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. 2011 Sep;21(9):1438-49.
doi: 10.1101/gr.121830.111. Epub 2011 Jul 12.

Genome-scale Analysis of Metazoan Replication Origins Reveals Their Organization in Specific but Flexible Sites Defined by Conserved Features

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Free PMC article

Genome-scale Analysis of Metazoan Replication Origins Reveals Their Organization in Specific but Flexible Sites Defined by Conserved Features

Christelle Cayrou et al. Genome Res. .
Free PMC article

Abstract

In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5' and 3' of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates.

Figures

Figure 1.
Figure 1.
Genome-scale mapping of replication origins by nascent strand (NS) chip. (A) NS isolation schematic. 0.5–2.5-kb NS were isolated from total genomic DNA by denaturation and sucrose gradient centrifugation. NS enriched by lambda exonuclease treatment were hybridized against total genomic DNA on high-density tiling arrays (see Supplementary Information). (B) Example of the distribution of replication origins in mouse (upper panel) and Drosophila cells (lower panel) along a 200-kb region. The log2-ratio between NS and total genomic DNA is shown. For genes, the position of the start site (high bar bordering the gene), exons (large gray boxes), and introns (thin gray boxes) are indicated (see Supplemental Fig. 2F). (C) Origin number and density per genome. (D) Immunoprecipitation of chromatin associated with ORC2 was carried out in P19 cells as described in Methods. Compilation of ORC2 signal strength data and correlation with the NS peaks is shown.
Figure 2.
Figure 2.
Replication origins in metazoans are linked to expressed genes. (A) Replication origins are significantly associated with transcribed genes (*: P < 0.001) in both mouse MEF and Drosophila Kc cells. (B) Association of replication origins with gene partitions in MEFs (left panel) and Drosophila Kc cells (right panel). Replication origins are found more frequently at gene promoters (mouse cells) and exonic sequences (mouse and Drosophila cells, *: P < 0.001). (C) Distribution of mouse replication origins along a gene. The position of each origin is allocated depending on the length of the gene adjusted to 100%. (D) Nascent strand signal strength at TSS in ES cells and (E) Drosophila Kc cells. The enrichment value is the log10 of the combined P-value associated with NS signal (see Supplementary Information). (F) NS signals in mouse ES cells are associated with CGI-positive TSS but not with CGI-negative TSS.
Figure 3.
Figure 3.
Association of replication origins with CGI in metazoans. Example of replication origins associated with CGI in (A) mouse ES cells and (C) Drosophila cells. The percentage of CGI/replication origin association is also shown. (B) NS signal strength around all CGI in mouse ES cells and (D) CGI-like regions in Drosophila Kc cells. The average size of CGI is shown in scale. (E) Common origins in mouse cells are strongly associated with CGI regions. The proportion of CGI-positive origins in the indicated groups of origins is shown.
Figure 4.
Figure 4.
Nucleotide skew and GC-rich elements at replication origins. (A) Origins were centered on Drosophila CGI-like regions. The mean AT and GC percentages of centered Oris are shown. Genome-scale NS signal strengths are represented by a black line. Note that the NS peaks (putative replication initiation sites) are not enclosed in the central CG-rich region. (B) Genome-scale nucleotide distribution of all Drosophila origins centered on the NS peak. Note the skew in nucleotide distribution with GT and AC enrichment at the 5′ and 3′ end of the origin peak, respectively. (C) Nucleotide distribution at and around the origin peak for origins in Drosophila Kc cells; 200 bp sequences of 300 replication origins were stacked and aligned around the NS peak. Four colors were used: green for A, red for T, yellow for G, and blue for C. The exact sequence can be read by enlarging the figure in Supplemental Data. A clear bias is observed for C or G, and A or T around the NS peak. (D) Motifs frequently found in Drosophila (top panel) and mouse (bottom panel) replication origins. The E-value is indicated (see Methods).
Figure 5.
Figure 5.
Positive link between HP1 and origin firing/early replication in heterochromatic regions. (A) Density of CGI-like regions in the whole genome and on chromosome 4 in Drosophila. (B) Positive correlation between HP1 binding and early S phase replication timing in Drosophila chromosome 4. Scatter plots between experimental and randomized HP1 data sets and replication timing are shown. The P-value is indicated at the bottom of the panels. (C) Significant association between HP1 binding and origins on the entire Drosophila chromosome 4. (D) A 300-kb region of chromosome 4 showing the relationship between origin firing, HP1 binding and early replication timing.
Figure 6.
Figure 6.
Early replication domains are characterized by high origin density. (A) Shown is the origin density in the three mouse cell lines calculated using a 100-kb sliding window along the chromosomal region. The computed gene and CGI densities are also illustrated. Origin density is positively correlated with early replication domains (*: P < 0.001). (B) Origin number is also positively correlated (*: P < 0.0001) with the early replication timing observed in mouse ES cells (Hiratani et al. 2008).
Figure 7.
Figure 7.
Replication origins are organized in a functional hierarchical manner along the chromosome. (A) DNA combing analysis performed in Drosophila Kc (top panel) and mouse (bottom panel) cells after two consecutive labeling pulses of IdU and CldU. (B) Summary of the experimental and simulated inter-origin distance distributions for MEF cells. For the “Increasing Ori efficiency” model, the values for the firing efficiency represent the initial and final origin firing efficiency during simulations. (C) “Random Ori firing” model. In this model, origins are completely independent and are activated randomly (red circles). Very short and long inter-origin distances are observed. (D) In the “Increasing Ori efficiency” model, origins are completely independent and activated randomly, but with increasing firing efficiency throughout S phase progression. (E) “Flexible Replicon” model. In this model, origins are linked within functional units where activation of one origin silences the others in the same group. The bottom panels present the computer-simulated results for each model. The gray profile is the distribution of inter-origin distances obtained by DNA combing of MEF cells. The red line represents the simulated distribution of inter-origin distances according to each model. The “Flexible Replicon” model is the only model to yield a simulated distribution of inter-origin distances that is statistically indistinguishable from the DNA combing data.
Figure 8.
Figure 8.
Origins, replicons, and replicon clusters. (A) The presence of a CpG island or CGI-like region allows the positioning of two potential initiation sites upstream of and downstream from the region. (B) Replicons are organized as functional units containing several potential DNA replication origins. Activation of one origin within a replicon silences the others. The origin choice within each replicon can occur either stochastically or be dictated by specific cell fates. Replicon clusters include several consecutive replicons which are activated simultaneously (Berezney et al. 2000). (C) Representation of replicons as chromatin loops where activation of one origin silences the other origins contained in the same replicon.

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