Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features

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 59 and 39 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.

Regulating DNA Replication in Plants

“…This is consistent with the depletion in CG methylation found in early and mid-S-phase replicons of Arabidopsis chromosome 4 (Lee et al 2010). Moreover, the higher than average C þ G content found in Arabidopsis origin regions (midpoint + 100bp), associated with a low CG methylation might favor a more open chromatin that facilitates pre-RC assembly and origin activation, in agreement with results obtained in Drosophila ) and mammalian cells (Karnani et al 2010;Cayrou et al 2011;Valenzuela et al 2011).…”

Chromatin and DNA Replication

“…Similar broad correlations between time of replication and activating histone modifications have been reported in numerous independent studies from a number of eukaryotic model systems (Hiratani et al 2008;Schwaiger et al 2009;Lee et al 2010). The apparent coordination between the transcription and replication programs may, in part, be caused by the frequent localization of replication origins near the start sites of transcription (Cadoret et al 2008;Cayrou et al 2011).…”

Epigenetic regulation of the human genome: coherence between promoter activity and large-scale chromatin environment

“…In contrast, progress in elucidating the chromatin-mediated control of replication origin usage and efficiency as well as of the maintenance of the spatio-temporal replication program in higher eukaryotes has been rather slow (Berezney et al 2000;Bogan et al 2000;Gilbert 2001Gilbert , 2010Méchali 2001Méchali , 2010Bell & Dutta 2002;McNairn & Gilbert 2003;Aladjem 2007;Courbet et al 2008;Hamlin et al 2008). Only very recently nascent DNA strands synthesized at origins were purified by various methods to map replication origins genome-wide in mouse (Sequeira-Mendes et al 2009;Cayrou et al 2011) and human (Lucas et al 2007;Cadoret et al 2008;Karnani et al 2010;Martin et al 2011;Mesner et al 2011;Valenzuela et al 2011). The set of replication origins identified so far are strongly associated with annotated promoters and seem to be enriched in transcription factor binding sites (Cadoret et al 2008;Karnani et al 2010;Besnard et al 2012) and in CpG islands (Cadoret et al 2008;Sequeira-Mendes et al 2009;Cayrou et al 2011).…”