Mutation of chromatin modifiers in vivo often results in embryonic lethality and impaired development (Table 1). Despite this, murine ES cell lines lacking individual modifiers have been established and, in many cases, shown to retain multi-lineage potential. Using a panel of mutant ES cell lines (described in detail in Table 1) we examined whether a lack of specific histone methyltransferases, DNA methyltransferases, the NuRD nucleosome remodeling complex or Dicer activity was sufficient to alter the temporal profile of locus replication in ES cells. All ES cell lines examined displayed ES cell morphology, expressed markers that are characteristic of murine ES cells (such as Oct4, alkaline phosphatase and SSEA-1) and had cell cycle profiles that were comparable with wild-type ES cells (supplementary Table 1 in Additional data file 1, and supplementary Figure 1 in Additional data file 2).

The replication timing profiles of Oct4, Esg1, Nkx2.9 and Mash1 for four independently derived wild-type (white bars) and eight mutant ES cell lines that lack Mll, Eed, Dnmt 1, Dnmt 3a/3b, Mbd3, G9a, Suv39 h1/h2 or Dicer (gray bars) are shown in Figure 1a. Histograms indicate the abundance of newly synthesized DNA corresponding to each locus in samples prepared from sequential stages of the cell cycle (G1-S, S1, S2, S3, S4 and G2/M) for all the wild-type and mutant ES cell lines. Oct4, which replicates early in S-phase in all cell types analyzed, showed only minor differences between wild-type and mutant cell lines (upper panel). Similarly, there was little variation in the early replication of Esg1 and late replication of Mash1 in wild-type and mutant ES cells, even though these loci are capable of switching replication timing upon differentiation; Esg1 has been shown to shift to late replication upon neural induction while Mash1 shifts to become early replicating 216. Nkx2.9, a neural specific gene that replicates in mid S-phase in undifferentiated ES cells showed some variation between wild-type and mutant cells. This analysis was extended to include a wider set of candidate loci that also have been shown to be permissive for changes in replication timing 2417. Figure 1b summarizes the data for 14 genes (shown in supplementary Figure 2 in Additional data file 2), in which replication timing is color-coded according to peak abundance in G1-S and/or S1 (early, green), S2 (mid-early, lime), S2 and S3 (middle, yellow), S3 (mid-late, orange), S4 and/or G2/M (late, red). Early replication of Nanog, Zfp57, Oct4, Esg1, Sox2 and Rex1 was unaffected in mutant ES cells lacking either a chromatin activator (Mll) or repressive chromatin modifiers (Eed, Dnmt1, Dnmt3a/3b, Mbd3, G9a, Suv39h1/h2 and Dicer) compared to wild-type cells (OS25 and WT). The replication of several middle- and late-replicating genes was also unchanged in chromatin modifier mutant ES cells, although three loci (Mage a2, Ebf, Sox3), in addition to Nkx2.9, showed some changes in replication patterns in the mutant lines. Sox3 replicated earlier in ES cells that lacked Dnmt1, Dnmt 3a/3b or Dicer but slightly later in Eed-deficient ES cells. Mage a2 was sensitive to loss of G9a (supplementary Figure 3 in Additional data file 2). This gene is transcriptionally regulated by G9a 18 (supplementary Figure 2 in Additional data file 2), and belongs to the Mage genes that are DNA methylated in adult somatic tissues 19. Replication of Ebf, a gene that replicates earlier in pro- and pre-B cells than in ES cells 17, showed slight shifts in replication in G9a, Suv39 h1/2 and Dicer-deficient ES cells. From a total of 14 loci analyzed in Figure 1b, four showed a temporal shift in one or more mutant ES cell lines. We analyzed the sequence context of the genes (supplementary Table 2 in Additional data file 1) but neither GC content nor Line density was obviously different between genes that change replication timing or those that remain unchanged in the mutant cells. Bivalent genes were represented among loci that showed shifts in response to loss of chromatin modifiers (such as Nkx2.9 and Msx1) as well as those that did not change their timing of replication (such as Math1 and Sox1; supplementary Figure 2 in Additional data file 2). Some replication timing changes were in the predicted direction (that is, an advance upon loss of a repressive chromatin modifier) whereas others were counter-intuitive, which we cannot explain. Importantly, however, we did not observe consistent shifts towards earlier or later replication in response to removal of a specific chromatin modifier. These results suggest that while some loci may be more sensitive to chromatin changes than others, none of the chromatin modifiers studied here is capable of overt de-regulation of the temporal order of gene replication in ES cells. This was true even for cells lacking Eed (and hence devoid of methylated H3K27), a factor previously shown to be important for transcriptional repression and chromatin bivalency in ES cells 15. Thus, our data do not support a model where methylation of specific histone residues or CpG dinucleotides confers replication at a certain time in S-phase. To explore this further, we also analyzed a large contiguous region surrounding Rex1 (Figure 1c), a gene that is expressed and early replicating in ES cells, but switches to late replication in differentiated cells and concomitantly looses histone acetylation as the gene is silenced 2. Chromosome walking has previously identified two domains within this 5 Mb region that replicate early in ES cells and switch to late replication upon differentiation (marked by red lines in Figure 1c) 2 (P Perry and VA, unpublished). Analysis of this entire region in each of the chromatin modifier mutants showed that the boundaries of early and late replication were retained.

