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Address correspondence to G. Almouzni, Institut Curie/Research section, UMR218 du Centre National pour la Recherche Scientifique, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Tel.: 33 1 42 34 67 01. Fax: 33 1 46 33 30 16. email: almouzni{at}curie.fr
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Abstract |
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Key Words: centromere; cohesion; replication; nuclear organization; cluster
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Introduction |
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In the mouse, Mus musculus domesticus, two types of repetitive DNA sequences are associated with centromeres. These are the major satellite repeats (6 megabases of 234 bp units) and minor satellite repeats (600 kb of 120 bp units; Choo, 1997). In situ hybridization on metaphase chromosomes has shown that major satellite sequences are located pericentrically, whereas minor satellite sequences coincide with the centric constriction (Wong and Rattner, 1988; Joseph et al., 1989). In interphase nuclei, the association of centromeres of different chromosomes results in an organization in clusters. These highly condensed clusters are easily detectable cytologically (Hsu et al., 1971). It is believed that ectopic pairing of repetitive sequences and/or association of heterochromatin components produces this organization (Comings, 1980; Manuelidis, 1990). However, it has yet to be elucidated whether this association occurs through major and/or minor satellite domains, and whether the nature of the heterochromatin in these domains differs in any way. To date, several typical marks have been associated with centromeric heterochromatin: histones are generally hypoacetylated (Jeppesen et al., 1992) and specifically methylated on lysine 9 in the NH2-terminal tail of histone H3 (Me-K9 H3; Peters et al., 2001). In mouse cells, both of these characteristics (Peters et al., 2001; Taddei et al., 2001), in conjunction with an RNA component (Maison et al., 2002; Muchardt et al., 2002), are necessary for the maintenance of heterochromatin protein 1 (HP1) within centromeric regions. The presence of the latter is thought to contribute to centromere function (Eissenberg and Elgin, 2000). Most importantly, how these heterochromatic marks distribute between the two subdomains in interphase to contribute to a mitotic function is still unknown. This is particularly critical given that in mitosis a clear specialization is already evidenced by the fact that kinetochore-associated proteins can be found at centric regions, whereas heterochromatin-associated proteins are associated mainly with pericentric domains (Craig et al., 2003). These findings prompted us to explore the (three-dimensional) organization of the minor and major satellite domains in the interphase nucleus and to compare this to their organization on mitotic chromosomes to gain insights into their spatio-temporal dynamics and potential importance for centromere function.
We find that in mouse nuclei, centromeric heterochromatin clusters ("chromocenters") are formed by the coalescence of the major satellites, whereas the corresponding minor satellites are located in a surrounding domain as several separate entities. By combining immunofluorescence and DNA FISH, we observed that HP1 specifically accumulates on the major satellites, whereas centromeric protein (CENP) distribution is associated only with the minor satellites. Although both of these regions contain Me-K9 H3, these domains display different micrococcal nuclease (Mnase) sensitivity. Furthermore, chromatin immunoprecipitation reveals the existence of distinct Me-K9 H3containing dinucleosomes within major satellite regions. Thus, each of these regions appears to be associated with a distinct higher order chromatin organization. In addition, we find that these domains replicate asynchronously: major satellites replicate in the middle of S-phase, and minor satellites replicate later during S-phase. Another important feature is detected in mitosis when we find that chromatid cohesion is sustained for a longer time in major satellites compared with minor satellites. Remarkably, in cells lacking the Suv39h histone methyltransferases, which is important for HP1 localization at centromeric regions, such prolonged cohesion in major satellite regions is lost while minor satellite regions remain unaffected.
Thus, we conclude that two spatially distinct domains can be defined with specific marks and differential timing in both replication and chromatid separation. Such spatio-temporal organization is proposed to be critical to ensure a proper kinetochore function with coordination between centromeric cohesion and dissociation necessary during chromosome segregation.
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Results |
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We then assessed the nuclear locations of K9 methylation of H3 by immunofluorescence. The anti-di-Me-K9 antibody revealed a broad granular staining through the nucleoplasm (Maison et al., 2002). In contrast, the anti-tri-Me-K9 stained nuclear spots that colocalized with HP1 at pericentric heterochromatin (Fig. 4 A, left). This staining was maintained on metaphase chromosomes (Fig. 4 D). Immunofluorescence followed by DNA FISH showed that tri-Me-K9 labeling colocalized clearly with major satellites and to a lesser extent with minor satellites (Fig. S2 for three-dimensional analysis, available at http://www.jcb.org/cgi/content/full/jcb.200403109/DC1). In contrast, we could not detect any specific enrichment for di-Me-K9 (Fig. 4 A, middle and right). These data suggest that H3-K9 tri-methylation is generally enriched across the whole centromere.
