Replication Stress Interferes with Histone Recycling and Predeposition Marking of New Histones

Molecular Cell

Short Article Replication Stress Interferes with Histone Recycling and Predeposition Marking of New Histones Zuzana Jasencakova,1 Annette N.D. Scharf,2 Katrine Ask,1 Armelle Corpet,3 Axel Imhof,2 Genevie`ve Almouzni,3 and Anja Groth1,* 1Biotech

Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark for Integrated Protein Science at Adolf-Butenandt Institute, Schillerstrasse 44, 80336 Munich, Germany 3Institut Curie UMR218-CNRS, 26 Rue d’Ulm, 75248 Paris, Cedex 05, France *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.01.033 2Center

SUMMARY

To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows that replication stress interferes with predeposition marking and histone recycling with potential impact on epigenetic stability.

INTRODUCTION Packaging of nucleosomal DNA into distinct structures relies on complex interplays between modifications of histones and DNA, histone variants, chromatin-binding proteins, and noncoding RNAs (Kouzarides, 2007; Probst et al., 2009). During cell division, this organization must be reproduced to maintain genome function and stability in cell progeny. In S phase, chromatin undergoes genome-wide disruption and restoration. Nucleosomes are disrupted ahead of the replisome, in a process likely coupled to unwinding of the double helix by the replicative helicase (Groth, 2009). Behind the fork, nucleosomal organization is restored through recycling of old parental histones and de novo deposition of new histones onto nascent DNA (Annunziato, 2005; Groth et al., 2007b). How histones are recycled remains a central question in epigenetics, as posttranslational modifications (PTMs) on old H3-H4 could serve

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important roles in chromatin restoration along with replicationcoupled recruitment of specific chromatin modifiers (Jasencakova and Groth, 2009). New histone H3.1-H4 dimers are diacetylated at lysines 5 and 12 of H4 (Loyola et al., 2006; Sobel et al., 1995), an evolutionary conserved mark imposed by HAT1 (Parthun, 2007). In yeast, all new H3 histones are also acetylated at lysine 56 (Rocha and Verreault, 2008), but whether this is the case in mammals remains open. With the exception of H3K9me1 imposed prior to assembly (Loyola et al., 2006), soluble histones H3.1-H4 are not methylated to any significant extent. The histone chaperone anti-silencing function 1 (Asf1) handles both replication-dependent histones H3.1-H4 and replacement variants H3.3-H4 (Tagami et al., 2004). Asf1 binds histone H3-H4 dimers and sterically precludes formation of histone (H3-H4)2 tetramers (De Koning et al., 2007). During chromatin replication, Asf1 provides histones to chromatin assembly factor 1 (CAF-1) localized at the replication fork via interaction with PCNA (Shibahara and Stillman, 1999; Tyler et al., 1999). Multicellular organisms have two Asf1 homologs, Asf1a and Asf1b, with largely redundant functions in S phase histone dynamics. Recently, we reported that Asf1 (a and b) form a complex with the replicative helicase MCM2-7 on chromatin and are required for replication fork progression (Groth et al., 2007a). Furthermore, histones together with Asf1 showed two typical chromatin marks, H4K16ac and H3K9me3, giving rise to the hypothesis that Asf1 handles both new and parental histones during replication (Groth et al., 2007a). We therefore reasoned that a comprehensive profiling of PTMs on Asf1-bound histones should provide insight into how chromatin is replicated. We took advantage of our human cell lines expressing epitope-tagged (e-) Asf1 to purify complexes, and analyzed H3 and H4 PTMs by quantitative mass spectrometry and western blotting. Given that Asf1 handles excess histones during replication stress (Groth et al., 2005), we also addressed changes in complexes and histone marks resulting from acute replication arrest.

