Dynamic Alterations of Replication Timing in Mammalian Cells

SUMMARYMammalian genomes replicate in a cell-type-specific order during the S phase, correlated to transcriptional activity, histone modifications, and chromatin structure. The causal relationships between these features and DNA replication timing (RT), especially during cell fate changes, are largely unknown. Using machine learning, we quantify 21 chromatin features predicting local RT and RT changes during differentiation in embryonic stem cells (ESCs). About one-third of the genome shows RT changes during differentiation. Chromatin features accurately predict both steady-state RT and RT changes. Histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C and KMT2D (KMT2C/D), emerges as a top predictor. Loss of KMT2C/D or their enzymatic activities impairs RT changes during differentiation. This correlates with local H3K4me1 loss and reduced replication origin firing, while transcription remains largely unaffected. Our findings reveal KMT2C/D-dependent H3K4me1 as a key regulator of RT and replication initiation, a role that likely impacts diseases associated with KMT2C/D mutations.

Recent evidence suggests that the timing of DNA replication is coordinated across megabase-scale domains in metazoan genomes, yet the importance of this aspect of genome organization is unclear. Here we show that replication timing is remarkably conserved between human and mouse, uncovering large regions that may have been governed by similar replication dynamics since these species have diverged. This conservation is both tissue-specific and independent of the genomic G+C content conservation. Moreover, we show that time of replication is globally conserved despite numerous large-scale genome rearrangements. We systematically identify rearrangement fusion points and demonstrate that replication time can be locally diverged at these loci. Conversely, rearrangements are shown to be correlated with early replication and physical chromosomal proximity. These results suggest that large chromosomal domains of coordinated replication are shuffled by evolution while conserving the large-scale nuclear architecture of the genome.

Genomes in eukaryotes normally undergo DNA replication in a choreographed temporal order, resulting in early and late replicating chromosome compartments. Leishmania, a human protozoan parasite, displays an unconventional DNA replication program in which the timing of DNA replication completion is chromosome size-dependent: larger chromosomes complete replication later then smaller ones. Here we show that both R-loops and RNase H1, a ribonuclease that resolves RNA-DNA hybrids, accumulate in Leishmania major chromosomes in a pattern that reflects their replication timing. Furthermore, we demonstrate that such differential organisation of R-loops, RNase H1 and DNA replication timing across the parasite’s chromosomes correlates with size-dependent differences in chromatin accessibility, G quadruplex distribution and sequence content. Using conditional gene excision, we show that loss of RNase H1 leads to transient growth perturbation and permanently abrogates the differences in DNA replication timing across chromosomes, as well as altering levels of aneuploidy and increasing chromosome instability in a size-dependent manner. This work provides a link between R-loop homeostasis and DNA replication timing in a eukaryotic parasite and demonstrates that orchestration of DNA replication dictates levels of genome plasticity in Leishmania.

The organization of mammalian DNA replication is poorly understood. We have produced high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal, and embryonic stem (ES) cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy-number variations during S phase to produce replication timing profiles. We first obtained timing maps of 3% of the genome using high-density oligonucleotide tiling arrays and then extended the TimEX method genome-wide using massively parallel sequencing. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation, where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by single molecule analysis of replicated DNA (SMARD). Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarters of S phase. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue-specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid-firing origins are located near moderately expressed genes, and that late-firing origins are located far from genes.

DNA replication in the human beta-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the beta-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5'HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5'HS2-5 in the normal chromosomal context of the human beta-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5'HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5'HS2-5 in the classically defined LCR do not control replication in the human beta-globin locus. Recent studies also show that targeted deletion of 5'HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

DNA replication is a tightly regulated process that follows a strict, yet poorly understood, temporal program [12, 9]. This timing program is intricately linked to many aspects of cell biology [1], it is cell type specific [11, 6] and altered in cancer cells [4, 5]. Although on the genome scale DNA replication appears as a highly orchestrated process, at the level of individual initiation events it is found to be stochastic [3, 8]. The mechanisms controlling global DNA replication timing remain largely unknown. Recently, stochastic DNA replication models, where global timing emerges from the collective action of unregulated initiation events, have been proposed [7, 2] yet there is still no quantitative model that can explain complex timing patterns observed in metazoan genomes. Contributing to the dearth of such models is the incomplete characterization of replication initiation sites in these genomes. We show that this issue does not prevent building a successful DNA replication timing model because we find that (a) the replication timing program is so robust that knowledge of exact firing probabilities is unnecessary and (b) high efficiency replicators are sufficiently localized by a specific chromatin mark.

