Correspondence to M. Cristina Cardoso: cardoso{at}mdc-berlin.de
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Introduction |
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In mammals, heterochromatin is characterized by high levels of specifically methylated forms of histone H3, deacetylated histone H4, and DNA methylation. Both methylation of histone H3 (at lysine 9) and methylation of cytosines (at CpG dinucleotides) are binding sites for chromatin modifiers such as the HP1 proteins and the methyl CpGbinding domain (MBD) proteins, respectively. The latter "translate" the DNA methylation signal into transcriptional repression at least partially by recruiting silencing complexes and histone deacetylases, thereby stabilizing and consolidating the heterochromatic state (for review see Bird and Wolffe, 1999; Leonhardt and Cardoso, 2000). Both HP1 (Furuta et al., 1997) and MeCP2 (Lewis et al., 1992) (the founding member of the MBD family) have been shown to be highly concentrated at pericentric heterochromatin. Binding of MeCP2 to pericentric heterochromatin is dependent on DNA methylation and requires the MBD (Nan et al., 1996). Mutations in the MeCP2 gene were linked to Rett syndrome, a common neurodevelopmental disorder in humans (Amir et al., 1999). The MeCP2 protein level has been shown to increase during neuronal differentiation (Jung et al., 2003) and was suggested to be critical for synaptogenesis (Mullaney et al., 2004), maturation, and maintenance of neurons (Kishi and Macklis, 2004).
Studies on mouse neurons (Manuelidis, 1985; Martou and De Boni, 2000; Solovei et al., 2004) indicated a specific rearrangement of centromeric domains in terminally differentiated cells. We have set out to test whether large-scale reorganization of heterochromatin within the nucleus is a feature of terminal differentiation and whether histone H3K9 or DNA methylation and its translation by MBD proteins play an important role in this process.
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Results |
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Clustering of pericentric heterochromatin during terminal differentiation
Because MeCP2 and DNA methylation have been implicated in heterochromatin formation and maintenance, we tested whether pericentric heterochromatin, which is highly enriched in both, undergoes structural changes during differentiation. To visualize the nuclear organization of pericentric heterochromatin during terminal differentiation, we used 3D-FISH (Solovei et al., 2001) with a major satellite-specific probe. Mouse pericentric heterochromatin consists of large arrays of tandem major satellite repeats. It accounts for 10% of the genome (Mitchell, 1996) and shows a tendency to form clusters, so-called chromocenters (Hsu et al., 1971). The mean number of chromocenters in terminally differentiated cells (11.1) versus undifferentiated precursors (20.4) was markedly reduced (Fig. 2), whereas the size of the clusters increased concomitantly. The decrease in numbers was statistically highly significant (P < 0.001). Moreover, the variability in chromocenter number within myotube nuclei diminished, as the SD dropped from 6.1 to 2.9 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200502062/DC1). This substantial increase in heterochromatin clustering is probably a continuous process in myoblast-to-myotube transition, as myocytes, which represent an intermediate differentiation state, showed an intermediate number of chromocenters (average 14.5 SD = 4.4; see Fig. 5 B and Fig. S1).