RESULTS Distinct Cytosolic and Nuclear Asf1 Complexes in S Phase We isolated e-Asf1 complexes from cells synchronized in mid-S phase before and after treatment with the replication inhibitor

Molecular Cell Histone Modifications in Asf1 S Phase Complexes

Figure 1. Characterization of Asf1 S Phase Complexes (A) Strategy for cell synchronization (right) and purification of e-Asf1 complexes (left). Cells were harvested in mid-S phase (S) or after 1.5 hr HU treatment (S + HU) for FACS analysis of DNA content and complex purification. (B) Coomassie staining of e-Asf1b complexes. Recombinant Asf1a (rAsf1a) was used to estimate quantity. Mass spectrometry identified the indicated proteins. MCM2, -4, -6, and -7 were described (Groth et al., 2007a). For e-Asf1a complexes, see Figure S1. (C) Analysis of e-Asf1a and e-Asf1aV94R complexes. (D) Overview of Asf1-H3-H4 interactors and their cellular distribution. (E) Gel filtration analysis of nuclear extracts from HeLa S3 cells in S phase. Two separate Asf1 complexes are indicated.

hydroxyurea (HU) (Figure 1A). Prior to complex purification, cells were fractionated into cytosolic and nuclear extracts to separate soluble material from nuclear chromatin-associated proteins retrieved by high-salt extraction (see the Supplemental Information). Asf1a and Asf1b complexes were overall similar (Figure 1B and Figure S1), as previously described for the nuclear complexes (Groth et al., 2007a). Mass spectrometry analysis of the cytosolic complexes identified Importin-4, MCM2, NASP, and RbAp46/48 (Figure 1B), all factors known to interact with histones. We thus asked whether their interaction with Asf1

depends on histones, taking advantage of cells expressing a mutated version of Asf1a abrogating H3 binding, Asf1aV94R (Groth et al., 2007a). In this histonebinding mutant, all Asf1 partners in the cytosolic complexes were lost (Figure 1C). We conclude that H3-H4 dimers form the central part of these complexes, being shielded from spurious interactions while in transit. We also identified the chaperones NASP and RbAp46/48 in nuclear Asf1 complexes (Figures 1B and 1D), along with MCM2, -4, -6, and -7 as expected (Groth et al., 2007a). Size-exclusion chromatography revealed that Asf1 (a and b) are part of two separate nuclear complexes (Figure 1E), a larger complex with MCM6 and a smaller one containing NASP. Since the latter is distinct from the previously described chromatin-bound Asf1-H3-H4-MCM2-7 complex, we envision that this ‘‘multichaperone’’ complex acts as histone donor upstream of CAF-1. Acute inhibition of replication triggered accumulation of almost all components including histones in the complexes (Figure 1B). This likely reflects that Asf1 stores excess histones H3.1-H4 building up when histone deposition is blocked (Groth et al., 2005). PTM Profile of Histones H3-H4 in Asf1b Complexes We then analyzed histones bound to Asf1b, the isoform most dedicated to replication (Tagami et al., 2004; Tang et al., 2006), by MALDI-TOF and LTQ Orbitrap, a high-mass accuracy mass spectrometer. Histone PTMs were quantified relative to total amount of the respective peptide in samples from four biological

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Molecular Cell Histone Modifications in Asf1 S Phase Complexes

Figure 2. Profiling of Marks on Histones H3-H4 in Asf1b Complexes Histones H3 and H4 were purified from cytosolic (cyt) and nuclear (nuc) Asf1b complexes as shown in Figure 1B. (A–E) Analysis by quantitative mass spectrometry. All graphs show averages of four biological replicates with error bars indicating SEM. For further details, see Supplemental Information, Figures S5–S12 and Tables S1 and S2. (A) Quantification of acetylation on the H4 peptide aa 4–17. The major diacetylated species, K5K12diAc, was identified by MS/MS (Table S1 and Figure S7). (B) Quantification of histones H3.1 and H3.3. (C) Quantification of modifications on the H3 peptide aa 54–63. MS/MS identified K56ac (Figure S12). (D) Quantification of modifications on the H3 peptide aa 18–26. MS/MS identified the acetylated species as mainly H3K18ac (Table S2 and Figure S10). (E) Quantification of modifications on the H3 peptide aa 9–17. MS/MS identified the acetylated species as mainly H3K14ac and the methylation as H3K9me1 (Table S2 and Figure S9). (F) Western blot analysis of histone PTMs. Asf1 complexes were analyzed in parallel with serial dilution of chromatin pellet from the same number of S phase cells. The antibodies did not crossreact with unmodified histones (Figure S2).

