Table 1.
Cells, DNA combing and replication timing datasets used in this study.
Figure 1.
DNA combing analysis of DNA replication in HeLa cells.
Cells were pulsed with IdU (20 min) followed by CldU (20 min) and sorted into four temporal compartments of S phase (S1, S2, S3 and S4). After DNA combing, DNA was stained in red, IdU in blue and CldU in green with fluorescent antibodies. (A) An exemplary DNA fiber and interpretative diagram. The blue-to-green transitions (indicated by arrows head) show the position and orientation of mobile forks at the time CldU was added (t = 20 min). This allows us to map origins that fired before (noted as Ori(1)) or after CldU addition (Ori(2)). Black solid lines show the intra-fiber distances between forks at the time of CldU addition. The dotted lines mark segments excluded from measurements of intra-fiber fork-to-fork distances. (B) Replication fork velocity analysis. Histograms of replication fork velocities in S1 to S4 fraction are shown. The five types of labeling patterns that could be unambiguously assigned to the progression of a single fork during 20 minutes labeling interval (white solid line) used to compute velocities are also presented. Each track length (L) was divided by the labeling time (20 min) to calculate the velocity of a single fork. (C) Distributions of local fork-to-fork distances in S1–S4. Only existing forks at the time of CldU addition were scored. For example, forks emanating from the leftmost origin in panel A, which fired after CldU addition, were not scored. Distribution of inter-origin (D) and inter-termini (E) distances in S1–S4. (F) Fibers containing ≥1 fork at the time of CldU addition were selected and the distribution of the number of forks per fiber was determined in S1–S4. (G) IdU-labeled fibers were selected and the distribution of the IdU-labeled length fraction of each fiber was determined in S1–S4.
Table 2.
Statistical analysis of the parameters of DNA replication determined by DNA combing.
Figure 2.
The global rate of DNA replication increases during S phase in HeLa cells.
(A) Histogram of replication timing values (TR50, hours) in the whole genome. (B) Histogram of global fork densities in S1, S2, S3 and S4 as determined by DNA combing. (C) Flow cytometry profile of cells pulsed labeled with 25 µM IdU/CldU for 20/20 min. IdU/CldU was stained with fluorescent antibodies. Fluorescence was plotted against total DNA content. Cells in S1, S2, S3 and S4 appear respectively in green, red, purple and blue. Four windows indicate cells in S1–S4 and labeled with IdU/CldU.
Figure 3.
Replication timing profiles segmented in CTRs/TTRs and multiscale analysis of apparent replication speeds in HeLa cells.
(A) Profile of replication timing (TR50, hours) along a 15 Mb segment of chromosome 17. Small TR50 values correspond to early replicating regions; large TR50 values correspond to late replicating regions. The replication timing profile was segmented into regions that replicate at apparent speed >10 kb/min (CTRs: Constant Timing Regions, red horizontal lines) and <10 kb/min (TTRs: Timing Transition Regions, green oblique lines) at scale 100 kb. (B) Multiscale analysis of apparent replication speeds along the same chromosome segment. Replication speeds determined by wavelet transform analysis (see Material and Methods) at scales indicated on the y-axis are shown in three colors (blue, <2 kb/min; green, from 2 to 10 kb/min; red, >10 kb/min). (C) Distribution of apparent replication speed at the 100 kb scale in the whole genome (pdf: probability density function). (D) Distribution of apparent replication speeds in the four temporal compartments of S phase: S1, S2, S3 and S4 (respectively: blue, green, pink, and red curves).
Figure 4.
Characteristics of CTRs and TTRs.
Blue, green and red curves respectively depict parameters defined at scales 100, 200 and 500 kb. (A, B) Size distribution of CTRs and TTRs (pdf: probability density function). (C, D) Genome coverage of CTRs and TTRs of length>Size (in Mb). (E) Mean TR50 of CTRs. (F) Apparent replication speeds of TTRs (pdf: probability density function). Note that different scales are used on the X-axis for CTRs and TTRs because their sizes are different.
Figure 5.
Replication Speeds along TTRs.
(A) Size and replication time of individual TTR. The time difference, Dt, between the early and the late side of each TTR is plotted along its length, Dx, for each of the 7504 TTRs (open circles). By definition, the maximum replication speed of TTRs is 10 kb/min (dark blue line). The mean apparent replication speed, Dx/Dt, is 3.63 kb/min (green dashed line). The mean (v = 0.64 kb/min, red line) and maximum (v = 2 kb/min, orange line) velocities of single forks measured by DNA combing are indicated. Only 24 TTRs lie between the orange line and the vertical axis. (B) Evolution of apparent replication speed along the 774 TTRs>400 kb. The apparent speed measured at the distance D4 = 400 kb from early edge of each TTR is plotted against the apparent speed measured at the distance D1 = 100 kb. Replication accelerates for 62% of TTRs. (C) Distribution of apparent replication speeds along the 774 TTRs>400 kb. The apparent speed has been measured at different distances D1–D4 from early edge of each TTR: D1 = 100 kb, red curve; D2 = 200 kb, green curve; D3 = 300 kb, blue curve; D4 = 400 kb, grey curve.
Figure 6.
Analysis of apparent replication speeds in multiple cell types.