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Ectopic expression of MeCP2-YFP induces clustering of pericentric heterochromatin independent of the histone H3K9 methylation pathway
To test whether MeCP2 plays a role in the aggregation of chromocenters, we transfected mouse myoblasts with a MeCP2-YFP fusion construct (Fig. 3, A and B) and performed a correlation analysis comparing expression levels of MeCP2-YFP with the number of chromocenters. 86 nuclei were first imaged for MeCP2-YFP fluorescence by confocal microscopy, followed by post-fixation and 3D-FISH with a major satellite-specific probe to visualize chromocenters. The correlation analysis revealed a significant (P < 0.01) negative correlation resulting in a coefficient of 0.52 (Fig. 3 D). Fig. 3 C shows two nuclei, one with low levels of MeCP2-YFP having many chromocenters (top), whereas the other (bottom), with high amounts of the fusion protein, shows only a few clusters. In addition to this reduction, also the variability in the number of chromocenters decreased with increasing MeCP2-YFP expression, similar to the results in differentiating myoblasts (Fig. 2). Control transfections using only YFP showed no effect on the clustering of chromocenters (see Fig. 6 B). Furthermore, expression of high levels of MeCP2 fused to other tags (GFP or DsRed variants) showed likewise clustering of chromocenters (see Fig. 6 A). To investigate whether other proteins with a similar nuclear localization as MeCP2 would be able to induce heterochromatin clustering, we transfected mouse myoblasts with constructs coding for fluorescently tagged versions of CENPB and HP1. Although CENPB has been shown to localize at centromeric sites, in mouse chromosomes encompassing a region made up by the so-called minor satellite repeat (Amor et al., 2004), HP1
is mainly found in pericentric heterochromatin just as MeCP2, and represents one of the major constituents of constitutive heterochromatin (for review see Singh and Georgatos, 2003; Maison and Almouzni, 2004). In both cases we did not find an increased clustering of chromocenters in cells expressing high levels of the fusion proteins (see Fig. 6 B and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200502062/DC1). These results clearly argue against a general intrinsic clustering potential of centromeric or heterochromatin-associated proteins when expressed at high concentrations.
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Our experiments show that increased clustering of pericentric heterochromatin can be artificially induced by ectopic expression of MeCP2 in the absence of differentiation. These results indicate that the increased expression of endogenous MeCP2 during terminal differentiation (Fig. 1) is sufficient for inducing the observed aggregation of pericentric heterochromatin.
Fusion of chromocenters occurs throughout interphase
Earlier reports have suggested a cell cycledependent redistribution of centromeric regions within the nucleus (Manuelidis, 1985; Vourc'h et al., 1993). We therefore investigated when during the cell cycle the fusion of chromocenters would take place. For that purpose, we doubly transfected C2C12 myoblasts with MeCP2-YFP and DsRed-Ligase I (Fig. 4 A) as a live-cell cell cycle progression marker (Easwaran et al., 2004, 2005). S-phase cells could be recognized simply by the subnuclear pattern of DsRed-Ligase Ilabeled DNA replication foci (Cardoso et al., 1997; Leonhardt et al., 2000), whereas mitotic cells could be identified by chromosome condensation. G1 cells were identified by a previous mitosis or by a subsequent S-phase and G2 cells by a previous S-phase or a successive mitosis (Fig. 4 C). Of 14 nuclei analyzed, 9 showed fusions of chromocenters (example in Fig. 4 B and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200502062/DC1). A total of 30 fusions could be traced, with 15 occurring in G2, 10 in G1, and 5 in S-phase (Fig. 4 D).
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MeCP2-YFPexpressing cells exhibit enhanced chromocenter clustering during differentiation
Because MeCP2-YFPtransfected myoblasts were able to differentiate and many myotubes contained nuclei with high levels of the fusion protein, we conclude that overexpression of the protein in no way disturbs differentiation. When we compared the number of chromocenters in myotubes/myocytes showing a high MeCP2-YFP expression with that of nontransfected controls, we found significantly higher values in the control cells (P < 0.01 for myocytes and P < 0.05 for myotubes; Fig. 5 A). The mean number dropped from 14.5/11.1 in nontransfected myocytes/myotubes to 9.6/9.5 in transfected and highly expressing cells (Fig. 5 B). This means that high level of expression of MeCP2-YFP is not only compatible with the differentiation of transfected myoblasts, but it actually enhances pericentric heterochromatin clustering during terminal differentiation.
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Clustering of pericentric heterochromatin occurs in muscle tissue of MeCP2-deficient mice
MeCP2 loss of function has been linked to the neurodevelopmental disorder Rett syndrome (Amir et al., 1999), in which maturation of neuronal cells seems to be impaired, possibly causing the severe neurological phenotype (for review see Kriaucionis and Bird, 2003). Concerning muscle development, Rett syndrome patients as well as MeCP2 knock-out mice (Guy et al., 2001; Shahbazian et al., 2002) show no severe defects (Jellinger, 2003).