replicates (see the Supplemental Information). Histone H4 was almost exclusively diacetylated at lysines 5 and 12 (Figure 2A), consistent with the presence of HAT1 in the cytosolic complex (Figure 1C). Western blot analysis confirmed high enrichment of H4K12ac in S phase Asf1b complexes as compared to chromatin (Figure 2F). H4K5K12diAc is an established mark of newly synthesized histones, present on about 70% of soluble histones H3.1-H4 in asynchronous cells (Loyola et al., 2006). Our data show that >95% of new H3-H4 dimers delivered through the Asf1 pathway during replication carry this mark. For histone H3, we first looked at peptides distinct for H3.1, the canonical replication-dependent histone, and H3.3, a replacement variant expressed at low levels throughout the cell cycle. In S phase, Asf1-H3.1-H4 complexes were around 5-fold more abundant than those containing H3.3 (Figure 2B). Upon HU treatment this ratio increased significantly, verifying at the level of endogenous histones that Asf1 buffers excess H3.1-H4 accumulating when histone demand suddenly drops (Groth et al., 2005). Yeast Asf1 is required for ubiquitous acetylation of new histones H3 at lysine 56. We could detect H3K56ac in human Asf1 complexes, but only at very low levels close to the technical

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limit for quantification (less than 1.5%, Figure 2C). This argues that H3K56ac is not a general mark of new histones comparable to H4K5K12diAc in human cells. In fact, other histone H3 acetylations were more prominent in our S phase Asf1 complexes, in particular tail acetylations at K14 and K18 (Figures 2D and 2E). These modifications were most abundant in the nuclear complexes and did not change after HU treatment. In contrast, H3K9me1, the most prevalent methylation mark with Asf1b, increased considerably by about 3-fold in both cytosolic and nuclear complexes after acute replication arrest (Figure 2E). Western blotting confirmed this and illustrated how the mark upon HU treatment became enriched in Asf1 complexes relative to chromatin (Figure 2F). H3K9me1 can be imposed on new histone H3.1 prior to deposition, as a first step in establishing H3K9me3-mediated silencing (Loyola et al., 2006, 2009). Our data suggest that Asf1 functions in this assembly line and that replication stress deregulates K9 monomethylation. Asf1b complexes also contained a number of less abundant modifications, including H3K18me1, H3K27ac, H3K27me1, H3K36ac, H3K79ac, H3K79me1, H4K31ac, and H4K77ac (Tables S1 and S2 and Figures S6–S12). With respect to H3K27 marks, both acetylation and monomethylation were mainly detected in the nuclear complex after HU treatment (Table S2). Interestingly, MS1 scans also showed H3 peptides aa 9–17 and aa 27–40 carrying di- or trimethylation within these complexes (Figure S5), but the peptides were not sufficiently

Molecular Cell Histone Modifications in Asf1 S Phase Complexes

Figure 3. Asf1 Is Retained with MCM2-7 on Chromatin upon Replication Arrest (A) Analysis of Asf1 in cytosolic and nuclear extracts from cells treated as in Figure 1A. 2x indicates a double input of the corresponding lysate, 1x. (B) Analysis of Asf1 in detergent-soluble and insoluble (chromatin-bound) fractions. Cells were treated as in (A) and fractionated as shown. Histone H4 was used as a loading control for chromatin. (C) Analysis of chromatin-bound Asf1b complexes. Following extraction of soluble proteins, chromatin-bound proteins were released by DNase I and e-Asf1b complexes isolated under high-stringency conditions. (D) Analysis of Asf1b and MCM2 localization by confocal microscopy. HeLa S3 cells were pre-extracted with 0.5% Triton on ice prior to fixation and immunofluorescence staining. (Top) Representative field of cells in mid-S phase treated with or without HU for 2 hr. Scale bar, 20 mm. (Bottom) Representative S phase cell treated with HU for 2 hr. Scale bar, 5 mm. Specificity of the Asf1b antibody was confirmed in RNAi experiments (Figure S3).

