Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS), suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation, unveiling a new hallmark in ORI efficiency regulation in mammalian cells. The duplication of the genetic information of a cell starts from specific sites on the chromosomes called DNA replication origins. Their number varies from a few hundred in yeast cells to several thousands in human cells, distributed along the genome at comparable distances in both systems. An important question in the field is to understand how origins of replication are specified and regulated in the mammalian genome, as neither their location nor their activity can be directly inferred from the DNA sequence. Previous studies at individual origins and, more recently, at large scale across 1% of the human genome, have revealed that most origins overlap with transcriptional regulatory elements, and specifically with gene promoters. To gain insight into the nature of the relationship between active transcription and origin specification we have combined a genomic mapping of origins at 0.4% of the mouse genome with detailed studies of activation efficiency. The data identify two types of origins with distinct regulatory properties: highly efficient origins map at CpG island-promoters and low efficient origins locate elsewhere in association with transcriptional units. We also find a remarkable parallel organisation of the replication initiation sites and transcription start sites at efficient promoter-origins that suggests a prominent role of transcription initiation in setting the efficiency of replication origin activation.

CpG islands are G+C‐rich regions ∼1 kb long that are free of methylation and contain the promoters of many mammalian genes. Analysis of in vivo replication intermediates at three hamster genes and one human gene showed that the CpG island regions, but not their flanks, were present in very short nascent strands, suggesting that they are replication origins (ORIs). CpG island‐like fragments were enriched in a population of short nascent strands from human erythroleukaemic cells, suggesting that islands constitute a significant fraction of endogenous ORIs. Correspondingly, bulk CpG islands were found to replicate coordinately early in S phase. Our results imply that CpG islands are initiation sites for both transcription and DNA replication, and may represent genomic footprints of replication initiation.

Chromatin is the template for the basic processes of replication and transcription, making the maintenance of chromosomal integrity critical for cell viability. To elucidate how dividing cells respond to alterations in chromatin structure, here we analyse the replication programme of primary cells with altered chromatin configuration caused by the genetic ablation of the HMGB1 gene, or three histone H1 genes. We find that loss of chromatin compaction in H1-depleted cells triggers the accumulation of stalled forks and DNA damage as a consequence of transcription–replication conflicts. In contrast, reductions in nucleosome occupancy due to the lack of HMGB1 cause faster fork progression without impacting the initiation landscape or fork stability. Thus, perturbations in chromatin integrity elicit a range of responses in the dynamics of DNA replication and transcription, with different consequences on replicative stress. These findings have broad implications for our understanding of how defects in chromatin structure contribute to genomic instability.

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

Eukaryotic DNA replication initiates at multiple points along the chromosomes known as replication origins (ORIs). We have developed a strategy to identify ORIs directly from replication intermediates in the fission yeast Schizosaccharomyces pombe . Mapping of a selection of the novel ORIs onto the genome reveals their preferential localization at intergenic regions upstream from genes. These results are supported by the observation that a large proportion of regions overlapping gene promoters contain active ORIs. Mapping of the genomic ars1 replication origin at nucleotide resolution shows that replication initiates at a defined position immediately upstream from the hus5+ promoter. Deletion analysis indicates that the regulatory elements required to initiate transcription and replication lie in close proximity, suggesting a possible relationship between both processes in vivo .

DNA replication origins are poorly characterized genomic regions that are essential to recruit and position the initiation complex to start DNA synthesis. Despite the lack of specific replicator sequences, initiation of replication does not occur at random sites in the mammalian genome. This has lead to the view that DNA accessibility could be a major determinant of mammalian origins. Here, we performed a high‐resolution analysis of nucleosome architecture and initiation sites along several origins of different genomic location and firing efficiencies. We found that mammalian origins are highly variable in nucleosome conformation and initiation patterns. Strikingly, initiation sites at efficient CpG island‐associated origins always occur at positions of high‐nucleosome occupancy. Origin recognition complex (ORC) binding sites, however, occur at adjacent but distinct positions marked by labile nucleosomes. We also found that initiation profiles mirror nucleosome architecture, both at endogenous origins and at a transgene in a heterologous system. Our studies provide a unique insight into the relationship between chromatin structure and initiation sites in the mammalian genome that has direct implications for how the replication programme can be accommodated to diverse epigenetic scenarios.