In Situ Replication Assays

5-bromo-2′-deoxyuridine (BrdU; Sigma Chemical Co.) incorporation was performed by incubating exponentially growing cells in the presence of 40 μM BrdU for 10 min. Cells were then washed in PBS and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. For pulse–chase experiments, cells were washed twice with prewarmed PBS and once in prewarmed medium after the BrdU pulse, and then cultured for different time periods. The cells were further processed for isolation of nuclei or fixed with 2% paraformaldehyde in PBS.

Biotin-16-deoxyuridine (BiodU) incorporation on isolated nuclei was performed essentially as described by Krude et al. 1997. In brief, after nuclei isolation in vitro run-on was initiated by adjusting the nuclear preparation to final concentrations: 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 bio-dUTP; 20 mM creatine phosphate; 0.5 mM DTT; and 2.5 μg phosphocreatine kinase (all Boehringer Mannheim Corp.). After a 10-min incubation at 37°C, reactions were stopped by a 25× dilution in PBS and immediately fixed in 2% paraformaldehyde. Nuclei were then deposited onto polylysine-coated coverslips by gentle centrifugation through a 30% cushion of glycerol in PBS, rinsed twice with PBS, and permeabilized in 0.2% Triton X-100 in PBS for 5 min. For the BiodU detection FITC-conjugated streptavidin (Enzo) was used, and fluorescence analysis carried out as described.

Immunolabeling

Paraformaldehyde-fixed cells or nuclei were washed twice in PBS and then incubated in 0.2% Triton X-100 in PBS for 5 min and blocked in 5% BSA, 0.1% Tween 20 in PBS (blocking buffer) for 10 min. Specific antibodies were then added at the appropriate dilution in blocking buffer. Incubation was carried out for 45 min at room temperature. To visualize the staining, FITC or Texas red-conjugated goat anti–rabbit or anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) were used. During the final washing steps with 0.1% Tween 20 in PBS, 0.25 μg/ml, 4,6-diamino-2-phenylindole (DAPI; Sigma Chemical Co.) was added for DNA staining.

In experiments involving BrdU immunodetection, a denaturation step in 4 N HCl for 10 min at room temperature was incorporated, after the permeabilization treatment, to render BrdU incorporated into DNA accessible. Neutralization was achieved by washes in PBS, and the slides were further processed as described above. Since detection of the nuclear proteins studied here is sensitive to HCl extraction, a specific protocol was used for double staining with BrdU. Proteins were first revealed (first and second antibodies) on paraformaldehyde-fixed cells as described. After 3 washes in PBS and 0.1% Tween 20, a second fixation step was included using either 2% paraformaldehyde in PBS for 15 min or 5 mM ethylene glycol-bis(succinimidylsuccinate; Pierce Chemical Co.) in PBS for 30 min at 37°C, followed by several washes in 0.3 M glycine in PBS. After this step, DNA was denatured with 4 N HCl, before BrdU revelation. The coverslips were mounted in Vectashield (Vector Laboratories Inc.). Despite postfixation, some loss of signal could still occur. Incorporation of BiodU (on isolated nuclei) into DNA was used instead of BrdU as an alternative (not shown). This allowed us to avoid the HCl denaturation since the biotinyl residue is accessible in the double helix of DNA.

Antibodies

Rabbit polyclonal antibodies (pAbs) against the different acetylated isoforms of histones H4 and H2A have been characterized in detail (Turner et al. 1989). They recognize H4 isoforms acetylated on one of the four lysines (K) acetylated in vivo or H2A acetylated at lysine 5 (H2A Ac 5). They were used at final dilutions of 1:500 for R101/12 (K12), R14/16 (K16), and R123 (H2A Ac 5), and 1:1,000 for R232/8 (K8) and R41/5 (K5). mAbs against HP1α (2HP1H5) and HP1β (IMOD-1A97), kindly provided by Dr. R. Losson and Dr. P. Chambon (CNRS, Strasbourg, France), were used at final dilutions of 1/400. The mAb against human CAF-1 p150 (mAb1) was kindly provided by Dr. B. Stillman (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Smith and Stillman 1991). The rabbit pAb1, directed against the human CAF-1 p60, was kindly provided by Dr. Marheineke (Ecole Normale Superieure, Ulm, France; Marheineke and Krude 1998; Martini et al. 1998). The specificities of the antibodies were tested by Western analysis on all sources of material studied. Monoclonal rat anti-BrdU was purchased from Harlan; Sera-Labo, Texas red goat anti–rabbit immunoglobulin (Ig), FITC goat anti–rabbit Ig, and Texas red goat anti–rabbit Ig antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.

