Sequencing newly replicated DNA reveals widespread plasticity in human replication timing

Abstract

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 cytometryfractionated 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 highresolution 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, genomewide picture of the epigenetic compartmentalization of the human genome and suggest that cell-lineage specification involves extensive reprogramming of replication timing patterns.chromatin structure | gene expression | tissue specificity

Global delay in nascent strand DNA methylation

“…To investigate the acquisition of CpG methylation on nascent DNA, we combined Repli-seq 21 (immunoprecipitation of bromodeoxyuridine (BrdU) labeled nascent strands followed by sequencing) with bisulfite treatment to measure post-replication cytosine methylation at base pair resolution (Repli-bisulfite seq: Repli-BS, Fig. 1a, Supplementary Fig.…”

DNA replication timing and selection shape the landscape of nucleotide variation in cancer genomes

“…For 1Mb windows, we examined the following genomic features: genomic GC content, recombination rate, distance to the telomeres, and the replication timing during the S phase [22][23][24] . We focussed on functionally neutral or nearly neutral regions, in which the observed mutation frequencies would be influenced by the mutation rate only.…”

“…We selected intergenic regions and removed from them potentially functional regions, such as transcription factor binding sites (TFBSs), open chromatin regions, and CpG islands. We removed regions with different replication timings in different cell types 24 . We conducted an ANOVA (type III) analysis to isolate contributions of individual genomic features to germ-line and cancer somatic SNVs while accounting for other features (Fig.…”

“…Replication timing is highly plastic and often changes, depending on the cell type 24 . Replication timing data with matching cell types are not available for most of the cancer genomes examined in this study.…”

“…We obtained genome-wide maps of replication timing 24 , which were generated by sequencing genomic DNAs replicated during six cell-cycle stages (G1, S1, S2, S3, S4 and G2) for four Encyclopedia of DNA Elements (ENCODE) cell lines: embryonic stem cell (BG02), fibroblast (BJ), lymphoblastoid cells (GM06990), and myelogenous leukaemic cells (K562). We used a wavelet-smoothed, weighted average signal whose high and low values indicate early and late replication during the S phase, respectively (http://genome.…”