An explanation for why the replication times of several loci are unchanged in mutant ES cells might be that other modifications compensate for this loss - for example, increased DNA methylation might compensate for loss of Eed-mediated repression. To address this possibility we knocked-down Eed (using short hairpin RNA) in ES cells that already lacked Dnmt1 (supplementary Figure 4a in Additional data file 2) but were unable to detect additional changes in the replication profiles of early (Oct4, Rex1), middle (Nkx2.9) or later replicating loci (Sox3, Mash1, β-Globin) (supplementary Figure 4b in Additional data file 2). Collectively, these data suggest that only a minority of loci (5/23; supplementary Figure 2 in Additional data file 2) change their replication timing in response to severe reduction of DNA methylation (Dnmt 1 knock out (KO)), methylation of H3K27 (Eed KO), euchromatic H3K9 methylation (G9a KO) or NuRD activity (Mbd3 KO), despite being sensitive to changes that occur during normal differentiation 2416.

To assess whether histone acetylation levels are indicative of early replicating regions in ES cells, as has been suggested for other cell types 911, we compared the abundance of histone acetylation at the candidate loci using the chromatin-immunoprecipitation (ChIP) assay. Replication timing domains are very large (0.2-2 Mb) compared to promoter regions that are conventionally analyzed by ChIP. We therefore applied custom-made tiling arrays to examine approximately 200 kb regions surrounding the loci for enrichment of acetyl-H3K9. Early replicating loci, such as Sox2, Nanog and Rex1, contained numerous peaks of acetylation (eight- to ten-fold enrichment relative to H3; Figure 2a and Additonal data file 3). Loci that replicated in the second half of S-phase showed much fewer peaks and the enrichment was less pronounced (one- to two-fold). Basal histone acetylation levels were, however, relatively constant across each of the regions analyzed, irrespective of whether they replicated early or late.

To assess whether enhanced histone acetylation was sufficient to determine early replication, we treated ES cells for 24-48 h with doses of the HDAC inhibitor Trichostatin A (TSA), which raised the global levels of histone acetylation in nuclei (as judged by immunofluorescence; data not shown) without compromising cell viability, proliferation or morphology. TSA treatment of wild-type OS25 ES cells did not affect the replication timing of any of the loci tested, including the region surrounding Rex1 (Figure 2b). Similar treatment has been reported to advance replication of the cystic fibrosis transmembrane conductance (CFTR) gene in cell lines 20. The failure of TSA treatment to impact on replication of these genes in ES cells suggests that either temporal shifts are highly gene specific or that HDAC inhibition by TSA treatment merely increases histone acetylation at sites that are already acetylated and early replicating in ES cells. Consistent with the latter explanation, TSA treatment was recently shown to increase histone acetylation and expression of genes such as Hox B1 and Brachyury that replicate early in ES cells (L Mazzarella and HFJ, unpublished) 21.

