken erythrocytes and quiescent mouse lymphocytes, and show that the native interphase conformation is a closed chromatin loop.
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Results and discussion |
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Telomere loops from chicken erythrocyte nuclei in 75 mM NaCl varied in contour length from 200 to
600 nm, with a mean of 317 nm (Fig. 4). Telomere DNA with a modal length of
20 kb (Fig. 1 D), would require a nucleosome packing ratio of
1:20 to yield a 317 nm length of chromatin fiber. This is somewhat lower than the
1:30 packing ratio measured for bulk chromatin from these nuclei under similar ionic conditions (Woodcock et al., 1984; Gerchman and Ramakrishnan, 1987), suggesting a lower level of compaction for telomere chromatin. Like bulk chromatin, telomeres appeared in an open beads-on-a-string conformation at low ionic strength (Fig. 2 A), and rough counts of the number of nucleosomes in the loops (75150) also indicate a DNA loop size in the 1530 kb range. Fibers which respond to changes in ionic strength by varying in diameter (and compaction) are characteristic of histone H1-containing chromatin (Widom, 1989), supporting the suggestion that telomere chromatin contains H1 (Bedoyan et al., 1996).
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Studies of the location of telomere repeats in HeLa nuclei in which telomere DNA ranges in size from 14 to 31 kb have identified discrete circular or ring-shaped structures 120 nm in diameter (Luderus et al., 1996; Pierron and Puvion-Dutilleul, 1999), which persist in nuclear matrix preparations and are presumed to represent the native telomere chromatin conformation at in vivo anchorage sites. The close correspondence in contour length between the perimeter of these sites (
375 nm) and the chromatin loops observed here (Fig. 4) suggests that they may represent in situ and in vitro manifestations of the same structure. The retention in nuclear matrix preparations of TRF1 as well as telomere chromatin has led to the hypothesis that TRF1 has an anchoring function (Luderus et al., 1996), and it is perhaps significant in this respect that we find the chicken homologue of TRF1 (De Rycker et al., 2003) to be undetectable in chicken erythrocyte nuclei under Western blotting conditions that give a strong signal from chicken liver nuclei. Similarly, using an antibody against mouse TRF1, we were able to detect the protein in nuclei from cycling 3T3 fibroblasts and mouse ES cells, but not from quiescent lymphocytes (unpublished data), although TRF1 mRNA has been detected in this tissue (Broccoli et al., 1997). Luderus et al. (1996) noted that a small fraction of TRF1-depleted telomeric chromatin could be released from HeLa nuclei, and suggested that telomeres might be transiently released from their anchorage sites. The present data confirm the lack of TRF1 in RE-solubilized telomeric chromatin, and further suggest that TRF1 abundance in nuclei is correlated with cell proliferation and telomere anchoring.
TRF1 plays an important role in telomere length control (van Steensel and de Lange, 1997; Smogorzewska et al., 2000), in conjunction with tankyrase, TIN1 and POT1 acting to repress telomerase activity in cycling cells (Loayza and de Lange, 2003). At present, the implications of the low abundance of TRF1 in quiescent cells are unclear, although as there is little or no detectable telomerase activity in unstimulated lymphocytes (Liu et al., 2001), it is possible that TRF1-mediated regulation is not needed. As shown here, TRF1 will bind stably and specifically to telomeric chromatin in vitro (Fig. 1 E), and, in light of its ability to bend DNA (Bianchi et al., 1997, 1999) and induce side-by-side DNA pairing (Griffith et al., 1998), it will be interesting to examine in detail any changes in chromatin morphology accompanying TRF1 binding.
It has been suggested that a primary function of DNA t-loops is to protect the chromosome end from recognition as a breakpoint by DNA repair machinery (Griffith et al., 1999), and in this context, the histone association and compaction afforded by chromatin is likely to enhance the protective role. The distinct morphology of telomeres reported here will allow detailed structural and compositional studies of these essential chromosome components. It will be particularly important to investigate the relationship between NRL and fiber architecture, and the 3D chromatin organization at the looptail junction where the single-stranded terminus is inserted.
