Transcription Initiation Activity Sets Replication Origin Efficiency in Mammalian Cells

Joana Sequeira-Mendes,Ramón Díaz-Uriarte,Derek Huntley,Neil Brockdorff,Anwyn Apedaile,María Gómez,Wendy A Bickmore

doi:10.1371/journal.pgen.1000446

Abstract

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.

Highlights

DNA replication initiation is thought to be the most highly regulated process in genome duplication as cells must ensure that replication origins (ORIs) fire precisely once before cell division
We 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
Preparations of short nascent strands purified from asynchronously growing cells are preferentially enriched in regions close to ORIs and less enriched in their immediately adjacent sequences, showing a pine-tree distribution peaking at the ORI that allows their fine mapping by quantitative real-time PCR methods (Q-PCR) [26,27,28]

Summary

Introduction

DNA replication initiation is thought to be the most highly regulated process in genome duplication as cells must ensure that replication origins (ORIs) fire precisely once before cell division. A large number of studies during the last twenty years have provided a good understanding of the molecular mechanisms that regulate the initiation of DNA synthesis to occur at specific chromosomal sites and during a specific window in the cell cycle to avoid undesired re- or under-replication of any part of the eukaryotic genome [1,2,3]. Recent high-throughput studies in various experimental systems have confirmed the long observed link between early replication timing and active transcription [14,15,16,17,18]. Despite these findings, the steps in the initiation process that are influenced by transcription are poorly understood. It is possible that changes in transcriptional status could modulate the initial selection of potential ORIs either during the G1 phase of the cell cycle (pre-RC formation) or during the activation of pre-RC in S-phase

Methods

Results

Conclusion