The transmission of genetic information relies on a coordinated network of cell cycle controls. Abnormalities in this network can result in genomic instability and lead to the transformation of normal cells into cancer cells. Chromosomal DNA replication is not only central to cellular division but also plays a crucial role in the maintenance of genomic integrity. DNA replication errors increase genetic instability, and may be a causative factor in diseases such as cancer and neuronal disorders. Replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict, often tissue-specific, temporal program. The genetic program that controls activation of replication origins in mammalian cells has still not been elucidated. There is evidence that specification of replication sites and timing of replication are dynamic processes that are regulated by tissue-specific and developmental cues and that are responsive to epigenetic modifications. Here, we focus on the spatiotemporal regulation of DNA replication in the human genome. There is growing evidence that chromosome band patterns and epigenetic transformation of chromatin influence the timing of replication. On the basis of this evidence, we propose that the chromatin regions showing switches in replication timing from early to late in S phase are correlated with chromosome band boundaries. These chromatin regions generally display transitions in GC contents and include more non-B-form DNA structures than other genomic regions. We also examine here the effect of changes in replication timing on genomic stability and the possible role of replication timing in the etiology of diseases such as cancer. Replication timing assays are one of many promising techniques under investigation that may in future allow much earlier cancer detection than is possible today.

Induced pluripotent stem cells (iPSC) are a widely used cell system and a foundation for cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which have the potential to affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing – a process linked to both genome regulation and genome stability – is efficiently reprogrammed to the embryonic state. To answer this, we profiled and compared genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicated their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibit delayed replication at heterochromatic regions containing genes downregulated in iPSC with incompletely reprogrammed DNA methylation. DNA replication delays were not the result of gene expression and DNA methylation aberrations and persisted after differentiating cells to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and lead to undesirable phenotypes in iPSCs, establishing it as an important genomic feature to consider when evaluating iPSC lines.

Histone modifications are thought to regulate gene expression in part by modulating DNA accessibility. Here, we measured genome-wide DNA accessibility in Drosophila melanogaster by combining M.SssI methylation footprinting with methylated DNA immunoprecipitation. We show that methylase accessibility demarcates differential distribution of active and repressive histone modifications as well as sites of transcription and replication initiation. DNA accessibility is increased at active promoters and chromosomal regions that are hyperacetylated at H4K16, particularly at the male X chromosome, suggesting that transcriptional dosage compensation is facilitated by permissive chromatin structure. Conversely, inactive chromosomal domains decorated with H3K27me3 are least accessible, supporting a model for Polycomb-mediated chromatin compaction. In addition, we detect higher accessibility at chromosomal regions that replicate early and at sites of replication initiation. Together, these findings indicate that differential histone-modification patterns and the organization of replication have distinct and measurable effects on the exposure of the DNA template.

Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.

Replication timing is a crucial aspect of genome regulation that is strongly correlated with chromatin structure, gene expression, DNA repair, and genome evolution. Replication timing is determined by the timing of replication origin firing, which involves activation of MCM helicase complexes loaded at replication origins. Nonetheless, how the timing of such origin firing is regulated remains mysterious. Here, we show that the number of MCMs loaded at origins regulates replication timing. We show for the first time in vivo that multiple MCMs are loaded at origins. Because early origins have more MCMs loaded, they are, on average, more likely to fire early in S phase. Our results provide a mechanistic explanation for the observed heterogeneity in origin firing and help to explain how defined replication timing profiles emerge from stochastic origin firing. These results establish a framework in which further mechanistic studies on replication timing, such as the strong effect of heterochromatin, can be pursued.

The human beta globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous beta globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

Studies on DNA replication during synchronized vegetative growth and gametic differentiation in Chlamydomonas reinhardtii