To directly test whether MeCP2 is required for clustering of pericentric heterochromatin during mouse development, we compared chromatin topology in nuclei from muscle fibers of MeCP2 knock-out mice with that of control mice. Muscle fibers were stained with DAPI to highlight pericentric heterochromatin and to investigate chromocenter clustering (Fig. 8). Clustering of pericentric heterochromatin in skeletal muscle tissue of MeCP2-deficient mice was comparable to that in wild-type mice (Fig. 8) and in vitro differentiated myotubes (Fig. 2). These results clearly indicate that clusters of chromocenters can form in the absence of MeCP2, and we hypothesized that another member of the MBD protein family might be capable to reorganize chromocenters in a similar fashion as MeCP2.
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Discussion |
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A possible mechanism to explain how increasing levels of MeCP2 and MBD2 may contribute to aggregation of heterochromatin could involve oligomerization of these proteins bound to chromatin. These factors are not likely to be involved in movements of chromocenters or other chromatin regions, per se, but rather act as a sort of "glue" stabilizing random encounters of chromocenters within the nucleus. This is supported by a recent report (Georgel et al., 2003) showing that MeCP2 has the ability to interconnect nucleosomal arrays in vitro, creating oligomers consisting of several units. The authors have proposed DNAMeCP2MeCP2DNA or DNAMeCP2DNA bridges to be responsible for the observed chromatin condensation activity. Such a mechanism could account for the clustering of pericentric heterochromatin during terminal differentiation simply by an increased interconnection due to elevated MeCP2 levels bound to methylated DNA. In this respect it should be noted that recombinant MeCP2 treated with the cross-linkers glutaraldehyde or EGS did not support a self-association of MeCP2 monomers (Klose and Bird, 2004). However, it remains to be tested whether mouse MeCP2 is capable of forming multimers under other conditions, i.e., in a nuclear environment and bound to methylated satellite repeats.
Because MeCP2 and MBD2 are highly basic proteins (with pI 10, similar to histones), they could act in a similar way as proposed for linker histones or inorganic bivalent cations (for review see Horn and Peterson, 2002) by neutralizing negative charges on the DNA, and thereby enabling or enhancing interactions between major satellite DNA located on separate chromocenters. Increased methylation of CpGs in pericentric regions (Fig. 1, C and E), creating a higher number of binding sites for MeCP2 and MBD2, would increase the probability of these proteins to bind, thereby augmenting their aggregation effect. However, the moderate degree of DNA methylation in the cell types analyzed suggests that extensive DNA methylation is not necessary for pericentric heterochromatin clustering. This is in agreement with a recent report showing that the compaction of oligonucleosomes by MeCP2 in vitro is not dependent on DNA methylation (Georgel et al., 2003).
Alternatively or in addition, a differentiation-dependent increase of MeCP2 could lead to a raise in the local concentration of histone deacetylases and/or of other chromatin-remodeling factors, which could bring about the observed aggregation effect. However, our results showing that the MBD domain alone, in the absence of the coRID, is sufficient for chromocenter reorganization (Fig. 6 and Fig. 7), argue against a role of the recruitment of deacetylase-containing complexes in the large-scale heterochromatin reorganization during differentiation. Also, the recently described recruitment of a histone H3K9 methylation activity by MeCP2 (Fuks et al., 2003) is unlikely to play a major role because mouse Suv39h double-null fibroblasts still showed increased clustering of pericentric heterochromatin upon MeCP2 overexpression, just as wild-type fibroblasts (unpublished data) or myoblasts did (Fig. 3 E). Altogether, our data favor a more direct and structural role of methyl CpGbinding proteins in chromatin reorganization rather than an indirect role through recruiting corepressor complexes.