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Materials and methods |
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DNA extracted from fractions was separated on 1% agarose gels and stained with ethidium bromide. Selected fractions were dialyzed against 25 mM NaCl, 1 mM EDTA, 5 mM Hepes, pH 7.5, and concentrated using 100,000 MW cutoff Centricon units (Amicon Inc.). To examine telomere enrichment, chromatin fractions were briefly digested with micrococcal nuclease (1.5 U of enzyme per 1 mg DNA for 5 min at 20°C in 1 mM CaCl2, 1 mM PMSF, 10 mM Tris, pH 7.5) to provide a size range convenient for transfer and blotting. DNA was then extracted, separated on 1% agarose gels, transferred to membranes and Southern blots with a telomere probe performed as described for dot blots. To examine the DNA size distribution of telomere DNA, fractions were separated by pulsed field gel electrophoresis on 1% FastLane agarose (FMS BioProducts), at 6 V/cm for 15 h with an excluded angle of 120° in 45 mM sodium borate, 1.0 mM EDTA, 45 mM Tris, pH 8.3, together with low range PFG Marker (New England Biolabs, Inc.) for DNA size calibration. To facilitate transfer of high MW DNA, gels were treated with 0.25 M HCl for 60 min, rinsed with H2O, and treated with 0.5 M NaOH, 1.5 M NaCl for 30 min before neutralizing in 1.5 M NaCl, 1 M Tris-HCl, pH 7.4, and probing for telomere sequences as described above.
For EM, concentrated samples were equilibrated to the desired NaCl concentration and fixed with 0.1% glutaraldehyde for 4 h, before applying to glow-discharged carbon films and staining with aqueous uranyl acetate or alcoholic phosphotungstic acid (Woodcock and Horowitz, 1998). Grids were examined in a FEI Tecnai 12 Transmission EM at 100KV with LaB6 filament, and micrographs recorded with a 2048 x 2048 CCD camera (TVIPS). Images were typically recorded in a 16-bit TIFF format. Contour lengths were measured on unprocessed images using the ImageJ image processing suite. For presentation purposes, images were converted to 8-bit TIFF files in ImageJ, and cropping and adjustment of contrast and brightness performed with FotoCanvas v 1.1 (ACD Systems, Ltd.).
hTRF1 was prepared in Sf21 cells (provided by J. Burand, University of Massachusetts) from a baculovirus plasmid (provided by T. de Lange, The Rockefeller University, New York, NY) containing the His-tagged gene (Chong et al., 1995), purified using the TALON affinity resin system (BD Biosciences) according to the manufacturer's directions, and stored in 500 mM KCl, 0.2 mM PMSF, 20 mM Hepes, pH 7.5, with 20% glycerol at 80°C. hTRF1 was biotinylated using the NHS-PEO-biotin reagent (Pierce Chemical Co.) according to the manufacturer's recommendations. The extent of biotinylation was measured using the EZ Biotin Quantitation Kit (Pierce Chemical Co.). RE-digested chromatin was mixed with biotinylated hTRF1 (hTRF1-bio) at a ratio of 16 µg hTRF1-bio to 1 µg telomere DNA (assuming telomere DNA constitutes 0.2% of total chicken erythrocyte DNA; Lejnine et al., 1995) for 30 min at 37°C followed by 1 h at 0°C, before separation on sucrose gradients as described above. Chromatin fractions enriched in telomeric sequence were fixed briefly in 75 mM NaCl, applied to glow-discharged carbon films, rinsed, and the grids floated on a drop of 5-nm streptavidin-gold beads (Electron Microscopy Supplies) for 5 min before rinsing with H2O and staining. Micrographs of looptail structures were analyzed by dividing each loop or tail into 50-nm segments, and the number of gold beads on each segment recorded. These values for 71 loops and tails were subjected to a nonpaired t test to determine the probability that loops and tails had identical bead distributions. PSI-Plot v. 7.0 (Poly Software International) was used for statistical analysis and histogram plotting. To determine the distribution of hTRF1 in the gradients, fractions were dialyzed and concentrated, aliquots solubilized in SDS-sample buffer, separated by SDS-PAGE, and the proteins electrophoretically transferred to membranes. Membranes were probed with anti-TRF1 (Imgenex) and the blots developed with the ECL system (Amersham Biosciences). Stained gels and developed films were recorded and analyzed using the Gel-Doc system and Quantity 1 software (Bio-Rad Laboratories). To examine the relative abundance of TRF1 in nuclei and RE-solubilized chromatin, Western blots were probed for chicken TRF1 with a specific antibody (De Rycker et al., 2003) supplied by C.M. Price (University of Cincinnati, Cincinnati, OH), and for mouse TRF1 with an antibody purchased from Alpha Diagnostics Inc. For all Western blots, the loading of chromatin was adjusted to be constant by staining duplicate lanes with Coomassie blue and recording the stain intensity of the core histones.
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Acknowledgments |
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This work was supported in part by National Institutes of Health GM43786 to C.L. Woodcock.
Submitted: 22 March 2004
Accepted: 3 June 2004
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