Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

The Hospital for Sick Children and University of Toronto, Canada
PLoS Computational Biology (Impact Factor: 4.62). 12/2011; 7(12):e1002322. DOI: 10.1371/journal.pcbi.1002322
Source: PubMed


Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.


Available from: Arach Goldar
    • "A recent study of 18 human and 13 mouse cell types [58] has further confirmed that early TTR borders share a near one-to-one correlation with TAD boundaries strongly suggesting that these structural domains are stable units of replication-timing regulation. As experimentally noticed in Ref. [71], the replication rate of TTRs is not always compatible with the unidirectional progression of one replication fork. In these cases, the coherent ''wave'' of replication from the early to the late TTR borders necessarily implies a more complex replication program. "
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    ABSTRACT: Recent analysis of genome-wide epigenetic modification data, mean replication timing (MRT) profiles and chromosome conformation data in mammals have provided increasing evidence that flexibility in replication origin usage is regulated locally by the epigenetic landscape and over larger genomic distances by the 3D chromatin architecture. Here, we review the recent results establishing some link between replication domains and chromatin structural domains in pluripotent and various differentiated cell types in human. We reconcile the originally proposed dichotomic picture of early and late constant timing regions that replicate by multiple rather synchronous origins in separated nuclear compartments of open and closed chromatins, with the U-shaped MRT domains bordered by "master" replication origins specified by a localized (∼200-300kb) zone of open and transcriptionally active chromatin from which a replication wave likely initiates and propagates toward the domain center via a cascade of origin firing. We discuss the relationships between these MRT domains, topologically associated domains and lamina-associated domains. This review sheds a new light on the epigenetically regulated global chromatin reorganization that underlies the loss of pluripotency and the determination of differentiation properties. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
    FEBS letters 04/2015; 589(20). DOI:10.1016/j.febslet.2015.04.015 · 3.17 Impact Factor
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    • "50 kb) early constant timing region and is surrounded by two stretches replicated just before it (Fig 1C and Supplementary Fig S1B) (Sarkies et al, 2010). This indicates that the BU-1 locus is either passively replicated equally from the left or from the right, or that is replicated by multiple, synchronous internal initiations (Guilbaud et al, 2011). Given that the average inter-origin distance in DT40, determined by molecular combing, is 76 +/À 7 kb (Supplementary Fig S1C), it is most likely that the locus is bidirectionally replicated, with the fork entering from the 3 0 end of the locus in about 50% of S phases. "
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    ABSTRACT: REV1-deficient chicken DT40 cells are compromised in replicating G quadruplex (G4)-forming DNA. This results in localised, stochastic loss of parental chromatin marks and changes in gene expression. We previously proposed that this epigenetic instability arises from G4-induced replication fork stalls disrupting the accurate propagation of chromatin structure through replication. Here, we test this model by showing that a single G4 motif is responsible for the epigenetic instability of the BU-1 locus in REV1-deficient cells, despite its location 3.5 kb from the transcription start site (TSS). The effect of the G4 is dependent on it residing on the leading strand template, but is independent of its in vitro thermal stability. Moving the motif to more than 4 kb from the TSS stabilises expression of the gene. However, loss of histone modifications (H3K4me3 and H3K9/14ac) around the transcription start site correlates with the position of the G4 motif, expression being lost only when the promoter is affected. This supports the idea that processive replication is required to maintain the histone modification pattern and full transcription of this model locus.
    The EMBO Journal 09/2014; 33(21). DOI:10.15252/embj.201488398 · 10.43 Impact Factor
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    • "Indeed, origins can be either activated or passively replicated by adjacent forks, in a stochastic pattern leading to cell-to-cell plasticity [2]. Replication timing has been deeply investigated with special emphasis for its possible modulation along development and differentiation [24], and robust evidence was recently collected that the timing may be cell-type specific and reprogrammable [3] [13] [12]. Finally, in mammalian cells a tight coordination between replication and transcription has been demonstrated [4] [21]; this regulation may be crucial for genome stability [14]. "
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    ABSTRACT: Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unravelled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates, organization of the replication clusters, proportion of unidirectional forks, fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harbouring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets.
    Experimental Cell Research 10/2013; 319(20). DOI:10.1016/j.yexcr.2013.10.001 · 3.25 Impact Factor
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