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Major and minor satellites replicate asynchronously in S-phase
Replication of DNA late in S-phase of the cell cycle has been considered a hallmark of heterochromatin (Goldman et al., 1984; Hatton et al., 1988; Schubeler et al., 2002), which could play an important role in centromere function (Csink and Henikoff, 1998). Data from most organisms, with the exception of S. cerevisiae, favor the notion of generally mid to late replication timing patterns for centromeric regions (Ten Hagen et al., 1990; O'Keefe et al., 1992; Shelby et al., 2000; Sullivan and Karpen, 2001). Given the aforementioned distinct properties of centric and pericentric heterochromatin, we wished to examine their relative timing of DNA replication. We decided to combine BrdU immunostaining with DNA FISH on synchronized cells to address this issue. Synchronization of NIH 3T3 cells at the G1/S border was achieved using aphidicolin, an inhibitor of DNA polymerase. Cells were released into S-phase by washing away the inhibitor at different time points (hourly between 1 to 8 h) and pulse labeled for 10 min with the nucleotide analogue BrdU. Immunostaining of BrdU incorporation combined with either major or minor satellite DNA FISH was performed (Fig. 5). S-phase usually takes 78 h to complete in NIH 3T3 cells. Different replication patterns could be distinguished corresponding to early, mid, and late S-phase, as revealed by detection of BrdU incorporation (Fox et al., 1991; Dimitrova and Berezney, 2002). Typical replication patterns (Fig. 5, red) are shown combined with specific probing (Fig. 5, green) for major (Fig. 5, top) or minor (Fig. 5, bottom). In early S-phase, replication begins with discrete punctuate sites distributed throughout the nucleus and no colocalization is found at this time point for any of the probes. In middle S, around 3 to 6 h after release, the cells show BrdU rings around the major satellite clusters (Fig. 5, top, Merged), possibly reflecting newly synthesized DNA occurring at the periphery of the domain (Quivy et al., 2004). At this stage, no colocalization between minor satellites and BrdU is observed (Fig. 5, bottom). Finally, in late S, 78 h after release, BrdU incorporation again became more granular with small foci in the interior of the nucleus. Only at this stage could we detect some minor satellite colocalization with BrdU (Fig. 5, bottom). These data indicated that major and minor satellite domains replicate at different times during S-phase, with replication of major satellites in middle S, followed by replication of minor satellites in later S-phase.
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Discussion |
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Importantly, minor and major satellite domains show distinct timing for chromatid separation during mitosis. Minor satellite chromatids, which are associated with CENPs, dissociate early at the metaphase stage. The major satellites associated with HP1, which is also important for centromeric function in mouse (Peters et al., 2001; Taddei et al., 2001), constitute the latest centromeric part to separate. These data suggest that these domains possess distinct roles in centromere function. Minor satellite domains may provide a nucleation center for kinetochore assembly, which is required for the proper segregation of sister chromatids during mitosis, whereas major satellites may help to ensure cohesion between sister chromatids next to centromeres. Indeed, in yeast S. pombe outer centromere heterochromatin components associated to the outer centromeres such as the HP1 homologue Swi6, H3 Lys 9 methylation and more recently the RNA interference machinery have been involved in sister chromatid cohesion at centromere and proper chromosome segregation (Hall et al., 2003). In mammals, several cytological observations support a role of pericentric heterochromatin in centromere cohesion (Vig, 1982). However, it is the first time that a direct link is made between H3 Lys 9 methylation/HP1
and centromere cohesion in mammals. Indeed, our data suggest that the loss of HP1
at major satellites pericentric heterochromatin as observed in Suv39h dn cells leads to a defect in centromeric cohesion in mouse cells. Mechanistically, the maintenance of cohesion between major satellite sisters may be advantageous when tension is imposed at the most centric part, which nucleates kinetochore formation and microtubule anchoring.
