To get insights into the regulation of replication initiation, we systematically mapped replication origins along 1% of the human genome in HeLa cells. We identified 283 origins, 10 times more than previously known. Origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no origins in large GC-poor regions. Origin sequences are evolutionarily conserved, and half of them map within or near CpG islands. Most of the origins overlap transcriptional regulatory elements, providing further evidence of a connection with gene regulation. Moreover, we identify c-JUN and c-FOS as important regulators of origin selection. Half of the identified replication initiation sites do not have an open chromatin configuration, showing the absence of a direct link with gene regulation. Replication timing analyses coupled with our origin mapping suggest that a relatively strict origintiming program regulates the replication of the human genome.chromatin structure ͉ DNA replication origin ͉ ENCODE regions ͉ genome-wide mapping ͉ CpG island C ontrolling the number of origins from which replication begins in a given chromosome is necessary to protect it from instability (1, 2). Mapping DNA replication starting points, known as ''origins of replication,'' would make a large contribution to understanding how genome replication is coordinated. Identification of a large number of replication origins is necessary to decipher the rules of origin specification. However, fewer than 30 origins have been identified in human cells (3), and they were mapped mostly in well-characterized transcribed regions, leaving gene-poor regions unexplored. The Encyclopedia of DNA Elements (ENCODE) project (4), launched to develop high-throughput methods for identifying functional elements, now provides a comprehensive view of gene expression and chromatin structure along 1% of the human genome (30 Mb, 44 regions) (5); it thus provides a powerful model for studying interactions among chromosome organization, gene regulation, and initiation of DNA replication. Origin selection is initiated by the binding of the origin recognition complex (ORC) to origin-proximal DNA sequences (6). In contrast to Saccharomyces cerevisiae, characterized metazoan origins do not conform to a clear consensus sequence, and the ORCs from higher eukaryotes exhibit no sequence specificity in vitro (7). Transcription factors at sites of replication initiation have been shown to stimulate replication in many systems, including viruses, yeast, Drosophila, and Xenopus (8, 9). This stimulation may be a consequence of direct interaction with components of the replication machinery or of facilitating the access of the replication complexes to DNA through recruitment of chromatin remodeling complexes (10-12). Here we present a high-resolution map of replication origins in HeLa cells based on hybridization of short nascent strands (SNS) on DNA microarrays covering ENCODE regions. To construct this map we use one of the most string...

The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.

A combination of cis-regulatory elements can impose the formation of an early replicating domain in a naturally late replicating region and might constitute the basic unit of early replicating domains.