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.

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins.

Transfer of somatic cell nuclei to enucleated eggs and ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, these methods are poorly efficient, and other unknown factors might be required to increase their success rate. Here we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development, and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4, and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M-phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies, and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M-phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergizes with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial, because none of these effects is detected when using interphasic Xenopus egg extracts. Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei. Murine induced pluripotent stem (iPS) cells have a complete developmental potential as demonstrated by their capacity to form teratomas, generate chimeras, and contribute to the germline. However, the efficiency of both iPS cell production and NT remains low, and most of the reprogrammed cells appear to be reprogrammed only partially. The epigenetic memory of the cell is one key barrier that must be overcome to reprogram differentiated cells (5), and additional factors may be needed to improve reprogramming efficiency (6, 7). Several attempts have been made to reprogram somatic cells by using cellular extracts, but they failed to reproduce the range of effects obtained by NT.In NT experiments, reprogramming is induced by exposure of transplanted nuclei to the cytoplasm of the receiving oocyte. However, NT reprogramming appears hard to study in vitro because of the difficulty of obtaining large quantities of mammalian oocytes. Xenopus eggs, which can be obtained in large amounts, can remodel the nuclear lamina of mammalian cells (8), and Xenopus egg extracts can up-regulate Oct4 expression in cells that already express Oct4 (9), similar to what is observed when adult mouse nuclei are injected in Xenopus oocytes (10). The replication origin pattern and chromosome organization of Xenopus erythrocyte nuclei also could be remodeled by metaphase-arrested extracts (M-phase extracts) from Xenopus eggs (11). We show here that such extracts increase the efficiency of NT and iPS cell production from mouse embryonic fi...