Enrichment of H4Ac 5 and 12 and Exclusion of H4Ac 8 and 16 Replication Foci Is Visible only in Late S Phase

From mammals to yeast, it has been demonstrated that H4 underacetylation is a characteristic feature of heterochromatin (Jeppesen and Turner 1993; O'Neill and Turner 1995; Belyaev et al. 1996). To examine the state of H4 acetylation at both early and late replication foci in HeLa cells, we combined pulse labeling with BrdU and immunodetection using antibodies specific for H4 acetylated at lys 5, 8, 12, or 16 (Turner et al. 1989). An antibody to acetylated H2A was used as a control for an acetylation group unrelated to de novo chromatin assembly. Confocal analysis showed that the different H4 and H2A antibodies displayed a general punctate nuclear staining (enriched in some regions and excluded from the nucleolus). In early S cells, replication foci appeared equivalently associated with the four acetylated isoforms of H4. In contrast, in late S cells, we distinguished two different staining patterns (Fig. 2 A): H4 isoforms acetylated at lys 5 or 12 were specifically enriched at late replication foci, whereas isoforms acetylated at lys 8 or 16 were excluded from these particular regions as is acetylated H2A. Similar results were obtained using mouse L929 or Xenopus A6 cells (not shown). This was further confirmed using a less destructive method (not shown, see Materials and Methods) that reveals the replication foci by in vitro run-on in the presence of Bio-16-dUTP (Krude 1995). To quantify these observations, we developed a program to estimate the degree of colocalization between two markers in sets of confocal optical sections. This program and experimental controls to validate its application are described in Materials and Methods. Thus, we could analyze the average behavior of several hundred foci, providing a more accurate estimation of the statistical significance of our observations (Fig. 2 B). From these data, we conclude that H4 acetylated at lys 5 or 12 is specifically enriched at late replicating foci, whereas forms acetylated at lys 8 or 16 are excluded. Since acetylation at lys 5 and 12 is specific to newly synthesized histone H4, this enrichment probably corresponds to the deposition of H4 on newly replicated heterochromatin regions. It is remarkable, however, that this specific enrichment could only be detected at late replication foci. The absence of specific enrichment of H4Ac 5 and 12 at early replication foci could be due to the multiple acetylation/deacetylation events occurring concurrently in euchromatin regions that may mask changes associated specifically with replication. Alternatively, the difference between the early and late replication profile could reflect a different mechanism for H4 deposition.

Although Equally Associated at Early and Late Replication Foci, CAF-1 Colocalizes with H4Ac 5 and 12 While Excluding H4Ac 8 and 16 only at Late Replication Foci

CAF-1 previously had been reported to be generally located at replication foci (Krude 1995). However, a strict comparison between early and late replication foci was not available. Using an mAb directed against p150-CAF-1 (Smith and Stillman 1989), a general localization of this subunit at both late and early replication foci was observed by confocal analysis (Fig. 3 A). Quantification of these data in sets of confocal sections, demonstrated that newly replicated regions are equally associated with CAF-1 in either early or late replication foci (Fig. 3 B). Importantly, we observed that not all the p150-CAF-1 foci were associated with replication foci (green spots), prompting us to analyze the temporal association of CAF-1 with DNA synthesis. In early S phase cells (Fig. 3 A), double staining patterns revealing each specific acetylated isoform of H4, together with the p150-CAF-1 subunit, were similar. In contrast, in late S phase p150-CAF-1 could be found associated with H4Ac 5 or 12, but never with H4Ac 16 or 8. Since H4Ac 8, in addition to H4Ac 5 and 12, were found in a complex with CAF-1 in human nuclear extracts (Verreault et al. 1996), these findings suggest that the CAF-1 complex including H4Ac 8 is not involved at late replication foci. The association of CAF-1 with early and late replication foci argues for a general chromatin assembly mechanism throughout S phase. Remarkably, H4Ac 5 and 12 association with CAF-1 reproduces the pattern of H4 acetylation associated with DNA replication in heterochromatin regions (Fig. 2).

The Temporal Relationships between Two DNA Synthesis Events Can Be Reflected by their Spatial Overlap

