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 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.

Fig. 1.

Repli-Seq approach. (A) Cell-cycle fractionation of newly synthesized DNA. Exponentially growing cells are pulse-labeled with BrdU, stained with DAPI, and sorted into different fractions of the cell cycle according to DNA content as shown for this normal lymphoblastoid cell line (LCL). Fractionation is continuous across the cell cycle (see Materials and Methods). Antibody-purified BrdU-labeled DNA is made into sequencing libraries and sequenced on the Illumina platform, and the sequence tags are mapped to the hg18 reference genome. (B) Visualization of replication patterns as exemplified at the LRMP locus in the GM06990 LCL. Mapped sequence tags in this region of chromosome 12 reveal a very early peak of replication at the lymphoid-specific LRMP gene. Sequence tag densities for each cell-cycle fraction (below) were calculated over 50-kb windows, normalized according to their genome-wide sequence tag counts, and further normalized at each genomic position by calculating the percentage of total replication (to avoid biases due to nucleotide composition or mapability).

Fig. 2.

Validation of Repli-Seq with STS-based replication timing. (A) Repli-Seq replication timing measurements compared to a standard PCR-based assay at specific genomic locations in the distal portion of 11p15.4 for erythroid and lymphoblastoid cells. Using standard STS-based replication timing, 12 markers define a 2-Mb domain (shaded) containing the β-globin gene cluster that is early-replicating in erythroid cells that express globin genes (K562) and late-replicating in lymphoid cells where globin genes are silent (GM08729). (B) Sequence-based replication timing. The Repli-Seq whole-genome sequence-based data closely match the replication patterns found for specific STSs, but provide more comprehensive coverage of this 3-Mb region.

Fig. 3.

Cell-lineage-specific replication timing. (A) Comparison of replication timing profiles from four cell types across chromosome 4, illustrating unique lineage patterns. (B) Lineage-specific early-replication patterns. Cell-lineage-specific early patterns are highlighted in expanded chromosome 4 subregions. The lymphoid-specific CENTD1 gene in the 34.6–37.1 Mb region is at the apex of an early-replication peak in the GM06990 LCL, whereas this region is uniformly late-replicating in the other three lineages. Similar patterns are seen in the other expanded regions: LPHN3 in the 60.9–63.4 Mb region (hESC-specific), GYPA-GYPB-GYPE in the 143.8–146.3 Mb region (erythroid-specific), and PDGFC in the 156.7–159.2 region (fibroblast-specific). (C) Stereotypical replication timing patterns. Shown are major patterns of DNA replication timing observed across the genome, including (i) regions of constant early replication across cell lineages; (ii) regions of constant late replication; (iii) regions with cell-specific early replication; (iv) regions with lineage-specific late replication (i.e., one cell type late, all others early); and (v) complex patterns that vary considerably between lineages.

Fig. 4.

Replication timing patterns versus annotated genomic features. (A) Major annotated genomic features across a 50 Mb portion of the long arm of chromosome 11 including the densities of L1 LINEs and Alu SINEs; G+C content; and gene density. (B) Total RNA output measured by Affymetrix exon arrays across four cell types. (C) Constant early, constant late, and plastic replication time regions. (D) Repli-Seq data from four cell types (see Fig. 1 for description). (E) Regions of nuclear lamina association recently reported for human fibroblasts (23) correlate well with the regions of late replication we found in BJ fibroblasts. (F) Chromatin accessibility and density of DNaseI-hypersensitive sites in BJ fibroblasts. Note the extremely close correspondence with early-replicating regions in fibroblasts (D above, lower track), and the inverse relationship with lamin-associated late-replicating regions.

Fig. 5.

Biphasic replication timing. (A) Biphasic replication timing (simultaneous early and late replication of the same genomic region) associated with X chromosome inactivation. Shown are two examples of biphasic replication timing associated with X chromosome inactivation in females. In the chrX:121.5–124 Mb region, male LCLs (H0287) have an early peak associated with several genes known to be subject to X inactivation in females (38) (depicted in red; X inactivation status of genes in black is unknown). The region is biphasic in female LCLs (GM06990) with the early component resembling the male pattern (active X) and the very late component (G2) thus representing the inactive X. In the chrX:53.1–54.7 Mb region there is a transition from a more synchronous region containing many genes that escape X inactivation (in green) to a biphasic region that is subject to X inactivation (in red). (B) Biphasic replication in the imprinted Prader-Willi/Angelman syndrome region on chromosome 15. Replication is markedly biphasic in hESCs over a 2–3 Mb region associated with Prader-Willi syndrome (27), including the imprinted SNRPN gene. The early component is specific to hESCs and correlates with the much higher level of RNA expression in this region relative to other cell types. (C) Biphasic patterns indicate allele-specific replication programs. Early and late hESC fractions were examined for the presence or absence of an informative restriction site polymorphism in the SNRPN gene (BstUI) to confirm that biphasic patterns are allelic. Early-replicating DNA is exclusively the cleaved allele that is expressed (paternal), whereas the late-replicating DNA is exclusively the uncleaved allele that is repressed (maternal). Controls shown include BG02 DNA (for an equal allelic contribution) and a homozygous-cleaved DNA control for digestion.