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 mechanisms regulating the coordinate activation of tens of thousands of replication origins in multicellular organisms remain poorly explored. Recent advances in genomics have provided valuable information about the sites at which DNA replication is initiated and the selection mechanisms of specific sites in both yeast and vertebrates. Studies in yeast have advanced to the point that it is now possible to develop convincing models for origin selection. A general model has emerged, but yeast data have also revealed an unsuspected diversity of strategies for origin positioning. We focus here on the ways in which chromatin structure may affect the formation of pre-replication complexes, a prerequisite for origin activation. We also discuss the need to exercise caution when trying to extrapolate yeast models directly to more complex vertebrate genomes.