DNA Replication Timing Research Articles

Defining the separation landscape of topological domains for decoding consensus domain organization of the 3D genome.

Topologically associating domains (TADs) have emerged as basic structural and functional units of genome organization and have been determined by many computational methods from Hi-C contact maps. However, the TADs obtained by different methods vary greatly, which makes the accurate determination of TADs a challenging issue and hinders subsequent biological analyses about their organization and functions. Obvious inconsistencies among the TADs identified by different methods indeed make the statistical and biological properties of TADs overly depend on the chosen method rather than on the data. To this end, we use the consensus structural information captured by these methods to define the TAD separation landscape for decoding the consensus domain organization of the 3D genome. We show that the TAD separation landscape could be used to compare domain boundaries across multiple cell types for discovering conserved and divergent topological structures, decipher three types of boundary regions with diverse biological features, and identify consensus TADs (ConsTADs). We illustrate that these analyses could deepen our understanding of the relationships between the topological domains and chromatin states, gene expression, and DNA replication timing.

The evolution of the human DNA replication timing program

DNA is replicated according to a defined spatiotemporal program that is linked to both gene regulation and genome stability. The evolutionary forces that have shaped replication timing programs in eukaryotic species are largely unknown. Here, we studied the molecular causes and consequences of replication timing evolution across 94 humans, 95 chimpanzees, and 23 rhesus macaques. Replication timing differences recapitulated the species' phylogenetic tree, suggesting continuous evolution of the DNA replication timing program in primates. Hundreds of genomic regions had significant replication timing variation between humans and chimpanzees, of which 66 showed advances in replication origin firing in humans, while 57 were delayed. Genes overlapping these regions displayed correlated changes in expression levels and chromatin structure. Many human-chimpanzee variants also exhibited interindividual replication timing variation, pointing to ongoing evolution of replication timing at these loci. Association of replication timing variation with genetic variation revealed that DNA sequence evolution can explain replication timing variation between species. Taken together, DNA replication timing shows substantial and ongoing evolution in the human lineage that is driven by sequence alterations and could impact regulatory evolution at specific genomic sites.

Epigenomic signatures associated with spontaneous and replication stress-induced DNA double strand breaks.

Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced in vitro by mild level of DNA replication stress, such as DNA polymerase inhibition or nucleotide pool disturbance. The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.

Replication Timing Dictates the Frequency of Chromosomal Translocations.

Synchronized DNA replication timing influences the rate of oncogenic translocations between Myc and Igh.

DNA replication timing: Biochemical mechanisms and biological significance.

The regulation of DNA replication is a fascinating biological problem both from a mechanistic angle-How is replication timing regulated?-and from an evolutionary one-Why is replication timing regulated? Recent work has provided significant insight into the first question. Detailed biochemical understanding of the mechanism and regulation of replication initiation has made possible robust hypotheses for how replication timing is regulated. Moreover, technical progress, including high-throughput, single-molecule mapping of replication initiation and single-cell assays of replication timing, has allowed for direct testing of these hypotheses in mammalian cells. This work has consolidated the conclusion that differential replication timing is a consequence of the varying probability of replication origin initiation. The second question is more difficult to directly address experimentally. Nonetheless, plausible hypotheses can be made and one-that replication timing contributes to the regulation of chromatin structure-has received new experimental support.

DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations.

Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT) directly regulates lymphomagenic Myc translocations during antibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion of minichromosome maintenance complexes alters replication origin activity, decreases translocations, and deregulates global RT. Ablating a single origin at Myc causes an early-to-late RT switch, loss of translocations, and reduced proximity with the immunoglobulin heavy chain (Igh) gene, its major translocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT also reduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanism in translocation biogenesis linking DSB formation to DSB ligation.

Genome architecture plasticity underlies DNA replication timing dynamics in cell differentiation.