In conclusion, we have characterized how centromeric domains are organized into distinct types of heterochromatin that can self-perpetuate. This functional organization may be conserved in human. The next challenge will be to address how these domains can be established de novo. Neocentromere formation in human cells, which is accompanied with the association of several CENPs and chromatin components (Warburton, 2001), is a challenge of particular interest. Given that they do not necessarily contain repeats found in pericentromeric regions, one may speculate that the cohesion property could be acquired "in trans," especially because specific centromeric clusters are also observed in human (Alcobia et al., 2000). Alternatively, it is possible that other means could be used in human cells to ensure this cohesion property. Nevertheless, the conceptual advance in mouse cells in the present study should promote future work to elucidate how other organisms have solved the puzzling issue of constructing a centromeric region with dual properties: cohesion and segregation, and thus open avenues into the evolution of epigenetic identity of centromeres.
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Materials and methods |
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Two-color DNA FISH
Plasmids pCR4 Maj9-2 and pCR4 Min5-1 (Lehnertz et al., 2003) contain major and minor satellite DNA (provided by T. Jenuwein, Research Institute of Molecular Pathology, The Vienna Biocenter, Vienna, Austria). DNA fragments were labeled by nick translation (Life Technologies) with digoxigenin-11-dUTP or biotin-16-dUTP (Boehringer). Cells grown on coverslips were fixed and treated with 0.7% Triton X-100 and 0.1 M HCl for 10 min on ice followed by denaturation with 2x SSC/50% formamide 30 min at 80°C. Heat-denatured probes were hybridized overnight at 37°C. After hybridization and washes with 2x SSC/50% formamide and 2x SSC at 42°C, probe detection used a three-step procedure for amplification (Manuelidis et al., 1982). Biotin was revealed using Texas redconjugated streptavidin and biotinylated antistreptavidin antibody (Vector Laboratories), followed by Texas redconjugated streptavidin. Digoxigenin was detected using a sheep FITC-conjugated anti-digoxigenin serum (Roche), rabbit FITC-conjugated antisheep antibodies (Jackson ImmunoResearch Laboratories), and goat FITC-conjugated antirabbit antibodies (Jackson ImmunoResearch Laboratories). Coverslips were mounted in Vectashield containing 0.5 µg/ml DAPI (Vector Laboratories).
Combined protein immunolabeling and DNA FISH
Immunostaining was performed on Triton X-100 extracted cells (Taddei et al., 2001) using the following: anti-HP1 (Euromedex HP1
, 2HP-1H5-As, at a dilution of 1:400), anti-di-Me-K9 H3 (Upstate Biotechnology; 07212, at 1:2,000), anti-tri-Me-K9 H3 (Abcam; ab1186 at 1:500), di-Me K4 antibody (Upstate Biotechnology; 07030 at 1:200), ACA serum (provided by G. Steiner, Northwestern University, McGaw Medical Center, Chicago, IL; at 1:100), and anti-phospho H3-S10 (Upstate Biotechnology; 05598, at 1:400) and secondary antibodies coupled to FITC or Texas red (Jackson ImmunoResearch Laboratories). DNA FISH was performed after PFA post-fixation.
Metaphase chromosome spreads, protein immunolabeling, and DNA FISH
Mitotic cells collected after growth for 30 min in medium containing colcemid (GIBCO BRL; 0.1 µg/ml) were washed in PBS. After swelling in 75 mM KCl, they were recovered onto glass slides by cytocentrifugation. Protein immunolabeling was performed without fixation (Jeppesen et al., 1992). For DNA FISH procedure, slides were fixed after cytocentrifugation.
Replication timing analysis combining BrdU immunolabeling and DNA FISH
NIH 3T3 cells were synchronized at the G1/S border by aphidicolin arrest, and maintained at 100% confluence for 12 d, and then reseeded to 6080% confluence in media containing 3 µg/ml aphidicolin (Sigma-Aldrich) for 18 h. Aliquots were released from the block every hour (from 1 to 8 h) and pulse-labeled for 10 min with 40 µM BrdU (Sigma-Aldrich). BrdU was immunodetected after a denaturation step in 4 N HCl for 10 min using rat mAb (AbCys; OBT0030) at 1:200 dilution. Cells were subsequently post-fixed with 2% PFA and DNA FISH was performed.