abundant to get MS/MS identification. Nevertheless, this finding was intriguing, as we previously observed H3K9me3 in nuclear Asf1 complexes (Groth et al., 2007a). We thus applied western blotting to identify low-abundance PTMs using specific antibodies, not cross-reacting with unmodified recombinant H3 and H4 (Figure S2). We detected several PTMs, including H3K4me3, H3K9me2, H3K9me3, H3K27me3, H4K20me2, and H4K16ac, in nuclear Asf1b complexes after HU treatment (Figure 2F). Since this type and variety of modifications are typical for nucleosomal H3-H4 in chromatin (Loyola et al., 2006), it argues that evicted histones become trapped with Asf1 when histone deposition is blocked upon replication arrest. The low abundance of these marks in the complexes is anticipated, as evicted histones like nucleosomes in chromatin show a heterogenous modification pattern.

Asf1 Is Trapped with ChromatinBound MCM2-7 upon Replication Stress During HU treatment the level of Asf1b in nuclear extracts increased (Figure 3A), and this correlated with accumulation of Asf1 on chromatin (Figure 3B). Given that Asf1-H3-H4-MCM2-7 complexes can be isolated from chromatin (Groth et al., 2007a), we anticipated that more Asf1 was trapped in histone-dependent interactions with chromatin-bound MCM2-7. Consistently, DNase I treatment of chromatin from cells exposed to replication stress released higher amounts of Asf1 in complex with MCMs (Figure 3C). We then used antibodies highly specific in immunocytochemistry (Figure S3) to address Asf1b localization on chromatin in pre-extracted cells. Asf1b associated weakly with chromatin in unperturbed cells (Figure 3D), probably reflecting its highly dynamic behavior. Upon HU treatment, Asf1b was retained on chromatin and localized in distinct patterns overlapping considerably with MCM2 staining (Figure 3D and Figure S3). As expected, MCM2 showed typical S phase patterns that did not change upon HU treatment. The MCM2-7 helicase is mainly found in unreplicated regions (Dimitrova et al., 1999; Laskey and Madine, 2003), but a fraction of these complexes are presumably present at sites of active replication (Takahashi et al., 2005). Our data argue that Asf1 is trapped with MCM2-7 when replication is perturbed. However, the retention of Asf1 on chromatin is weak, as Asf1b staining was lost upon a short room temperature wash prior to fixation

