Li F et al. (2001), The replication timing program of the Chinese h...

XB-ART-8671

J Cell Biol 2001 Jul 23;1542:283-92.

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The replication timing program of the Chinese hamster beta-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase.

Li F , Chen J , Izumi M , Butler MC , Keezer SM , Gilbert DM .

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We have examined the dynamics of nuclear repositioning and the establishment of a replication timing program for the actively transcribed dihydrofolate reductase (DHFR) locus and the silent beta-globin gene locus in Chinese hamster ovary cells. The DHFR locus was internally localized and replicated early, whereas the beta-globin locus was localized adjacent to the nuclear periphery and replicated during the middle of S phase, coincident with replication of peripheral heterochromatin. Nuclei were prepared from cells synchronized at various times during early G1 phase and stimulated to enter S phase by introduction into Xenopus egg extracts, and the timing of DHFR and beta-globin replication was evaluated in vitro. With nuclei isolated 1 h after mitosis, neither locus was preferentially replicated before the other. However, with nuclei isolated 2 or 3 h after mitosis, there was a strong preference for replication of DHFR before beta-globin. Measurements of the distance of DHFR and beta-globin to the nuclear periphery revealed that the repositioning of the beta-globin locus adjacent to peripheral heterochromatin also took place between 1 and 2 h after mitosis. These results suggest that the CHO beta-globin locus acquires the replication timing program of peripheral heterochromatin upon association with the peripheral subnuclear compartment during early G1 phase.

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Species referenced: Xenopus
Genes referenced: avd dhfr hbg1 lmna

