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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    • "Rif1's distribution overlaps extensively with late-replicating regions, while it is generally depleted from early replicating domains (Figures 2A and 2B; Figures S2A and S2B). Because late-replicating regions associate with the nuclear lamina (Hansen et al., 2010; Peric- Hupkes et al., 2010), RADs largely correspond to genomic regions associated with the nuclear lamina (LADs) (Figure 2A) (Peric-Hupkes et al., 2010) and, more specifically, overlap with LADs that are invariant among cell types (cLADs) (Figure S2B) (Meuleman et al., 2013). We confirmed the association of Rif1 (E) Cell proliferation measured as the averages from triplicates of six independent Rif1 +/+ versus Rif1 À/À ESCs from three experiments (paired t test, ***p < 0.0001). "
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    ABSTRACT: DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.
    Full-text · Article · Jan 2016 · Molecular cell
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    • "Blocks of synchronously firing origins can also be visualized by genome-wide RT profiling as large synchronously replicated chromosome segments, and their boundaries can be identified by monitoring the coordinated switches in RT that occur across half of the genome during cell fate transitions, revealing 400 –800 kb units of replication regulation, or " replication domains (RDs) " (Hiratani et al. 2008, 2010; Hansen et al. 2010; Ryba et al. 2010). RDs are also likely to be the equivalent of the punctate " replication foci " that are seen whenever cells are briefly labeled with detectable nucleotide analogs . "
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    ABSTRACT: Chromosome architecture has received a lot of attention since the recent development of genome-scale methods to measure chromatin interactions (Hi-C), enabling the first sequence-based models of chromosome tertiary structure. A view has emerged of chromosomes as a string of structural units (topologically associating domains; TADs) whose boundaries persist through the cell cycle and development. TADs with similar chromatin states tend to aggregate, forming spatially segregated chromatin compartments. However, high-resolution Hi-C has revealed substructure within TADs (subTADs) that poses a challenge for models that attribute significance to structural units at any given scale. More than 20 years ago, the DNA replication field independently identified stable structural (and functional) units of chromosomes (replication foci) as well as spatially segregated chromatin compartments (early and late foci), but lacked the means to link these units to genomic map units. Genome-wide studies of replication timing (RT) have now merged these two disciplines by identifying individual units of replication regulation (replication domains; RDs) that correspond to TADs and are arranged in 3D to form spatiotemporally segregated subnuclear compartments. Furthermore, classifying RDs/TADs by their constitutive versus developmentally regulated RT has revealed distinct classes of chromatin organization, providing unexpected insight into the relationship between large-scale chromosome structure and function.
    Full-text · Article · Nov 2015 · Cold Spring Harbor Symposia on Quantitative Biology
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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.
    Full-text · Article · Jun 2015 · Genome Research
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