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(Figure S3). This suggests that the interaction of Asf1 and histones with MCM2-7 probably remains rather dynamic during replication fork stalling. Replication Stress Challenges Histone Recycling and Predeposition Marking Replication stress causes ssDNA formation at replication sites, likely because the DNA helicase continues unwinding after the polymerases arrest (Pacek and Walter, 2004). Nucleosomes are disrupted in this process (Hall et al., 2009), and as new DNA is not available for deposition, normal recycling of evicted histones would be compromised. This could explain the presence of evicted histones with Asf1 under stress conditions. To follow this potential link, we compared the kinetics of eviction and ssDNA formation. S phase cells responded homogenously to HU, generating ssDNA at replication sites marked by CAF-1 staining (Figure 4A). Globally, this resulted in a 3-fold increase in ssDNA within 1.5 hr with little change upon prolonged treatment (Figures 4A and 4B). Similarly, analysis of Asf1 complexes showed that chromatin marks accumulated mainly within the first 1.5 hr after HU treatment (Figure 4C), supporting the idea that these histones could be evicted from replication sites upon ssDNA formation. We noted that histones with these PTMs were maintained in the Asf1b complexes during prolonged replication arrest (Figure 4C) and thus asked whether they would be reused during recovery. After removal of HU, cells efficiently resumed replication and progressed in S phase (data not shown). Notably, the pool of Asf1-bound histone H3-H4, including the population carrying chromatin marks, decreased substantially in the recovery process (Figure 4D). This argues that histones, new and old, trapped with Asf1 during replication stress are used to fulfil the high demand arising when forks restart. In this scenario, histones H3 monomethylated at lysine 9 would also be deposited upon resumption of DNA synthesis. Indeed, during recovery H3K9me1 levels dropped significantly in Asf1 complexes as well as in nuclear extracts (Figure 4E). This mark has been implicated in heterochromatin assembly in mid to late S phase (Loyola et al., 2009), suggesting that H3K9me1 levels on new histones would reflect this temporal regulation. As anticipated, H3K9me1 was more abundant in Asf1 complexes from late S phase cells in comparison to cells in early S phase (Figure 4F). However, the levels of H3K9me1 increased dramatically in early S phase cells challenged with replication stress (Figure 4F), implying that this response could have effects on euchromatin restoration. DISCUSSION Here we present a detailed analysis of histone H3-H4 PTMs and core components of cell-soluble and nuclear Asf1 complexes involved in chromatin replication. We show that H4K5K12diAc marks all new histones delivered by Asf1, while 20%–30% carry H3 tail-acetylations at K14 and/or K18. This is consistent with previous studies of new histones (Benson et al., 2006; Loyola et al., 2006; Sobel et al., 1995; Tyler et al., 1999). Importantly, we provide evidence that (1) Asf1 is implicated in monomethylation of H3K9 prior to deposition and that perturbation

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of replication significantly increases the abundance of this mark in Asf1 complexes; (2) Asf1 handles old histones evicted upon replication fork stalling and ssDNA formation; and (3) Asf1-bound histones are rapidly used upon recovery from replication stress, raising the possibility that premarked histones could be incorporated at unscheduled sites. While in yeast acetylation at H3K56 is ubiquitous for new histones (Rocha and Verreault, 2008), we found this mark only on a minor fraction of histone H3 (about 1%) in human Asf1b complexes. Histones in Asf1a complexes from asynchronous cells also showed low abundance of H3K56ac (data not shown). This is consistent with the low level of H3K56ac in chromatin from HeLa cells (about 1%) (Das et al., 2009; Xie et al., 2009). Thus, if H3K56ac serves a general role in the mammalian Asf1-CAF-1 assembly pathway, it would have to be much more transient than in yeast. Alternatively, this mark could function in a specialized assembly pathway, as suggested from analysis of human ES cells (Xie et al., 2009). In contrast, H4K5K12diAc is imposed on all Asf1-bound histones by RbAp46-HAT1 in the cytoplasm (this work and Barman et al., 2008). It is notable that histone H3-H4 dimers in complex with Asf1 can interact stably with other proteins including chaperones (NASP, RbAp46/48) and import factors (Importin-4). This finding is consistent with the idea that multichaperone complexes handle new histones in transit (Groth et al., 2005). Given that a cytosolic Asf1 complex can provide histones to CAF-1 in vitro (Groth et al., 2005; Mello et al., 2002; Tyler et al., 1999), our cytosolic complex or the highly related nuclear NASP-containing complex probably represents the histone donor for CAF-1 during de novo deposition. Recent evidence shows that SETDB1, in complex with HP1a and CAF-1, monomethylates H3.1 at K9 during assembly of heterochromatin (Loyola et al., 2009). We detected H3K9me1 in S phase Asf1 complexes, suggesting that Asf1-H3-H4 may dock on to CAF-1 as this mark is established. Importantly, when chromatin replication is blocked, H3K9me1 levels increase substantially on available histones in Asf1 complexes regardless of whether cells are in early or late S phase. Given that this mark can be a precursor of H3K9me3 on nucleosomes (Loyola et al., 2009), incorporation of such premarked histones during fork restart could pose a danger of unscheduled silencing. We previously proposed that Asf1 accepts histones H3-H4 released upon nucleosome disruption during progression of the MCM2-7 helicase (Groth et al., 2007a). After acute replication stress, we find a variety of PTMs typical of chromatin on histones H3-H4 in nuclear Asf1 complexes. Histones carrying these marks appear at the time ssDNA is generated at replication sites and no new DNA is available for deposition. This further supports a role of Asf1 in recycling of parental histones and the model that histones H3-H4 pass through a transient dimeric state prior to restoration of ‘‘old’’ tetramers (Groth, 2009). At unperturbed forks, histone transfer to new DNA is probably rapid and highly efficient (Gruss et al., 1993). We suspect that histones, caught in transfer upon fork stalling, are initially maintained in Asf1H3-H4-MCM complexes on chromatin. Consistently, we find that Asf1 is retained and partly colocalizes with chromatin-bound MCM2 upon replication arrest. While some ssDNA foci