During the S-phase of eukaryotic cell cycle, DNA is replicated in a dedicatedly regulated temporal order, with regions containing active and inactive genes replicated early and late, respectively. Recent advances in sequencing technology allow us to explore the connection between replication timing (RT), histone modifications, and three-dimensional (3D) chromatin structure in diverse cell types. To characterize the dynamics during cell differentiation, corresponding sequencing data for human embryonic stem cells and four differentiated cell types were collected. By comparing RT and its extent of conservation before and after germ layer specification, the human genome was partitioned into distinct categories. Each category is then subject to comparisons on genomic, epigenetic, and chromatin 3D structural features. As expected, while constitutive early and late replication regions showed active and inactive features, respectively, dynamic regions with switched RT showed intermediate features. Surprisingly, although early-to-late replication and late-to-early replication regions showed similar histone modification patterns in hESCs, their structural preferences were opposite. Specifically, in hESCs, early-to-late replication regions tended to appear in the B compartment and large topologically associated domains, while late-to-early replication regions showed the opposite. Our results uncover the coordinated regulation of RT and 3D genome structure that underlies the loss of pluripotency and lineage commitment and indicate the importance and potential roles of genome architecture in biological processes.

Chromatin accessibility of primary human cancers ties regional mutational processes and signatures with tissues of origin.

Somatic mutations in cancer genomes are associated with DNA replication timing (RT) and chromatin accessibility (CA), however these observations are based on normal tissues and cell lines while primary cancer epigenomes remain uncharacterised. Here we use machine learning to model megabase-scale mutation burden in 2,500 whole cancer genomes and 17 cancer types via a compendium of 900 CA and RT profiles covering primary cancers, normal tissues, and cell lines. CA profiles of primary cancers, rather than those of normal tissues, are most predictive of regional mutagenesis in most cancer types. Feature prioritisation shows that the epigenomes of matching cancer types and organ systems are often the strongest predictors of regional mutation burden, highlighting disease-specific associations of mutational processes. The genomic distributions of mutational signatures are also shaped by the epigenomes of matched cancer and tissue types, with SBS5/40, carcinogenic and unknown signatures most accurately predicted by our models. In contrast, fewer associations of RT and regional mutagenesis are found. Lastly, the models highlight genomic regions with overrepresented mutations that dramatically exceed epigenome-derived expectations and show a pan-cancer convergence to genes and pathways involved in development and oncogenesis, indicating the potential of this approach for coding and non-coding driver discovery. The association of regional mutational processes with the epigenomes of primary cancers suggests that the landscape of passenger mutations is predominantly shaped by the epigenomes of cancer cells after oncogenic transformation.

A non-transcriptional function of Yap regulates the DNA replication program in Xenopus laevis.

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

Optimized Repli-seq: improved DNA replication timing analysis by next-generation sequencing.

The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type-specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.

From the archives: Polar auxin transport in nodule development, DNA replication timing, and developmentally light-regulated genes.

From the archives: Polar auxin transport in nodule development, DNA replication timing, and developmentally light-regulated genes.

Abstract 1508: Chromatin accessibility of primary human cancers ties regional mutational processes with tissues of origin

Abstract Cancer genomes are shaped by mutational processes with complex spatial variation at multiple scales. However, the underlying mechanisms of this mutagenesis and its functional and genetic determinants remain poorly understood. Somatic regional mutagenesis is known to correlate with DNA replication timing and chromatin accessibility; however, these studies have used epigenetic information from common cell lines while the epigenomes of primary human cancers remain uncharacterized in this context. Here we model megabase-scale mutation frequencies of single nucleotide variants (SNVs) in thousands of whole cancer genomes using hundreds of genome-wide profiles of chromatin accessibility and replication timing spanning primary cancer samples, normal tissues, and cell lines. Using a machine learning framework, we show that CA profiles of primary cancers, rather than those of normal cells, predict regional mutagenesis and single base substitution (SBS) signatures in most cancer types. Thus the majority of passenger mutations follow the epigenetic landscapes of transformed cells of matching cancer sites and tissues. Mutational signatures associated with carcinogen exposure, as well as signatures of unknown origin, show the strongest associations with epigenomes while associations with endogenous signatures are weaker. In most cases, overall mutation burden and SBS signatures inversely correlate with chromatin accessibility, however certain signatures are instead enriched in chromatin-accessible elements. Lastly, the genomic regions with excess mutations unexplained by the epigenetic profiles in our models likely represent a combination of localized mutagenesis and positive selection of functional oncogenic mutations. Our computational models highlight an enrichment of known driver mutations, point out novel intergenic regions with putative non-coding drivers, and indicate an over-representation of developmental and lineage-specific genes in these frequently mutated genomic regions. These results characterize the complex interplay of mutational processes, genome function, and somatic evolution in cancer and its tissues of origin, and also outline strategies for distinguishing driver and passenger mutations. Citation Format: Jüri Reimand, Oliver Ocsenas. Chromatin accessibility of primary human cancers ties regional mutational processes with tissues of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1508.