We used the BrdU/BiodU double labeling approach to relate a temporal link between two DNA synthesis events to the spatial overlap of their corresponding signals. Synchronized cells were allowed to progress to late S phase, pulsed first with BrdU (see Materials and Methods) and chased for various times in vivo before a second pulse labeling was performed on isolated nuclei by in vitro run-on in the presence of Bio-16-dUTP. We compared the localization of the BrdU-labeled patches with sites of DNA synthesis after various times, by merging both signals acquired by confocal microscopy (Fig. 4). In nuclei labeled immediately after the first pulse, both in vivo and in vitro replication sites clearly overlapped. Indeed, individual foci appeared mainly yellow, as shown in Fig. 4 B, even though the colocalization was not perfect, as expected for two consecutive events. Within 15 min, a partial separation of the two signals could already be observed. In the same domain we could visualize red (in vivo pulse with BrdU), green (in vitro pulse with BiodU), and yellow signals (both pulses). Although we cannot exclude that some red, green, and yellow domains could correspond to closely linked clusters of separate smaller foci, the frequency of this arrangement diminishing with time is in favor of a vast majority being the same foci. Foci in which only BiodU (green) or BrdU (red) was incorporated were likely to be those that had been switched on or off (respectively) during the chase. Consistent with this interpretation, we observed that the number of the latter increased with time, reflecting the progressive extinction of foci, whereas only a few new foci appeared during the chase, presumably due to the ending of S phase. In the same way, within double-labeled domains, separation between both signals increased with time and the respective BiodU and BrdU signals could be perfectly distinguished after 30 min. One hour after BrdU pulse, only a few BrdU foci were still associated with BiodU foci. In summary, the overlap between the two signals decreased as a function of time, due to both extinction and firing of replicating foci during the chase and to the progressive separation of clusters corresponding to DNA synthesized during the two pulses within one focus.

Quantification of BrdU-labeled regions associated with BiodU-labeled regions in sets of confocal sections showed a decrease in their spatial association, consistent with the visual observation (Fig. 4 C). These results are in agreement with previous in vivo studies in rodent cells using double pulse labeling in vivo and different methods of quantification (Manders et al. 1996; Ma et al. 1998a). The consistency of these data further supports the validity of our method. In this analysis, patches of synthesis occurring at 15 min intervals (corresponding approximately to the synthesis of 50 Kb between both pulses) can be distinguished. Thus, this analysis could be used as a reference to study the large scale relationships between factors of interest and DNA synthesis.

CAF-1 Localization at Heterochromatin Domains Is Temporally Linked to, but Not Superimposable with DNA Synthesis

We investigated how the association of recently replicated DNA with CAF-1 evolved as a function of time in a pulse–chase experiment. Synchronized cells were allowed to progress to late S phase, pulsed with BrdU (see Materials and Methods), and chased for various times in vivo. After the chase, CAF-1 and sites of BrdU incorporation were revealed, and their localization compared by merging both signals acquired by confocal microscopy. Immediately after the pulse, BrdU incorporation sites widely overlapped with CAF-1 sites (Fig. 5), although labeled sites were not exclusively yellow, as shown in Fig. 5 B. The signals corresponding to CAF-1 and BrdU had not separated within 20 min. This contrasts with the signals corresponding to two successive DNA synthesis events, which were separated at 15 min after BrdU pulse (Fig. 4). After 50 min, CAF-1 and BrdU signals had begun to separate, and reached complete separation at 80 min after pulse. As in the previous experiment, few foci with a single color appeared as chase time increased. Hence, CAF-1 association with BrdU incorporation sites appears related to DNA synthesis, but does not strictly follow the same dynamics.

Considering the association of BrdU signal with CAF-1, our quantitative analysis showed no significant change within the first 20 min after BrdU pulse (Fig. 5 C). After this time, following the extinction of replication foci, the percentage of colocalization decreased as a function of time. Furthermore, comparing the quantitative analysis of BrdU/CAF-1 and BrdU/BiodU pulse–chase experiments, a time difference of 20 min could be seen between release of CAF-1 and cessation of DNA synthesis. It is remarkable that after 80 min post-BrdU pulse, whereas replication had stopped within foci labeled at the time of the pulse, CAF-1 could still be found associated with BrdU incorporation sites. Further supporting these findings, we found that the two largest subunits of CAF-1, p60 and p150, detected respectively using a pAb and an mAb, behaved similarly in this assay. A similar analysis performed on early replicating foci gave comparable results (not shown), further suggesting a general behavior of CAF-1 throughout S phase. This behavior of CAF-1 is consistent with the rapid association of this assembly factor with newly replicated regions of DNA and its retention for at least 20 min at these locations.