Molecular Cell Histone Modifications in Asf1 S Phase Complexes

Figure 4. Replication Stress Challenges Histone Recycling and Predeposition Marking (A) (Top) Time course analysis of ssDNA by immunoflourescence. Scale bar, 50 mm. (Bottom) Colocalization of ssDNA and CAF-1 p60 using confocal microscopy. A representative mid-S phase cell is shown. Scale bar, 5 mm. HeLa S3 cells were prelabeled with BrdU, synchronized in S phase, and treated with HU as in Figure 1A. To reveal ssDNA, we exploited that the BrdU epitope is accessible in ssDNA and occluded in dsDNA. Some ssDNA foci also contained Asf1b (Figure S4). (B) Quantification of ssDNA by semiquantitative dot blot. DNA from cells prelabeled with BrdU and treated with HU was analyzed under native conditions. BrdU in ssDNA was detected by western blotting. Error bars represent standard deviation of two independent experiments performed in triplicates. (C) Time course analysis of PTMs in Asf1b complexes after HU treatment. We purified nuclear e-Asf1b complexes from cells in S phase (0) and after HU treatment as indicated. Chromatin from S phase cells was included for comparison. (D) Asf1 recycles histones during recovery. We purified e-Asf1b complexes from S phase cells treated as indicated. For recovery (R), HU was washed out and cells were allowed to progress in S phase for 1.5 hr. (E) Analysis of H3K9me1 in Asf1b complexes (left) and nuclear extracts (right) during recovery from HU. Cells were treated as in (D). (F) Comparison of H3K9me1 in Asf1b complexes from early and late S phase cells challenged with replication stress. (Left) FACS analysis of synchronized cells. (Right) Western blot analysis. 2x indicates a double input of the corresponding complex, 1x. (G) Hypothesis. Replication stress is known to promote genetic instability and cancer (Halazonetis et al., 2008). We show here that acute replication arrest interferes with normal histone dynamics and triggers accumulation of H3K9me1 and evicted histones in Asf1 complexes. We hypothesize that unscheduled incorporation of such premarked histones could present a hazard to chromatin restoration and potentially introduce epigenetic changes in cell progeny.

contained Asf1 (Figure S4), the overall pattern of Asf1 localization was distinct from that of ssDNA. This parallels the behavior of MCM2-7, which is present at high levels in unreplicated regions and cannot be localized to replication sites by immunofluorescence in vertebrates (Dimitrova et al., 1999; Laskey and Madine, 2003). To explain this ‘‘MCM paradox,’’ it has been argued that either MCMs work at a distance as pumps (Laskey and Madine, 2003) or helicases at active forks are below detection limit (Takahashi et al., 2005). Our analysis of histone PTMs implies that the majority of Asf1-MCM complexes contain newly synthesized histones H3.1-H4 diacetylated at K5 and K12. An attractive hypothesis is that new histones are stored with latent MCM complexes on chromatin, while those at active forks would be

involved with parental histones. Future studies will aim to address this issue. Our data indicate that evicted histones become trapped with Asf1 when replication is perturbed and remain available for deposition upon recovery. Impeding the normally rapid transfer process could increase the risk that old histones are not deposited at their proper site. Moreover, incorporation of premarked histones, including those carrying H3K9me1, at unscheduled sites could also pose a hazard. We thus speculate that replication stress, along with well-characterized effects on genome stability, also challenges integrity of the epigenome (Figure 4G). Future work should aim to address whether replication stress by jeopardizing proper chromatin restoration can trigger epigenetic changes in cell progeny.