Another aspect contributing to the clustering of pericentric heterochromatin in terminally differentiated, post-mitotic cells could be an intrinsic ability to aggregate during interphase, which in proliferating cells would be counteracted at each cell cycle by the dissociation of chromocenters as chromosomes condense and are separated during mitosis. With live-cell microscopy we could indeed follow such extensive dissociations of chromocenters in G2 nuclei before mitosis (Fig. S4 and Video 2). In post-mitotic, terminally differentiated cells, where chromosomes are no longer subjected to mitotic events, this "default" aggregation affinity would not be counteracted and might be further enhanced by MeCP2 and MBD2, finally leading to very large clusters.
A possible function of this nuclear reorganization of pericentric heterochromatin could be the establishment and/or stable maintenance of a specific transcriptional program in differentiated cells. The fact that heterochromatin, especially pericentric heterochromatin, conveys transcriptional silencing in many different settings, starting from position effect variegation (for review see Schotta et al., 2003) over transgene silencing (Francastel et al., 1999) to endogenous gene silencing (for review see Fisher and Merkenschlager, 2002), would support this hypothesis. It is conceivable that the silencing effects depend on a local threshold concentration of factors that are bound to or attracted by pericentric heterochromatin or some of its constituents. Forming bigger clusters would thus bring about an increase of such a critical concentration leading ultimately to the formation of effective silencing domains. Our results showing that MeCP2, which is known to act as a transcriptional repressor, plays an important role in inducing aggregation of heterochromatin clusters also favors this idea. Recently, it has been proposed that MeCP2 might be involved in the reduction of transcriptional noise (Hendrich and Tweedie, 2003). This function could be enhanced by a nuclear clustering that provides stringent control of leaky transcription via the creation of repressive subnuclear compartments. Moreover, our results showing that chromocenter clustering is maintained in MeCP2-deficient mice (Fig. 8) further strengthen the hypothesis that this large-scale topological chromatin reorganization might be of functional relevance, as it involves redundant pathways.
The finding that MeCP2 deficiency does not have a pronounced effect on gene expression pattern (Tudor et al., 2002) speaks in favor of MeCP2 being involved in stabilizing transcriptional silencing in terminally differentiated cells, rather than in regulating gene expression during differentiation. A recent report correlating the level of MeCP2 protein during central nervous system development in the mouse with the maturation of neurons further suggests that MeCP2 is involved in maintenance of the differentiated state, rather than in cell fate decisions (Kishi and Macklis, 2004). Alternatively, functional redundancy between MeCP2 and other methyl CpGbinding proteins such as MBD1 or MBD2, which have a similar pericentric distribution, could also explain the merely subtle changes in gene expression patterns observed in MeCP2-deficient mice (Tudor et al., 2002). Such a functional redundancy between MBD proteins is supported by our findings that MBD2 can likewise induce heterochromatin clustering and is expressed in a differentiation-dependent manner (Fig. 9). To which extent other MBDs can actually back up MeCP2 function has yet to be determined. Double or triple knockouts including MeCP2, MBD2, and MBD1 are required to further elucidate functional redundancies within the MBD protein family.
Both aspects of MeCP2 function (i.e., stabilization of transcriptional patterns and functional redundancy with other MBDs) would also explain why Rett syndrome patients as well as MeCP2 knock-out mice are viable and form differentiated tissues (for review see Kriaucionis and Bird, 2003), indicating that MeCP2 is not, per se, essential for cellular differentiation.
Our results clearly show that MeCP2 and MBD2 protein levels dramatically increase during differentiation and that either of them is sufficient to induce a large-scale chromatin reorganization during terminal differentiation, and thus represent a molecular link between nuclear genome topology and cellular differentiation.
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Materials and methods |
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Mouse tissue, cell culture, and transfection
Mouse muscle fibers from 50-d-old male MeCP2/y (Guy et al., 2001) and from 12-wk-old male C57BL/6J used as controls were dissected from the hind limb and immediately frozen.