The Association of H4Ac 5 and 12 With Heterochromatin Domains Is Transient

We then investigated how the association of H4Ac 5 with recently replicated regions of the genome evolved as a function of time in a pulse–chase experiment carried out as described above. Considering data in Fig. 2 and Fig. 3 showing that replication specific acetylated isoform of H4 (H4Ac 5 and 12) could not be detected enriched at early replication foci, we concentrated our study on late replication foci. Immediately after the pulse, BrdU incorporation sites were widely stained with H4Ac 5 antibody (Fig. 6), although labeled sites were not exclusively yellow, as shown in Fig. 6. The signals corresponding to BrdU were still associated with H4Ac 5 after a 20-min chase. Thus, clusters of newly synthesized DNA can been found associated with H4Ac 5 for at least 20 min. After 50 min, BrdU incorporation sites appeared partially dissociated from H4Ac 5 and both signals were completely distinct in G2 phase (after a 3-h chase). BrdU signal associated with H4Ac 5 was determined after different chase times on several optical sections from different nuclei to establish the statistical significance of our observations (Fig. 6 C). Similar data were obtained for H4Ac 12 in both human (Fig. 7) and Xenopus (not shown). Thus, H4 acetylation at lys 5 and 12 appeared to be transiently associated with late replicated sequences in both mammals and amphibians. This result contrasts with the maintenance of lys 12 acetylation reported for heterochromatin from both yeast and Drosophila (Turner et al. 1992; Braunstein et al. 1996). The loss of H4Ac 5 and 12 enrichment at late replicated sequences is likely to result from an enzyme catalyzed deacetylation or a loss of epitope accessibility. Preliminary immunolabeling experiments have failed to detect any specific enrichment of deacetylase catalytic subunits at late replication foci (not shown). This may be because the specific enzymes involved still have to be identified, though we cannot exclude the possibility that the deacetylase activity at replication foci is locally regulated by a cofactor or a modification, rather than by varying the amount of enzyme present. Alternatively, without any specific enrichment of HDAC, the lack of HAT in these regions could be sufficient to account for H4 deacetylation following replication as supported by Fig. 7.

Open in a separate window

Figure 7

Dynamics of H4 acetylation in heterochromatin regions is restricted to the time of their DNA replication. A, Sites of DNA synthesis in an asynchronous population of HeLa cells were first labeled in vivo by BrdU pulse (red), and chased for 90 min either in the presence or absence of TSA (50 ng/ml). Different acetylated isoforms of H4 were localized using specific pAbs (K5, K8, K12, and K16, green). B, Sites of DNA synthesis in an asynchronous population of HeLa cells were first labeled in vivo by BrdU pulse (red), and chased for 14 h to allow late replicating cells, labeled during the pulse, to exit mitosis, before prolonged incubation in the absence or presence of TSA (50 ng/ml) for 90 min. Different acetylated isoforms of H4 were localized using specific pAbs (green). Digital merge of both signals, corresponding to single optical sections, are shown in each case. Bars, 10 μm. Note that these images were processed differently to permit visualization, because the intensities recorded were very different + or − TSA. Enlarged inserts (top left of each images) correspond to selected foci.

Most importantly, these data strongly support the existence of a time window of ~20 min, during which heterochromatin regions are enriched in acetylated H4, followed by a deacetylation event.

Hp1α and -β Are Maintained through DNA Replication

The existence of local concentrations of specific proteins has been proposed to compartmentalize chromatin regions, creating an index system for the nucleus (Gasser and Laemmli 1987; Stone and Pillus 1998). The local concentration of specific proteins, such as Hp1α and -β, could also be critical for defining domains that should be repressed. In our study, no major changes in the distribution of Hp1α and -β were observed during DNA replication of heterochromatin regions (Fig. 1; and not shown). This was surprising at first, since the presence of Hp1α and -β may reflect a high level of DNA condensation (Cavalli and Paro 1998) that may not necessarily be compatible with the replication process. We thus conclude that, if the replication fork is destabilizing the nucleosome (Sogo and Laskey 1995), this is a local phenomenon that does not affect the stability of the larger scale organization in a detectable manner. The maintenance of the local concentration of Hp1α and -β as part of nucleoprotein complexes, either directly interacting or simply in close proximity with DNA, may ensure the rapid reassociation of the replicated regions into heterochromatin. Furthermore, the ability of HP1 proteins to oligomerize has led to the hypothesis that they can cross-link chromatin domains by the formation of concatenated multiprotein complexes (Cowell and Austin 1997). Thus, Hp1α and -β appear to be components that favor the maintenance of a heterochromatin region. Interestingly, in mouse cells, association of some genes with an HP1β domain has been shown to correlate with their transcriptional repression (Brown et al. 1997). One would thus predict that in vertebrates, overexpression of HP1β could result in a spreading or expansion of these domains, possibly producing phenotypes resembling PEV (Festenstein et al. 1996; Milot et al. 1996). Additional modification adding to stability in heterochromatin, such as methylation of DNA in vertebrates (Bird 1992), should also be incorporated into the overall picture. Future experiments will provide insights into these issues.

We thank Dr. B. Stillman, Dr. R. Losson, Dr. P. Chambon, and Dr. K. Marheinecke for antibodies. We thank Drs. G. Cavalli, P. Grandi, S. Holmes, C. Maison, J.G. Moggs, and P. Ridgway for critical reading. We are grateful to the anonymous reviewers for their useful suggestions.

A. Taddei was initially a Ministère de l'Education Nationale de l'Enseignement Supérieur de la Recherche fellow and then supported by the Association pour la Recherche sur le Cancer (ARC). This work was supported by a Training Mobility Research Network grant from the European Community (G. Almouzni and B. Turner) and funding from Fondation pour la Recherche Médicale, ARC, and Ligue contre le cancer.