Next we assessed the replication of three different murine repeat sequences. X141 is a complex X-linked repeat that is constitutively late replicating and heterochromatic 622. Minor and major satellites are simple direct repeats located around the centromeres of mouse chromosomes that, in wild-type ES cells, replicate in mid-early and mid-late stages of S-phase, respectively (Figure 3a). In mutant ES cells, late replication of X141 was retained but the timing of both minor and major satellites was altered. Minor satellite replication was selectively delayed in ES cells lacking Mll, which catalyses methylation of H3K4, an activating histone mark. The replication of both satellite sequences was delayed in Eed deficient ES cells, which lack repressive H3K27me3 (Figure 3a). Retarded replication of the major satellite was also seen in cells lacking the Suv39h1/h2 HMTases compared with matched wild-type controls. In contrast, major satellite replication was advanced in Dnmt1 KO and G9a KO ES cells. Interestingly, a comparison of matched mutant and wild-type ES cells showed advanced replication of major satellite sequences in the absence of Dicer, consistent with the proposed role of siRNA in silencing repetitive elements 23. In a recent study, an advance in the replication of the major satellite in Suv39h1/h2 double knockout (DKO) relative to wild-type fibroblasts was reported 24, although the authors noted that this advance was, in fact, not statistically significant. The apparent discrepancy between their observation and ours could be the result of intrinsic differences in the mutant cell lines used, or reflect secondary adaptations to loss of chromatin components. In this regard, compensatory chromatin modifications have been previously described, including an increase in H3K27me3 levels in Suv39h1/h2 deficient ES cells 25.

Minor and major satellites both carry DNA methylation and share some histone marks 26, but their chromatin structure is remarkably dissimilar. Major satellite DNA replicates in mid to late S-phase in ES and somatic cells and is characterized by hypoacetylation, trimethylation of H3K9 and H4K20 and DNA methylation 252728. The minor satellite contains the centromeric H3 variant CenpA, lacks appreciable amounts of the repressive H3K9me3 and does not bind HP1 2829. Instead, this repeat carries the permissive H3K4me2 mark 28 and it replicates in the first half of S-phase (Figure 3). We show that the replication timing of major and minor satellites responds very differently to mutation of Mll, Dnmt1, Suv39h1/h2, Dicer and G9a, supporting the view that the two repeats are regulated differently.

Earlier replication of the major satellite in Dicer KO cells might reflect increased repeat RNA accumulation, as has been reported in some cells upon loss of Dicer 2330, prompting us to measure transcript levels of the repeats in each of the mutant cell lines (Table 2). Despite variation among different lines of wild-type ES cells (major 0.7-4, minor 0.1-5), a significant increase in major satellite transcript levels was seen in Dicer KO cells (17 compared with 4 in matched wild-type cells). Increased minor and major satellite transcripts were also seen in Eed deficient ES cells but not in other mutant lines that also change satellite replication timing (for example, Dnmt 1 KO). These data suggest that while chromatin modifiers can influence satellite transcript levels, precocious replication is not an invariable consequence of satellite transcription.

To determine whether satellite sequences are particularly sensitive to loss of chromatin modifiers or if this is a general repeat-associated feature, we analyzed long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), which are found as single copies interspersed with genes and other unique sequences at many locations in the genome, as well as the tandemly repeated rDNA array. In wild-type ES cells, SINE B1 replicates early whereas LINE 1 elements show replication in all fractions of the S-phase (Figure 4), consistent with the known genomic distribution of these repeats; SINEs are primarily associated with gene rich regions (which replicate early), whereas LINEs are enriched in AT-rich, gene poor regions (which often replicate late, but can change replication timing depending on the cell type 3). The rDNA sequence, which in fibroblasts comprises an early replicating active and a late replicating silent fraction 31, replicated synchronous very early in S-phase in wild-type ES cells (Figure 4), possibly reflecting a high demand for biosynthesis in these rapidly dividing cells. Analysis of these three repeat sequences in the mutant ES cell lines revealed only very small changes with respect to wild-type cells. Replication of rDNA was extended in Eed deficient cells and slightly delayed in ES cells lacking Dicer.