Telomere-to-telomere human DNA replication timing profiles

The spatiotemporal organization of DNA replication produces a highly robust and reproducible replication timing profile. Sequencing-based methods for assaying replication timing genome-wide have become commonplace, but regions of high repeat content in the human genome have remained refractory to analysis. Here, we report the first nearly-gapless telomere-to-telomere replication timing profiles in human, using the T2T-CHM13 genome assembly and sequencing data for five cell lines. We find that replication timing can be successfully assayed in centromeres and large blocks of heterochromatin. Centromeric regions replicate in mid-to-late S-phase and contain replication-timing peaks at a similar density to other genomic regions, while distinct families of heterochromatic satellite DNA differ in their bias for replicating in late S-phase. The high degree of consistency in centromeric replication timing across chromosomes within each cell line prompts further investigation into the mechanisms dictating that some cell lines replicate their centromeres earlier than others, and what the consequences of this variation are.

High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control

DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We develop an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes in silico flow cytometry, a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyze up to 2437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression.

Single‐Molecule Analysis of in vivo DNA Replication Origin Licensing Stoichiometry

The organization of DNA replication location and timing is regulated by the establishment of replication origins. Origins are established in G1 by the loading of a pair of MCMs, the catalytic core of the replicative helicase, as an inactive double‐hexamer complex. Activation of MCM during S phase is the rate‐limiting step for replication initiation, and therefore regulates where and when replication occurs. We have previously shown that origins at which multiple MCM complexes are loaded have a higher probability of initiation and therefore replicate earlier, on average. Bulk biochemical experiments suggest the ARS1, a early‐firing budding yeast origin, loads, on average, about three MCM complexes in G1, whereas ARS316, a late‐firing origin, loads less than one. However, these bulk measurements have two major drawbacks. First, the required normalization controls make our absolute stoichiometry estimates uncertain. Second, they only reveal the average numbers of MCM's loaded; they provide no insight into the distribution of MCM stoichiometry, information which could elucidate how MCM loading stoichiometry is controlled.

Epigenetics: A Gatekeeper to DNA Amplification and Replication Control

DNA amplification is associated with pathological states such as neurological disorders, cardiac disease and cancer. At least 50% of the amplifications in cancer are transient extrachromosomal DNA (ecDNA). The transient behavior contributes to copy number plasticity, results in heterogeneous oncogene expression and alters therapeutic response. The unanswered question remains as to whether distinct mechanisms control ecDNA copy gains within cells and how they impact copy gain events associated with disease. Our laboratory aims to define the principles regulating selective DNA amplification and the associated plasticity, so we may control these events in disease. By resolving general mechanisms governing selective DNA amplifications, we will build a framework to further understand the molecular basis by which copy gains occur in neoplasia and other diseases.Recent studies have begun to uncover the importance of epigenetic states and histone lysine methyltransferases (KMTs) and demethylases (KDMs) in regulating extrachromosomal transient site‐specific DNA copy number gains (TSSGs). In fact, our laboratory discovered the first chromatin factor promoting TSSGs in the human genome. We have revealed a critical interplay between a myriad of KMTs and KDMs in modulating methylation balance in order to control rereplication and extrachromosomal amplification events. The methyl‐lysine modifier networks that control site‐specific DNA amplification were resolved through genetic and chemical treatments coupled to cytological approaches, epigenomic profiling and 3D genome organizational analyses. While interrogating their role in controlling DNA amplification, we have also begun to appreciate their impact on chromatin modification dynamics and global DNA replication timing. We have established that enhancers coupled to methylation changes in broad methylation domains strongly predicts replication timing changes when perturbed by specific lysine modifiers. Taken together, we have established that methyl‐lysine modifiers are key regulators promoting or preventing DNA amplification and play instrumental roles in regulating replication timing control through the combined regulation of methylation states. The most recent studies addressing these observations will be presented at the meeting.

Comprehensive analysis of DNA replication timing across 184 cell lines suggests a role for MCM10 in replication timing regulation.

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.

Delayed DNA replication in haploid human embryonic stem cells.

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

The genetic architecture of DNA replication timing in human pluripotent stem cells

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome’s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) – sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.

Loss of full-length DNA replication regulator Rif1 in two-cell embryos is associated with zygotic transcriptional activation

Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.