The distribution of apparent replication speeds at the 100 kb scale in the whole genome was determined for (A) BG02, a human embryonic stem cell line; (B) K562, a chronic myelogenous leukemia cell line; (C) BJ, normal fibroblasts; (D) GM06990, (E) TL010, and (F) H0287, lymphoblastoid cell lines, as described for HeLa cells in Figure 3 legend.
Figure 7.
Size and replication time of individual TTRs in multiple cell types.
The time difference, Dt, between the early and the late side of each TTR detected at scale 100 kb is plotted along its length, Dx, for each of the TTRs (dots) for (A) BG02, a human embryonic stem cell line (7657 TTRs); (B) K562, a chronic myelogenous leukemia cell line (3638 TTRs); (C) BJ, normal fibroblasts (5266 TTRs); (D) GM06990 (4017 TTRs), (E) TL010, (2492 TTRs) and (F) H0287 (3237 TTRs), lymphoblastoid cell lines. By definition, the maximum replication speed of TTRs is 10 kb/min (dark blue line). Lines corresponding to 5 kb/min (turquoise) and 2 kb/min are also provided as a guide to the eye. The mean apparent replication speed, Dx/Dt, is indicated by a green line (BG02, 2.34 kb/min; K562, 3.77 kb/min; BJ, 3.24 kb/min; GM06990, 4.04 kb/min; TL010, 4.21 kb/min; H0287, 4.17 kb/min). The mean (orange dashed line) and maximum (purple dashed line) velocities of single forks measured by DNA combing in identical or comparable cell lines (see text) are indicated. Except for BG02, practically no TTR is found between the orange dashed line and the vertical axis.
Figure 8.
DNA combing analysis of the IGH TTR.
(A) Replication timing profile of the IGH region and its surroundings in HeLa cells. (B) Map of the IGH region, position of the fosmid probes (red lines) and chromosome coordinates. Combed DNA molecules were hybridized either with Fos1-2-3 or with Fos4-5-6, allowing reliable detection and orientation of the combed IGH molecules. (C) Exemplary DNA molecules and interpretative diagrams showing probe hybridization (red), IdU (blue) and CldU (green) tracks and total DNA (white) and deduced origin locations (purple arrowheads). The complete set of analyzed molecules is shown on Figure S4. (D) Schematic representation of all replicative DNA molecules analyzed aligned along the locus using the detected hybridization patterns. Note that for one of these molecules (13th line) the orientation could not be unambiguously determined and one of the two possible orientations was arbitrarily chosen. (E) Distribution of detected origins along the locus.
Figure 9.
Replication mode of the FHIT locus in fibroblasts and lymphoid cells.
(A) Replication timing profile of the locus in BJ fibroblasts and (B) summary of initiation and termination events mapped in MRC5 fibroblasts (data from Figure 2c and Figure S7 in [58]). The FRA3B region is embedded into a 1.2 Mb CTR that replicates by evenly spread initiations. (C) Replication timing profile of the locus in GM06990 lymphoblasts and (D) summary of initiation and termination events and direction of single forks mapped in JEFF lymphoblastoid cells (data from Figure 2a and Figure S3 in [58]). The FRA3B region maps at the bottom of two converging TTRs where forks move in both directions.
Figure 10.
Analysis of replication bubble and λ-SNS coverage in ENCODE CTRs and TTRs.
Replication bubble data are from log-phase HeLa Rep4 library [62] and λ-SNS data are from [64]. (A) Replication bubble coverage is plotted against apparent replication speed of CTRs (blue circles) and TTRs (dark circles). (B) An exemplary ENCODE region. Replication timing profile (dark wavy line) of region ENm001 (grey line) and its surroundings with replication bubbles (cyan) and λ-SNS (orange) in CTRs (red) and TTRs (green) are shown. (C) Replication bubble coverage computed by 100 kb adjacent windows along ENCODE regions is plotted against replication time. (D) λ-SNS coverage is plotted against apparent replication speed of CTRs (blue circles) and TTRs (dark circles). (E) λ-SNS coverage computed by 100 kb adjacent windows along ENCODE regions is plotted against replication time.
Figure 11.
Representation of open chromatin markers along all TTRs relative to the corresponding genome-wide average value.
(A) Mean coverage by DNase I hypersensitive zones, as a function of the distance to the earliest TTRs border. TTRs have been detected at 200 kb scale and classified by size: in red TTRs<200 kb; in green 200 kb<TTRs<360 kb; in blue TTRs>360 kb. (B) Mean coverage by 1 kb-enlarged CpG islands as a function of the distance to the earliest TTRs border. Three size categories have been defined: in red TTRs<200 kb; in green 200 kb<TTRs<360 kb; in blue TTRs>360 kb.
Figure 12.
Two alternative models for origins activation along TTRs.
(A) Replication first initiates at early firing origins. Origins fire independently of each other and are specified by an open chromatin structure. (B) Domino model in which replication initiates at early efficient origins. Activation of later origins in less open chromatin is stimulated by approaching replication forks from upstream origins. In both cases (A and B), the rate of origin firing increases during S phase resulting in a U-shaped replication timing profile. Origins of replication are marked by purple circles and black arrows show the direction of replication forks. Color variation (green to red) depicts the chromatin openness. Blue arrows indicate origin stimulation by replication forks from upstream origins.