Combined in situ elongation assay and DNA FISH
in situ elongation was performed on Triton X-100 permeabilized MEFs. Slides were incubated for different time points at 37°C in buffer containing 40 mM K-Hepes, pH 7.8, 7 mM MgCl2; 3 mM ATP; 0.1 mM each of GTP, CTP, and UTP; 0.1 mM each of dATP, dGTP, and dCTP; 40 µM BiodU; 20 mM of creatine phosphate; 0.5 mM DTT; and 2.5 µg of phosphocreatine kinase (Boehringer). Reactions were stopped in 40 mM K-Hepes, pH 7.8, 7 mM MgCl2, 3 mM CaCl2, 0.5 mM DTT and fixed in 2% PFA. BiodU was detected with Texas redconjugated streptavidin (Vector Laboratories) and cells were subsequently refixed with 2% PFA. DNA FISH was performed using DNA probes labeled with digoxigenin-11-dUTP to avoid antibody cross-reactions. The assay was performed twice, in each independent experiment 200300 nuclei were analyzed.
Microscopy and image analysis
Image acquisition was performed with an epifluorescence photomicroscope (DMR; Leica) equipped with a chilled charge-coupled-device camera (C5985; Hamamatsu Photonics), in which the resolution is 200 nm in x-y. Confocal sections were obtained with a confocal scanning microscope (model TCS-4D; Leica) equipped with an acousto-optical tunable filter and with 100x numerical aperture 1.4 plan-apochromat oil-immersion objective. For two-color images, 2530 serial sections were collected at each imaging time point (256 x 256 pixels or 512 x 512; pixel size, 120200 nm; z-step, 0.40.5 µm). Data in 8-bit TIFF format series for each color were analyzed using Metamorph software (Universal Imaging Corp.). Selected images were assembled using Adobe Photoshop. Three-dimensional reconstruction of confocal image stacks was performed using AmiraTM 2.3 (TGS).
Mnase digestion and NChIP
Native oligonucleosomes were isolated by partial Mnase digestion on L929 cell nuclei and sucrose gradient purification and analyzed by gel electrophoresis followed by transfert and hybridization with probes. For NChIP, 10 µg of oligonucleosomes were incubated with 4 µg of either purified IgG antimouse (Jackson ImmunoResearch Laboratories), anti-di-Me-K9 (Upstate Biotechnology; 07212), anti-tri-Me-K9 (Abcam; ab1186), or anti-ac K9 (acetylated histone H3 on lysine 9; Upstate Biotechnology; 06942) antibodies in 1 ml of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5 mM EDTA at 4°C overnight. 100 µl of protein ASepharose slurry (50% wt/vol; Amersham Biosciences) was added for 3-h incubation at 4°C. Immunoprecipitated and nonimmunoprecipitated oligonucleosomes were recovered by centrifugation. After washes in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, both unbound and bound fractions were adjusted to 0.4% SDS. DNA was purified and analyzed by agarose gel electrophoresis, stained with ethidium bromide, and transferred onto a Hybond N+ membrane (Amersham Biosciences). For hybridization, sequences of the probes used were minor satellite (947: 5'-CACATTCGTTGGAAACGGGATTTGTAGAAC-3'; Kipling et al., 1994), major satellite (204: 5'-GTGAAATATGGCGAGGAAAACT-3'; Nicol and Jeppesen, 1996), and B2 repeats (Krayev et al., 1982).
Online supplemental material
Fig. S1 shows characterization of the tri-Me-K9 H3 antibody by slot-blot. Fig. S2 shows three-dimensional analysis of tri-Me-K9 H3 staining at minor and major satellites. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200403109/DC1.
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Acknowledgments |
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M. Guenatri was supported by Ministère de l'Education Nationale de l'Enseignement Supérieur de la Recherche and la Ligue Nationale contre le Cancer. G. Almouzni benefited from grants from Programme Collaboratif Institut Curie/Commissariat à l'Energie Atomique, la Ligue Nationale contre le Cancer (Equipe labelisée la Ligue), Euratom (FIGH-CT-1999-00010 and FIGH-CT-2002-00207), the Commissariat à l'Energie Atomique (LRC no. 26), and Research Training Network (HPRN-CT-2000-00078 and HPRN-CT-2002-00238).
Submitted: 19 March 2004
Accepted: 2 July 2004
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