Wu R , Singh PB , Gilbert DM .

GM-57233 NIGMS NIH HHS , R01 GM057233 NIGMS NIH HHS

	Figure 1. The replication timing program of chromocenters is established 2–3 h after mitosis. (a) Nuclei isolated from cells synchronized at different times during G1 phase were introduced into Xenopus egg extract, and DNA synthesized in vitro was pulse labeled with biotin-dUTP at 30, 60, and 120 min thereafter. Labeled nuclei were subjected to FISH with a γ-satellite DNA probe (green in b) and counterstained with Texas red streptavidin (red in b). Chromocenter replication was visualized as the colocalization of γ-satellite DNA with biotin-dUTP. (b) Optical sections through the center of each nucleus were obtained by dual-color confocal laser scanning microscopy. Separate green and red images and their merges are shown on the left (colocalization in yellow). Colocalizing pixels were then imaged in white, and the coefficient of colocalization (percentage of γ-satellite signal that colocalized with biotin signal) was quantified for each nucleus (value shown). (c) Box plot of the coefficient of colocalization for >100 nuclei per time point. Horizontal bars represent the 10th, 25th, 50th (median), 75th, and 90th percentiles, and the 25–75th percentiles are presented as gray boxes. Shown are the results from a single experiment. Similar results were obtained in two additional experiments in which anti-Me3K9H3 antibodies were used to localize chromocenters. (d) Aliquots of nuclei from panel c were introduced into Xenopus egg extract supplemented with α-[32P]dATP, and the percentage of input DNA replicated at the indicated times was determined. The means and SEM (error bars) of all three independent experiments are shown.
	Figure 2. Global replication timing and subnuclear repositioning. (a) Asynchronous cultures were pulse labeled with BrdU for 30 min, and mitotic cells were harvested 4–5 h later to create a population of cells in which late-replicating sequences were tagged with BrdU. At 1, 2, and 3 h after release into G1 phase, nuclei were introduced into Xenopus egg extract. The earliest DNA to replicate in vitro was pulse labeled with biotin-dUTP, and labeled nuclei were stained with Texas red streptavidin (red in b) and anti-BrdU antibodies (green in b). In parallel, aliquots of the same cells were fixed and stained with anti-BrdU antibody to visualize the subnuclear repositioning of labeled domains making up the easily identifiable late S peripheral spatial replication pattern. (b) Exemplary confocal images from G1 1- and 3-h nuclei displayed as in Fig. 1. (c) The percentage of BrdU-labeled nuclei displaying precocious synthesis of late-replicating sequences in vitro was scored manually as nuclei displaying yellow foci using a dual red/green filter. (d) Percentage of nuclei from the same cells that had repositioned late-replicating BrdU-labeled chromosome domains to the nuclear periphery. Under these conditions, ∼50% of BrdU-labeled cells display the label at the periphery. Data in panels c and d show the mean and SD (when >1%; error bars) of two independent experiments in which >100 nuclei for each time point were scored.
	Figure 3. Me3K9H3–HP1 association takes place before chromocenter assembly and the TDP. (a) Confocal images of γ-satellite DNA (γ-sat) by FISH and deconvolution images of HP1β or Me3K9H3 for G1 1- (before TDP) and 3-h (after TDP) nuclei. Immunofluorescence images were counterstained with DAPI, and the enrichment of HP1 and Me3K9H3 at chromocenters (DAPI-dense DNA; pseudocolored in red) is revealed in the merged images. (b) Whole cell extracts (WCE) prepared from equal numbers of G1-phase cells were analyzed by immunoblotting with anti-Me3K9H3, Me3K20H4, or anti-HP1β antibodies. The blot used to probe Me3K9H3 was reprobed with anti-histone H3 antibody as a loading control. (c) Protocol for chromatin extraction. (d) Aliquots of cells from panel b in mitosis (M phase), G1 1 h (before TDP), and G1 3 h (after TDP) were extracted as in panel c with the indicated salt concentration, and fractions representing equal cell numbers (half the cell number for M phase) were analyzed by immunoblotting with anti-HP1β antibody. No differences in salt lability were observed for HP1β between pre- and post-TDP cells. Virtually identical results were obtained when parallel immunoblots were probed for HP1α. Similar results were obtained in three independent experiments.
	Figure 4. Removal of HP1 proteins from chromatin does not permit early replication of chromocenters. Nuclei prepared from C127 cells synchronized at 3 h after mitosis were incubated for 2 h on ice in the presence of 50 μg/ml of either methylated or unmethylated peptide or buffer alone. (a) Peptide used for HP1 competition. The underlined amino acid is the attachment site for the methyl group. (b) Aliquots of nuclei were extracted, and fractions were analyzed by immunoblotting as in Fig. 3 (0.15 M NaCl). Adding additional methylated peptide or a combination of chromoshadow peptides (Smothers and Henikoff, 2000) and methyl-H3 peptides did not remove additional HP1 proteins (not depicted). WCE, whole cell extract. (c) Aliquots of the same nuclei were stained with anti-HP1 antibody (red) and counterstained with DAPI (pseudocolored green). The insets show higher magnification images of one chromocenter each. HP1 was not detected in the core of the chromocenters after extraction with methylated peptide, although the less DAPI-dense chromatin surrounding the chromocenters retained some HP1. (d) Additional aliquots of the same nuclei were introduced into Xenopus egg extract, and the replication timing of chromocenters was evaluated as in Fig. 1 except that the analysis was restricted to the 30-min time point. Shown is a box plot (displayed as in Fig. 1) for >200 nuclei from three independent experiments (40–80 nuclei/experiment). (e) Aliquots of the Xenopus egg extract reaction were supplemented with α-[32P]dATP, and the percentage of the total input genomic DNA synthesized was evaluated at each of the indicated time points as in Fig. 1. The means and SEM (error bars) for three independent experiments are shown.
