Marie-Noëlle Prioleau

Genome-wide studies highlight indirect links between human replication origins and gene regulation

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 origin-timing program regulates the replication of the human genome.

An insulator element and condensed chromatin region separate the chicken β‐globin locus from an independently regulated erythroid‐specific folate receptor gene

We have identified a folate receptor gene upstream of the chicken β‐globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU‐E) preceding the activation of β‐globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the β‐globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient to induce strong expression of a transgene in CFU‐E stage cells. We also find that the region between the β‐globin locus and the folate receptor gene is fully methylated and condensed at this stage of differentiation. Its 3′ boundary coincides with the 5′ β‐globin insulator. We speculate that the 5′ β‐globin boundary element might be important for the proper regulation of two adjacent domains activated at two different stages during differentiation.

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.

The Spatiotemporal Program of DNA Replication Is Associated with Specific Combinations of Chromatin Marks in Human Cells

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.

The Relationship between DNA Replication and Human Genome Organization

Assessment of the impact of DNA replication on genome architecture in Eukaryotes has long been hampered by the scarcity of experimental data. Recent work, relying on computational predictions of origins of replication, suggested that replication might be a major determinant of gene organization in human (Huvet et al. 2007. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17:1278-1285). Here, we address this question by analyzing the first large-scale data set of experimentally determined origins of replication in human: 283 origins identified in HeLa cells, in 1% of the genome covered by ENCODE regions (Cadoret et al. 2008. Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc Natl Acad Sci USA. 105:15837-15842). We show that origins of replication are not randomly distributed as they display significant overlap with promoter regions and CpG islands. The hypothesis of a selective pressure to avoid frontal collisions between replication and transcription polymerases is not supported by experimental data as we find no evidence for gene orientation bias in the proximity of origins of replication. The lack of a significant orientation bias remains manifest even when considering only genes expressed at a high rate, or in a wide number of tissues, and is not affected by the regional replication timing. Gene expression breadth does not appear to be correlated with the distance from the origins of replication. We conclude that the impact of DNA replication on human genome organization is considerably weaker than previously proposed.

DNA replication origins—where do we begin?

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages.

USF Binding Sequences from the HS4 Insulator Element Impose Early Replication Timing on a Vertebrate Replicator

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.

Interplay between DNA replication and gene expression: a harmonious coexistence

Multicellular organisms have evolved highly sophisticated machinery to that their genomes are accurately duplicated and that the various gene expression programs are established correctly. Recent large-scale studies have shed light on how these fundamental processes interact. Although the machinery mediating these processes share similar cis-regulatory elements, they are not strictly coregulated. Furthermore, studies of the replisome show that highly transcribed genes present a major obstacle to its operation. Further studies will be needed to identify key regulators of the spatio-temporal program of DNA replication, for the elucidation of the complex interplay between replication and transcription.

CpG Islands: Starting Blocks for Replication and Transcription

ORI sequences. A few months ago, common features shared by large groups of ORIs were extracted from a large-scale mapping of replication origins based on the resistance of short nascent strands (SNS) to l-exonuclease digestion [5]. This study, conducted in HeLa cells and covering approximately 1% of the human genome, showed that origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no or only few origins in GC-poor regions. Moreover, half of the origins were mapped within or near CpG islands. In this issue of PLoS Genetics, a study by Sequeira-Mendes et al. extends this obser

Genomic approaches to the initiation of DNA replication and chromatin structure reveal a complex relationship

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.