Three-dimensional nuclear organisation and the DNA replication timing program

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 temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

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

Replication timing alterations in leukemia affect clinically relevant chromosome domains

During the last decades it has been shown that the replication timing program in metazoans is related to chromosome structure, the nuclear positioning and AT/GC content of chromosomal loci, their patterns of histone modifications, and their transcriptional regulation. Here, the current state of knowledge concerning these relationships is reviewed. An integrated view on structure-function relationships in the nucleus is provided and the determination and functional role of the replication timing program is discussed in this context. A corresponding comprehensive model is developed and a key aspect of this model is the suggestion that mammalian chromosomes are organized into stable units equivalent to replicon clusters. It is proposed that the nuclear positions of these units would depend on their histone modifications and determine the replication timing of the whole unit. It is furthermore predicted that replication timing is only indirectly linked to transcriptional regulation and contributes to the maintenance of gene expression patterns. These clear predictions, and the fact that the tools are at hand now to further test them, open an avenue towards solving the long standing problem on how replication timing is determined in metazoan cells.

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.

Epigenome The temporal order of DNA replication is conserved from yeast to humans, but its biological significance remains unclear. Klein et al. eliminated the protein RIF1, a master regulator of replication timing, in several human cell lines. RIF1 loss during the G1 phase of the cell cycle resulted in a heterogeneous, nearly random replication timing program from the first S phase that persisted even in stable RIF1-null clones. Altered replication timing was followed by replication-dependent redistribution of active and repressive histone modifications and alterations in genome architecture. These results support a model in which replication timing orchestrates the epigenetic state of newly replicated chromatin. Science , this issue p. [371][1] [1]: /lookup/doi/10.1126/science.aba5545

1. Early Embryonic Cell Fate Decisions in the Mouse Yojiro Yamanaka and Amy Ralston Abstract Introduction Lineage Establishment and the Pre?stem Cell Program: Formation of the Blastocyst Lineage Maintenance and the Stem Cell Program: Beyond the Blastocyst The Second Lineage Decision: Subdividing the ICM Cell Signaling Regulates PE/EPI Specification Establishment and Modulation of Pluripotency in the EPI Lineage Conclusion 2. Nuclear Architecture in Stem Cells Kelly J. Morris, Mita Chotalia and Ana Pombo Abstract Introduction Functional Compartmentalization of the ES Cell Nucleus Stem Cell Features of Other Nucleoplasmic Subcompartments Chromatin Features Characteristic of ES Cell Nuclei Conclusion 3. Epigenetic Regulation of Pluripotency Eleni M. Tomazou and Alexander Meissner Abstract Introduction Epigenetic Modifications The Epigenome of ES Cells Conclusion 4. Autosomal Lyonization of Replication Domains During Early Mammalian Development Ichiro Hiratani and David M. Gilbert Abstract Introduction Replication Timing Program: An Elusive Measure of Genome Organization An Evolutionarily Conserved Epigenetic Fingerprint Replication Timing as a Quantitative Index of 3?Dimensional Genome Organization Replication Timing Reveals An Epigenetic Transition: Autosomal Lyonization at the Epiblast Stage Replication Timing and Cellular Reprogramming: Further Support for Autosomal Lyonization Maintenance and Alteration of Replication Timing Program and Its Potential Roles Conclusion 5. PRESERVATION OF GENOMIC INTEGRITY IN MOUSE EMBRYONIC STEM CELLS Peter J. Stambrook and Elisia D. Tichy Abstract Introduction and Historical Perspective Mutation Frequencies in Somatic Cells Protection of the Mouse ES Cell Genome Conclusion 6. Transcriptional Regulation in Embryonic Stem Cells Jian?Chien Dominic Heng and Huck?Hui Ng Abstract Introduction Embryonic Stem Cells as a Model to Study Transcriptional Regulation Transcription Factors Governing ESC Pluripotency TranscriptionalRegulatory Network Technologies for Dissecting the Transcriptional Regulatory Network The Core Transcriptional Regulatory Network: Oct4, Sox2 and Nanog Expanded Transcriptional Regulatory Network Enhanceosomes: Transcription Factor Complex Integration of Signaling Pathways to Transcriptional Network Interface Between Transcriptional and Epigenetic Regulation Conclusion 7. ALTERNATIVE SPLICING IN STEM CELL SELF?RENEWAL AND DIFERENTIATION David A. Nelles and Gene W. Yeo Abstract Introduction Introduction to Alternative Splicing Alternative Splicing of Genes Implicated in Stemness and Differentiation Genome?Wide Methods to Identify and Detect Alternative Splicing Events Regulation of Alternative Splicing by RNA Binding Proteins Conclusion and Perspectives 8. MicroRNA Regulation of Embryonic Stem Cell Self?Renewal and Differentiation Collin Melton and Robert Blelloch Abstract Introduction: The Self?Renewal Program Embryonic Stem Cells miRNA Biogenesis and Function ESCC miRNAs Promote Self?Renewal miRNAs Induced during ESC Differentiation Suppress the Self?Renewal Program Regulatory Networks Controlling miRNA Expression miRNAs Can Promote or Inhibit Dedifferentiation to iPS Cells miRNAs in Somatic Stem Cells miRNAs in Cancer Cells Conclusion 9. TELOMERES AND TELOMERASE IN ADULT STEM CELLS and PLURIPOTENT EMBRYONIC STEM CELLS Rosa M. Marion and Maria A. Blasco Abstract Introduction Regulation of Telomeres and Telomerase Role of Telomeres and Telomerase in Adult SC Compartments Telomeres and Telomerase Regulation During Reprogramming by SCNT Telomeres and Telomerase Regulation During iPS Cell Generation Telomerase Activation is Essential for the Good Quality of the Resulting iPS Cells Regulation of Telomere Reprogramming Conclusion 10. X Chromosome Inactivation and Embryonic Stem Cells Tahsin Stefan Barakat and Joost Gribnau Abstract Introduction Cis Acting Factors in XCI Trans Acting Factors in XCI Counting and Choice Silencing and Maintenance of