C2C12 mouse myoblasts and Pmi28 primary mouse myoblasts were cultured and differentiated as described previously (Cardoso et al., 1997; Kaufmann et al., 1999). The suv39h1/2 double-null and wild-type mouse fibroblasts (MEF-D15 and MEF-W9, respectively) were cultured as described before (Peters et al., 2001).
Pmi28 cells were transfected with PolyFect reagent (QIAGEN). For in vivo analysis, cells were transfected by the Ca3(PO4)2 coprecipitation method as described previously (Cardoso et al., 1997).
To relocate MeCP2-YFPexpressing cells after post-fixation and FISH procedures, cells were cultivated on coverslips that featured 500 photoetched alphanumerical squares (Bellco). Myocytes were identified as postmitotic single nucleated cells in differentiated myotube cultures. To exclude cell cycledependent influences we included only S-phase cells for the evaluation of cycling myoblasts identified by pulse-labeling with BrdU (10 µM) 30 min before fixation. To determine post-mitotic cells, i.e., G0 cells, fixation was preceded by a 24-h BrdU (10 µM) incubation period. Details on BrdU detection can be found in Solovei et al. (2001).
Western blotting
To compare the level of endogenous MeCP2, MBD2, and MBD3 protein in myoblasts versus myotubes, it was important to normalize for equivalent number of nuclei because myoblasts and myotubes have a very different cytoplasm/nucleus ratio and the methyl CpGbinding proteins are exclusively nuclear. For that purpose, DNA amounts from the different samples were measured using the Hoechst 33258 dye on a fluorimeter, and the arbitrary Hoechst fluorescence units from different cell suspension volumes were then compared to calculate equal nuclei amounts of cell suspension. Equivalent DNA-containing cell suspension aliquots were directly boiled in Laemmli loading buffer so that also insoluble proteins were solubilized and loaded onto the SDS-PAGE gels. This step was also relevant because methyl CpGbinding proteins are mostly bound to chromatin containing methylated DNA and therefore not efficiently extracted from the cells. MeCP2 was detected with a rabbit pAb (Abcam); MBD2 and MBD3 with a goat pAb (Santa Cruz Biotechnology, Inc.). Antibody specificity was tested by probing extracts of cells overexpressing tagged versions of all other family members with each individual antibody. Nuclear protein amounts were controlled by probing with an anti-histone mouse mAb (Roche clone H11-4).
FISH and immunofluorescence
FISH with a mouse major satellite-specific probe was performed as described in Weierich et al. (2003). In brief, cells were fixed with 4% PFA in 1x PBS for myoblasts and in 0.75x PBS for myotubes. Cells were permeabilized with 0.5% Triton X-100/1x PBS followed by incubation in 20% glycerol and a repeated freezing/thawing step in liquid nitrogen. Additional pretreatments included incubation in 0.1 N HCl and for myoblasts/myotubes a pepsinization step.
The probe was generated by PCR using 5'-GCGAGAAAACTGAAAATCAC-3' and 5'-TCAAGTCGTCAAGTGGATG-3' as primers and mouse genomic DNA as template and labeled by nick translation using TAMRA-dUTP.
For immunofluorescence, cells were fixed as described for FISH until the Triton X-100 step. Mouse muscle fibers were fixed and permeabilized as cultured cells, but incubating 15 min for fixation and 40 min for permeabilization.
Detection of methylated DNA was performed as described previously (Habib et al., 1999). The following primary antibodies were used: rabbit anti-MeCP2 (Upstate Biotechnology) 1:25; mouse anti-5mC (Eurogentec) 1:100; goat anti-MBD3 (Santa Cruz Biotechnology, Inc.) 1:25; and mouse anti-FLAG M2 (Kodak) 1:2,000. As secondary antibodies we used goat antirabbit IgG-FITC (Sigma-Aldrich), goat antimouse IgG-Alexa 488 (Molecular Probes, Inc.), and donkey antigoat IgG-Cy3 (Rockland).
