Sequencing newly replicated DNA reveals widespread plasticity in human replication timing

Department of Medicine, Division of Medical Genetics, University of Washington School of Medicine, Seattle, WA 98195, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2009; 107(1):139-44. DOI: 10.1073/pnas.0912402107
Source: PubMed


Faithful transmission of genetic material to daughter cells involves a characteristic temporal order of DNA replication, which may play a significant role in the inheritance of epigenetic states. We developed a genome-scale approach--Repli Seq--to map temporally ordered replicating DNA using massively parallel sequencing and applied it to study regional variation in human DNA replication time across multiple human cell types. The method requires as few as 8,000 cytometry-fractionated cells for a single analysis, and provides high-resolution DNA replication patterns with respect to both cell-cycle time and genomic position. We find that different cell types exhibit characteristic replication signatures that reveal striking plasticity in regional replication time patterns covering at least 50% of the human genome. We also identified autosomal regions with marked biphasic replication timing that include known regions of monoallelic expression as well as many previously uncharacterized domains. Comparison with high-resolution genome-wide profiles of DNaseI sensitivity revealed that DNA replication typically initiates within foci of accessible chromatin comprising clustered DNaseI hypersensitive sites, and that replication time is better correlated with chromatin accessibility than with gene expression. The data collectively provide a unique, genome-wide picture of the epigenetic compartmentalization of the human genome and suggest that cell-lineage specification involves extensive reprogramming of replication timing patterns.

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    • "Consistent with previous reports in mouse (Hiratani et al. 2008) and human (Hansen et al. 2010) cell types, constitutively early replicating regions were gene and GC-rich, with a lower density Figure 1. Genome-wide RT patterns are lineage specific. "
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    ABSTRACT: Duplication of the genome in mammalian cells occurs in a defined temporal order referred as its replication-timing (RT) program. RT changes dynamically during development, regulated in units of 400-800 kb referred as replication domains (RDs). Changes in RT are generally coordinated with transcriptional competence and changes in sub-nuclear position. We generated genome-wide RT profiles for 26 distinct human cell types including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures) and confirmed global consolidation of the genome into larger synchronously replicating segments during differentiation. Surprisingly, transcriptome data revealed that the well-accepted correlation between early replication and transcriptional activity was restricted to RT-constitutive genes, whereas two thirds of the genes that switched RT during differentiation were strongly expressed when late replicating in one or more cell types. Closer inspection revealed that transcription of this class of genes was frequently restricted to the lineage in which the RT switch occurred, but was induced prior to a late to early RT switch and/or down-regulated after an early to late RT switch. Analysis of transcriptional regulatory networks showed that this class of genes contains strong regulators of genes that were only expressed when early replicating. These results provide intriguing new insight into the complex relationship between transcription and RT regulation during human development. Published by Cold Spring Harbor Laboratory Press.
    Genome Research 06/2015; 25(8). DOI:10.1101/gr.187989.114 · 14.63 Impact Factor
    • "This comparison was very instructive since it revealed the existence of a strong correlation between the four prevalent chromatin states and the MRT, and this for both the pluripotent (H1hesc) and the differentiated (K562, Gm12878, Nhdfad) cell lines (Fig. 1) [90] [92]. The transcriptionally active euchromatin states EC1 and C1 replicate early in the S-phase in agreement with the previous studies of open chromatin marks in human and mouse [30] [32] [34] [61] [62] [117]. The bivalent EC2 state and the differentiated polycomb repressed C2 facultative heterochromatin state both replicate slightly later in mid-S phase, as recently confirmed by the sequencing of nascent DNA strands synthetized at replication origins in human [118]. "
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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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    • "Here we have used standard annotations derived from a variety of RNA sequencing approaches; however, the availability of large promoter databases determined using 5 ′ Cap Analysis of Gene Expression (CAGE) mapping, will improve our ability to resolve phasing patterns relative to precise transcriptional initiation sites (FANTOM Consortium and the RIKEN PMI and CLST [DGT] et al. 2014). Long (megabase-scale) domains of H3K9 methylation have been linked to topological domains (Dixon et al. 2012) and associated with the nuclear lamina territory (Guelen et al. 2008) and late replication timing (Hansen et al. 2010). In cancer, these domains tend to become hypomethylated (Hansen et al. 2011; Berman et al. 2012). "
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    ABSTRACT: The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes. © 2015 Lay et al.; Published by Cold Spring Harbor Laboratory Press.
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