Our data show that loss of Dnmt1 in ES cells (which causes genome-wide loss of CpG methylation; Table 1) results in early replication of the pericentric major satellite sequence without widespread changes in the replication timing of euchromatic loci or other repeat elements (Figures 3 and 4). To verify that reduction in DNA methylation is sufficient to precipitate this advance in major satellite replication, we experimentally demethylated wild-type ES cells. Exposure for three days to the Dnmt inhibitor 5-azacytidine reduced DNA methylation (from 0.88 in untreated to 0.21 in 5-azacytidine-treated cells, compared to 0.11 in the Dnmt1 KO ES cell line; Figure 5b) and caused an advanced replication of the major satellite (Figure 5a). The replication timing of the minor satellite as well as single copy genes (α-Globin, Mash1 and Myf5) was unaffected in treated cells. Collectively, these results suggest that DNA methylation per se is important for maintaining the correct temporal replication of the major satellite.

A role for DNA methylation in replication of heterochromatic foci has been previously observed in fibroblasts and during development 32. Here we show that DNA methylation is important in maintaining late replication specifically of major satellite repeats in undifferentiated ES cells. As DNA methylation of the major satellite is also reduced in Suv39h1/h2 DKO ES cells (Table 1), it is perhaps surprising that these mutant cells have delayed major satellite replication (Figure 3). It is possible that other chromatin modifications compensate for the loss of H3K9me3 to ensure heterochromatin formation in Suv39h1/h2 deficient cells, an idea that is consistent with enhanced H3K27me3 at pericentric regions in these cells 25.

ES cells used in this study were wild-type OS25 34, G9a knock out (KO) clone 2-3, G9a wild-type clone col4 (G9a WT), G9a transgene rescue clone 15-3 (G9a tg) 18, Suv39 h1/h2 double KO (DKO) clones DN57/DN72 and wild-type littermate clone wt26 (Suv39 h1/h2 WT) 35, Eed KO clones B1.3/G8.1 4, Dnmt1 c/c (Dnmt1 KO) 36, Dnmt3a/Dnmt3b DKO clone 10 (Dnmt3a/3b DKO) 2737, Mbd3 KO clone Fix2 38, Dicer KO clones D3-S5/D3-S6 and Dicer wild-type clone D3 (described below). ES cells were derived from Dicer flox/flox blastocysts 39 and transfected with the CRE-ER transgene to produce the Dicer flox/flox ES clone D3 (Dicer WT). The Dicer KO clones (D3-S5, D3-S6) were established after tamoxifen treatment (800 nM; Sigma, Poole, UK) of the D3 clone. Deletion of both alleles was confirmed by genotyping.

The ES cell lines were maintained in the undifferentiated state by culturing on gelatinized plates in KO-DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with leukemia inhibitory factor (LIF), 10% ES-tested fetal calf serum (GlobePharm, Surrey, UK), L-glutamine, 2-mercaptoethanol, non-essential amino acids and antibiotics. For Eed KO and Dicer cells (WT and KO), a feeder layer of mitotic inactivated fibroblasts was used and the medium was additionally supplemented with 5% knockout serum replacement (KSR, Invitrogen). OS25 cells were grown on gelatinized plates in Glasgow-MEM (Invitrogen) supplemented with LIF, FCS-gold (PAA, Yeovil, UK), L-glutamine, 2-mercaptoethanol, non-essential amino acids, sodium pyruvate, sodium bicarbonate and antibiotics. All ES cell lines examined in this study were Oct4 positive as determined by immunofluorescence (>92 %, data not shown). Undifferentiated ES cells (OS25) were treated with 10 nM (Sigma) for 48 h, 20 nM TSA for 24 h or 15 μM 5-azacytidine (Sigma) for 72 h.

The protocol described by Azuara et al. 6 was used. Briefly, asynchronous cell populations were pulse labeled with bromodeoxyuridine (BrdU; 30 minutes), fixed in 70% ethanol, stained with propidium iodide and fractionated according to DNA content by fluorescence assisted cell sorting (FACS). For ES cells grown on a feeder layer, the feeder cells were removed by differential attachment; less than 1% fibroblasts remained after 20-25 minutes plating in non-gelatinized plates. Pre-plating of feeder-dependent ES cells in this way may result in a slight delay in the apparent time of replication for genes that normally replicate very early in S-phase. Six cell cycle fractions were collected, G1-S, G2 and four fractions covering S-phase, S1-S4, where S1 corresponds to early S-phase and S4 to late S-phase. An equal amount of BrdU labeled Drosophila DNA was added to each fraction to control for equal recovery. After isolation of total genomic DNA, the DNA was sheared by sonication, denatured and newly replicated, BrdU-labeled DNA was immunoprecipitated using anti-BrdU antibody (BD, Franklin Lakes, NJ, USA). After purification, quantitative real-time PCR (qPCR) was employed to determine the relative quantity of specific loci in each fraction. The sequences of primers for qPCR analysis are given in Table 3. Locus replication was categorized based on the peak fraction(s) as early (peak in G1 or S1), middle-early (peak in S2), middle (peak in S2 and S3), middle-late (peak in S3) or late (peak in S4 or G2).