Nuclear counterstaining was done using DAPI, Hoechst 33258, or TO-PRO 3. Samples were mounted in Vectashield antifade (Vector Laboratories).
Southern blot
Genomic DNA from undifferentiated Pmi28 myoblasts and from differentiated cultures (6 d after application of differentiation medium) was isolated by spooling according to Sambrook and Russel (2001). Equal amounts of genomic DNA (5 µg) were digested overnight with the methylation-sensitive restriction enzyme HpyCH4 IV (5'-ACGT-3') (New England Biolabs, Inc.), analyzed by 1.2% agarose gel electrophoresis, and blotted onto Zeta-Probe membrane (Bio-Rad Laboratories). A major satellite-specific probe was generated by PCR as described for FISH, whereas a PCR fragment corresponding to a repeat monomer was used, which was extracted by gel elution and labeled radioactively by random priming method (Prime-It II; Stratagene). After overnight hybridization, the membrane was washed and exposed to a phosphor screen. Signals were detected on a phosphorimager.
Microscopy
Epifluorescence microscopy was performed at RT using an Axiophot 2 with 63x/1.4 oil and 100x/1.3 oil lenses (Carl Zeiss MicroImaging, Inc.), equipped with a Coolview CCD camera system (Photo Science Ltd.). Images were acquired with Cytovision software (Applied Imaging). Confocal image stacks were collected with an LSM410 and LSM510 microscope (Carl Zeiss MicroImaging, Inc.), equipped with 63x/1.4 oil and 63x/1.4 oil Ph3 lenses, respectively, at ambient temperature. For living-cell microscopy we used an FCS2 heated live cell observation chamber (Bioptechs) in combination with the Zeiss LSM510 microscope. The chamber was kept at a constant temperature of 37°C. The lateral resolution was between 0.05 and 0.1 µm. The axial resolution was between 0.2 and 0.5 µm in fixed cells and 0.75 µm in living cells. The temporal resolution of time series was 1 h.
Image analysis and evaluations
Endogenous MeCP2 levels and methylation of cytosines visualized by immunofluorescence were evaluated by wide-field epifluorescence microscopy. The fraction of cells showing pericentromeric staining was determined by visual inspection.
Chromocenters were counted from confocal stacks using Image Browser software (Carl Zeiss MicroImaging, Inc.) by scanning the xy plane plus additionally inspecting z planes to discriminate between signals on top of each other.
Quantification of MeCP2-YFP fluorescence for the correlation analysis was done by determining the intranuclear mean fluorescence intensity of YFP using Image J software. As a first step, a threshold-defined counterstain-derived binary stack was created that defined the nuclear volume. This was used to set the signal intensity of all extranuclear voxels within the MeCP2-YFP channel to zero. All the remaining voxels were defined as intranuclear and their mean voxel intensity calculated.
Correlation analysis was performed using SPSS 11.5 software assuming a linear correlation. Differences between chromocenter numbers in different cell types were tested for statistical significance by comparing cumulative frequencies within individual cell populations using a two-sample Kolmogorov-Smirnov test.
Line scan analysis was performed on confocal mid-section images using LSM 5 Image Examiner software (Carl Zeiss MicroImaging, Inc.).
Online supplemental material
Video 1 and Video 2 show dynamic behavior of pericentric heterochromatin in C2C12 mouse myoblasts visualized in vivo. Fig. S1 depicts chromocenter number during myogenic differentiation. Fig. S2 shows that high level expression of GFP-HP1 does not induce clustering of pericentric heterochromatin. Fig. S3 shows that HP1-ß does not localize at pericentric heterochromatin in Suv39h double-null fibroblasts. Fig. S4 depicts splitting of chromocenters in a G2 cell. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200502062/DC1.
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Acknowledgments |
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This work was funded by grants from the Deutsche Forschungsgemeinschaft to T. Cremer, H. Leonhardt, and M.C. Cardoso.
Submitted: 10 February 2005
Accepted: 2 May 2005
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References |
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