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.

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.

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.

Eukaryotic DNA replication follows a strict temporal program where genomic loci are replicated at precise times during the S phase of the cell cycle. Yet, the mechanism in control of the timing program in metazoan cells is poorly understood. In a recent publication, the authors proposed an intuitive stochastic model of DNA replication and showed that it predicts replication timing with an accuracy approaching the level of experimental biological repeats. Here, we discuss an extended software implementation of the mechanistic model: Replicon. This package allows interested researchers to predict the global replication timing program in human cells from chromatin data.

Eukaryotic cells have evolved a complex set of regulatory proteins to maintain genome stability by regulating DNA replication once and once per cell cycle. Despite intensive investigation, a precise understanding of the proteins that are phosphorylated during the initiation phase of DNA replication remains to be identified. Here we investigate the roles of cyclin E and cyclin A in the G1 to S-phase transition and reconstitute initiation of DNA replication in cyclin depleted nuclei using an in vitro DNA replication system. Using a murine 3T3 fibroblast model, chemical and genetic inhibition of CDK activity was used to evaluate the role of specific cyclin complexes in post-quiescent cells. CDK inhibition reduced levels of CDC6, MCM2 and PCNA in all contexts. Evaluation of the role of individual cyclin-CDK complexes was performed by siRNA mediated depletion. Cyclin E1/E2 depletion showed that Cyclin E regulates CDC6, MCM2 and PCNA at the transcriptional level; whereas Cyclin A regulates their accumulation though protection from the ubiquitin -proteasome system in late G1 phase. Furthermore, ubiquitination of CDC6 and MCM2 was confirmed using Histagged ubiquitin pulldown assays suggesting that the CDK and the UPS regulate their levels late G1 phase. Comparison of replication competence was performed using in vitro cell free DNA replication assays. Cyclin E depleted nuclei were unable to initiate DNA replication in S phase extracts; whereas cyclin A2 depleted nuclei retained full activity. Crucially, cyclin E depleted nuclei were unable to load CDK/DDK activated MCM2 proteins onto chromatin in G1 phase that correlated with replication competence in vitro. The data suggests that sustained cyclin activity is required to maintain pre-RC protein levels and facilitate formation of replication complexes in G1 phase. These results further suggest that perturbations in the CDK network results in destabilisation of replication proteins CDC6, MCM2, Ciz1 and PCNA that blocks replication complex assembly on chromatin, contributing to genomic stability.

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.