	Figure 5. Late replication of major and minor satellite DNA is independent of Suv39h1,2. (a) Each of the three indicated cell lines (wild type [WT], Suv39dn knockout [D15], and a derivative of D15 in which a Suv39h1 cDNA expression vector was stably integrated into the genome [D15 + Suv39h1; cell line described in Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200601113/DC1]) was pulse labeled with BrdU, stained for DNA content with propidium iodide, and separated by flow cytometry into four S-phase populations with increasing DNA content. (b) DNA was isolated from each fraction of each cell line, BrdU-substituted (nascent) DNA was immunoprecipitated with an anti-BrdU antibody, and aliquots of each of these nascent DNA preparations were immobilized on a nylon filter and hybridized with major and minor satellite DNA probes as well as with mouse mitochondrial DNA that is known to replicate throughout the cell cycle (Bogenhagen and Clayton, 1977; James and Bohman, 1981; Magnusson et al., 2003). (c) For each fraction, the total counts per minute for major and minor hybridizations were first normalized to that of mitochondrial DNA to control for any variation in sample preparation, and the percentage of the total signal from all four fractions of the cell cycle that was present in any given fraction was plotted. The mean values and SEM (error bars) for five (wild type), four (D15), and three (D15 + Suv39h1) independent experiments are shown.
	Figure 6. Suv39h1,2 knockout advances the replication timing of chromocenters. (a) The pulse-chase-pulse method to define the temporal order of spatial replication patterns. MEFs were labeled for 10 min with CldU, chased for various lengths of time, labeled for 10 min with IdU, and stained with fluorescent antibodies specific to CldU (green) and IdU (red). Exemplary confocal images of the dynamic changes in replication patterns observed with increasing chase times (from 0 min through 10 h) in D15, which were similar in all lines, are shown. (b) Displaying only the IdU stain within nuclei that display early CldU patterns reveals the temporal order in which each of the replication patterns take place, which were similar to other mouse cell lines (Wu et al., 2005). In brief, DNA synthesis begins at many small, discrete foci in the internal euchromatic region of the nucleus, excluding the nucleoli (and associated chromocenters) and nuclear periphery (pattern I). In pattern II, replication continues throughout the euchromatic region but is also observed in the perinucleolar and nuclear periphery regions so that a clear demarcation of the nucleoli is no longer apparent. Pattern III is characterized by decreasing euchromatic foci in the interior and increased replication foci at the nuclear (and nucleolar) periphery. Shortly thereafter, most euchromatic (small internal) foci have finished replication, and DNA synthesis begins within the chromocenters (pattern IV). Next, chromocenter replication is completed, whereas some replication at the nuclear periphery continues, coinciding with the replication of a few internal but nonpericentric domains (pattern V). Finally, a few large clusters of foci are observed in both the interior and periphery of the nuclei (pattern VI). All six of these patterns were observed in their proper temporal order in the wild-type MEFs, but in D15 the chromocenter replication pattern IV appeared to merge with pattern III, making it difficult to distinguish between these patterns (i.e., chromocenters were seen to begin replication coincident with the decrease in internal foci labeling). (c) The percentage of cells displaying early CldU-labeled patterns (pattern I) that also showed IdU-labeled chromocenters at each chase time was scored. At least 200 nuclei were scored for each data point. Because the total length of S phase varied (7–10 h) from experiment to experiment, each data point was normalized to the length of S phase, which was measured as the time it takes for 50% of cells to progress from CldU-labeled pattern I into G2 phase (unlabeled IdU), to give a relative chase time as the percentage of S phase (chase time/length of S × 100). After this normalization, the relative chase time for each data point is no longer the same for each experiment, so multiple experiments cannot be averaged. Instead, all data points from all experiments are shown, and a sixth order polynomial curve was fit to each dataset. Experiments were repeated four (wild type), six (D15), and two (D15 + Suv39h1) times. (d) Data on the rising end of each curve in panel c were plotted independently, and a linear curve as well as the slope and y intercept for each dataset are shown. This reveals a very similar slope for all cell lines, with the y intercept advanced by 15% in D15 versus wild-type cells.
	Figure 7. Chromocenters replicate after the Xi in wild-type cells but coincident with the Xi in Suv39dn. (a) A large synchronously replicating body (indicated with arrowheads) is highly enriched for Me3K27H3, a defining characteristic of the Xi. The Xi does not colocalize with DAPI-dense bodies that define chromocenters. (b) In wild-type (WT) cells, the Xi replicates during a distinctly different spatial replication pattern (pattern III) than do the chromocenters (pattern IV). In pulse-chase-pulse experiments like those shown in Fig. 6, 2 h of chase time are necessary to traverse from CldU-labeled Xi to IdU-labeled chromocenters. (c) In contrast to wild-type cells, in D15 cells, the Xi (arrowheads) can be seen to replicate synchronously with the replication of chromocenters in a single BrdU pulse (i.e., with no chase). (d) The percentage of cells displaying simultaneous colocalization of the Xi and chromocenters with BrdU label (after a 10-min pulse of BrdU) was scored for wild type, D15, and the D15 derivative rescued by the expression of a Suv39h1 cDNA. More than 100 cells showing Xi replication were scored for each cell line. The means and SD (error bars) for two independent experiments are shown.

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