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	Figure 1. Relative replication timing of DHFR and β-globin loci. (A) Asynchronously growing CHO cells were pulse labeled with BrdU and hybridized in situ with digoxigenin-labeled DHFR cosmid cSc26 and biotin-labeled β-globin phage λJHC2. Sites of hybridization were visualized with FITC-conjugated antidigoxigenin antibodies (DHFR, green) and Texas red avidin (β-globin, red). BrdU label was detected with AMCA-labeled anti-BrdU antibodies (blue). In the image shown, BrdU foci are not visible because nuclei were additionally stained with DAPI (blue) to highlight the entire nucleus. (B–D) The number of DHFR and β-globin singlets and doublets per nucleus was evaluated. (B) The percentage of total alleles scored that displayed doublets for each locus was calculated. (C) The percentage of nuclei that displayed more doublets for one locus than the other was calculated. (D) The percentage of nuclei that showed doublets for both alleles of one locus and singlets for both alleles of the other locus was calculated. (C and D) Schematic representations of the arrangement of the DHFR (dot) and β-globin (x) alleles are included. The mean values ± SEM are shown for three independent experiments in which >100 BrdU-positive nuclei each were scored. All sets of data in this report include hybridizations in which both DHFR and β-globin probes were labeled with both biotin and digoxigenin and used in either combination, to control for any differences in labeling and detection of different nucleotide analogues. Only nuclei showing clear signals for both DHFR and β-globin loci were scored.
	Figure 2. The CHO β-globin locus is replicated during the stage of peripheral DNA synthesis. (A) Examples of early (type I/II), middle (type III), and late (type and type V) replication patterns, revealed by pulse labeling CHO AA8 cells with BrdU and staining with fluorescent anti-BrdU antibodies. (Top) Shows cells fixed with ethanol and stained only for anti-BrdU, as described (Dimitrova and Gilbert, 1999b). (Bottom) Shows the pattern of anti-BrdU staining with cells that were first subjected to FISH. Conditions used for FISH distort the pattern of BrdU staining; however, it is still possible to distinguish the different pattern types. (B) CHO AA8 cells were synchronized in mitosis and released into medium containing aphidicolin to accumulate cells at the G1/S border. Cells were then released from the G1/S block, pulse labeled with BrdU at various times thereafter, and stained with anti-BrdU antibodies as in A (top). Shown are the percentage of BrdU-positive cells that exhibited early (▪), middle (▴), or late (•) replication patterns at each time point. (C) Aliquots of the same cells from B were hybridized with β-globin and DHFR probes by FISH, and the percentage of doublet alleles for each locus was determined. Results in B and C show the means ± SEM (when >2) for three independent experiments in which >100 nuclei each were scored. (D and E) Cells synchronized as in B were subjected to FISH with either a DHFR (D) or β-globin (E) probe and subsequently stained with anti-BrdU antibodies as in A (bottom). The percentage of doublet alleles was scored as a function of the early (▪), middle (▴), or late (•) BrdU patterns. The dashed lines for middle- and late-replication patterns indicate the time of appearance of these patterns during S phase (i.e., middle patterns first appeared between 2.5 and 4.5 h, and late patterns first appeared between 4.5 and 6.5 h).
	Figure 3. Colocalization of β-globin versus DHFR doublets with sites of DNA synthesis during middle to late S phase. CHO AA8 cells pulse labeled with BrdU were subjected to FISH with either a β-globin or a DHFR probe (red) and then stained with anti-BrdU antibodies (green). Analysis was concentrated on nuclei that displayed type III or IV BrdU patterns (described in legend to Fig. 2). (A) (Top) Show examples of merged confocal images. (Bottom) Show the same images after computer-assisted colocalization analysis. After background subtraction, those pixels still showing colocalization of red and green signals were colored in yellow, whereas pixels showing no colocalization were colored in white. (B) Sections of the nuclear periphery from exemplary nuclei showing the proximity of β-globin doublets to the peripheral heterochromatin. (C) The percentage of doublets within type III and IV nuclei that colocalized with BrdU signal was scored. Data were scored independently for doublets found either at the periphery (defined as <1 μm from the edge of DAPI staining) or the interior of the nucleus.
	Figure 4. Late replication of the β-globin gene locus in CHO cells is established 1–2 h after mitosis. (A) CHO AA8 cells were synchronized in mitosis and released into G1 phase. At the indicated times thereafter, cells were pulse labeled with BrdU, stained with anti-BrdU antibodies, and the percentage of BrdU positive nuclei was scored. (B) Nuclei isolated either at 1, 2, or 3 h after mitosis were introduced into Xenopus egg extracts. Aliquots of these nuclei were then subjected to FISH analysis at 30, 60, and 120 min thereafter. The percentage of total DHFR or β-globin alleles displaying doublets was calculated for >100 nuclei per time point. Shown are the means ± SEM for three independent experiments. (C–E) Results shown in B for 30, 60, and 120 min in vitro were averaged together and displayed by the same three methods described in the legend to Fig. 1, B–D.
	Figure 5. The repositioning of type III sequences and the peripheral localization of the β-globin locus are completed 1–2 h after mitosis. (A) Asynchronous cultures of CHO AA8 cells were pulse labeled with BrdU for 30 min. Metaphase cells were harvested 3.5 h thereafter, creating populations of cells in which late-replicating sequences were tagged with BrdU (∼1/3 of which were type III). At 1, 2, and 3 h after release into G1 phase, cells were fixed and stained with anti-BrdU antibody, and the percentage of nuclei displaying a type III spatial pattern, represented by peripheral and nucleolar BrdU staining, was calculated. (B) Nuclei were isolated from CHO AA8 cells synchronized at 1, 2, or 3 h after mitosis and introduced into Xenopus egg extracts. At the indicated time points, reactions were pulse labeled in vitro with biotin-11-dUTP, and nuclei were stained with Texas red streptavidin. The percentage of nuclei from each time point that displayed either early (many internal punctate foci) or middle (peripheral and nucleolar DNA synthesis) replication patterns was calculated. (C and D) Asynchronously growing cultures (Asyn.), as well as cells synchronized at 1, 2, or 3 h after metaphase, were subjected to FISH with DHFR or β-globin probes and then stained with an anti-lamin A/C antibody. Confocal images were collected and the distance from each allele to the nuclear lamina was measured and divided by the radius of the nucleus at its widest point. (C) Examples of images found for either DHFR or β-globin at either 1 or 2 h after mitosis. A copy of each image is shown to its immediate right, with white lines to denote the measurements taken for the diameter and the shortest distance from the FISH signal to the nuclear envelope. The lamina was used as an indicator of the nuclear periphery rather than DAPI (as in Fig. 1) due to the occasional invaginations of the nuclear envelope that are not always detectable with the use of DNA dyes. An example of such an invagination is shown (2, a and b). Two β-globin alleles at the periphery, appearing as yellow FISH foci, which would be scored as a distance of zero, are also shown (3a and 3b). (D) Box plot representing the shortest distance between DHFR or β-globin alleles and the nuclear lamina. Data for each cell population were collected from ≥37 nuclei. Horizontal bars represent the 10th, 25th, 50th (median), 75th, and 90th percentiles, and the p-values for each pair of samples are shown. This type of plot indicates that 50% of the population is found between the 25th and 75th percentiles, and is represented as a box. Gray boxes denote DHFR alleles, and white boxes denote β-globin alleles.

References [+] :

Abney, Chromatin dynamics in interphase nuclei and its implications for nuclear structure. 1997, Pubmed