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EXPERIMENTAL PROCEDURES

Das, C., Lucia, M.S., Hansen, K.C., and Tyler, J.K. (2009). CBP/p300-mediated acetylation of histone H3 on lysine 56. Nature 459, 113–117.

Cells, Synchronization, and Drug Treatment HeLa S3 stable cell lines expressing OneStrep-tagged (e-) Asf1a, Asf1b, and Asf1aV94R were described (Groth et al., 2007a). We synchronized cells by a single thymidine (2 mM) block and released them into S phase with deoxycytidine (24 mM) 3 hr prior to HU (3 mM) treatment. For recovery, we washed out HU and added fresh medium.

De Koning, L., Corpet, A., Haber, J.E., and Almouzni, G. (2007). Histone chaperones: an escort network regulating histone traffic. Nat. Struct. Mol. Biol. 14, 997–1007.

Complex Purification and Biochemistry We purified e-Asf1 complexes under high stringency from cytosolic and nuclear extracts or chromatin solubilized by DNase I digestion (Groth et al., 2007a; Jasencakova and Groth, 2008; Supplemental Information). For gel filtration and primary antibodies, see the Supplemental Information.

Groth, A. (2009). Replicating chromatin: a tale of histones. Biochem. Cell Biol. 87, 51–63.

Mass Spectrometry Excised protein bands were analyzed by liquid chromatography-MS/MS on a Qstar elite machine at the LSMP Platform, Curie Institute, as described (Groth et al., 2007a). Excised histone bands were analyzed on a Voyager DE STR spectrometer (Applied Biosystems) and LTQ-Orbitrap mass spectrometer (Thermo Fisher) as described in the Supplemental Information. ssDNA Detection and Immunofluorescence The BrdU epitope occluded in dsDNA is available for antibody recognition in ssDNA. We used dot blotting and immunofluorescence to detect BrdU under native conditions in cells treated with or without HU (see the Supplemental Information). MCM2 and Asf1b localization was analyzed by confocal microscopy in cells pre-extracted on ice with 0.5% Triton (see the Supplemental Information). SUPPLEMENTAL INFORMATION Supplemental Information includes 12 figures, 2 tables, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at doi:10.1016/j.molcel.2010.01.033. ACKNOWLEDGMENTS We thank C. Alabert, W. Faigle, and members of our labs for help and discussions. Z.J. is supported by a Benzon Investigator Fellowship. A.G.’s laboratory is supported by the Lundbeck Foundation, Fabrikant Vilhelm Pedersen og Hustrus Mindelegat, the Danish Cancer Society, and the Danish Research Council. G.A.’s laboratory is supported by la Ligue Nationale contre le Cancer, Epigenome Network of Excellence, and Cance´ropoˆle Ile-de-France. A.N.D.S. is supported by Boehringer predoctoral fellowship. A.I.’s laboratory is supported by the European Union (LSHG-CT2006-037415) and the Deutsche Forschungsgemeinschaft (SBF/TR5, M4). Received: July 23, 2009 Revised: November 25, 2009 Accepted: January 25, 2010 Published: March 11, 2010 REFERENCES Annunziato, A.T. (2005). Split decision: what happens to nucleosomes during DNA replication? J. Biol. Chem. 280, 12065–12068. Barman, H.K., Takami, Y., Nishijima, H., Shibahara, K., Sanematsu, F., and Nakayama, T. (2008). Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex. Biochem. Biophys. Res. Commun. 373, 624–630. Benson, L.J., Gu, Y., Yakovleva, T., Tong, K., Barrows, C., Strack, C.L., Cook, R.G., Mizzen, C.A., and Annunziato, A.T. (2006). Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange. J. Biol. Chem. 281, 9287–9296.

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