Exponentially growing wild-type ES cells (OS25) were paraformaldehyde (1%) fixed for 10 minutes at room temperature, lysed and chromatin immuno-precipitated essentially as described 40. Briefly, chromatin (50 μg) was pre-cleared 2 h at 4°C, incubated with antibodies (2 μl IgG (Z0259, DAKO, Copenhagen, Denmark); 2 μl anti-H3 (ab-1791-100, binds H3 independent of modification state, Abcam, Cambridge, UK), 5 μl anti-H3K9me2, 10 μl anti-H3K9me3 or 5 μl anti-H3K9ac (07-441, 07-442, 07-352, Upstate/Millipore, Billerica, MA, USA) at 4°C over night (ON) and the immune-complexes collected by adding protein-A sepharose (Sigma) (incubated 2 h at 4°C). Unbound chromatin was removed by washing 4× in ChIP wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris.Cl pH 8.1 and protease inhibitors) and 1× in high salt ChIP wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris.Cl pH 8.1 and protease inhibitors) after which 250 μl elution buffer was added (1% SDS, 0.1 M NaHCO₃, 100 μg/ml RNaseA, 500 μg/ml Proteinase K). After incubating at 37°C for 2 h and at 65°C ON, DNA was purified using a Gel purification kit (Qiagen, Crawley, UK), using 2 × 40 μl of 10 mM Tris.Cl pH 8 for elution. ChIP samples were analyzed by qPCR (sequences of primers are given in Table 3) or microarray hybridization.

Input and ChIP samples were amplified by LM-PCR as advised by Nimblegen, Reykjavik, Iceland. Labeling and hybridization was done by Nimblegen using a custom designed 50mer tiling array (100 bp average resolution) covering a region from 100 kb upstream to 100 kb downstream of the analyzed genes. Normalized and scaled Chip: input ratios for anti-H3K9ac and anti-H3 ChIP hybridizations were produced by Nimblegen. The log2(H3K9ac/H3) ratios were calculated from these data and plotted against the chromosomal position of the probes. Blue lines in Figure 2 indicate peaks in the dataset detected by a hidden Markov model-based algorithm using TileMap 41. Gaps in the profile arise from repetitive regions in the genome that are not represented on the array.

RNA was isolated from 3-5 × 10⁶ cells using the RNAeasy mini prep kit (Qiagen) with on-column DNase treatment. To remove residual genomic DNA, RNA (1.2 μg) was treated with RNAfree (Ambion, Austin, TX, USA) for 50 minutes before reverse transcription using SuperScript III (Invitrogen) and random primers in 20 μl reactions according to the manufacturer's instructions.

The sequence of primer pairs used in this study is given in Table 3. All primer pairs were tested for efficiency (>1.95) and linearity (R² > 0.99). Reactions (30 μl) were set up using a Qiagen SYBR green kit with the appropriate template (2 μl (corresponding to 200 cell equivalents) for replication timing, 1.5 μl of 1:5 diluted cDNA for gene expression, 2% of eluted DNA for ChIP, 2 μl (corresponding to 1.7 ng) genomic DNA for analysis of DNA methylation) and analyzed on Chromo4™ Real-Time PCR Detector (Bio-Rad, Hercules, CA, USA) with Opticon Monitor™ software.

Triplicate reactions of genomic DNA with or without the methylation sensitive restriction enzyme HpyCh4IV (New England Biolabs, Beverly, USA) were incubated for 3 h and the extent of digestion analyzed by qPCR. The primers for the major satellite span a HpyCh4IV site. The Sox2 primers, which do not span a HpyCh4IV site, were used to control for equal DNA content.