The temporal program of DNA replication: new insights into old questions

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

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.

Chromosomal replication results in the duplication not only of DNA sequence but also of the patterns of histone modification, DNA methylation, and nucleoprotein structure that constitute epigenetic information. Pericentromeric heterochromatin in human cells is characterized by unique patterns of histone and DNA modification. Here, we describe association of the Mi-2/NuRD complex with specific segments of pericentromeric heterochromatin consisting of Satellite II/III DNA located on human chromosomes 1, 9, and 16 in some but not all cell types. This association is linked in part to DNA replication and chromatin assembly and may suggest a role in these processes. Mi-2/NuRD accumulation is independent of Polycomb association and is characterized by a unique pattern of histone modification. We propose that Mi-2/NuRD constitutes an enzymatic component of a pathway for assembly and maturation of chromatin utilized by rapidly proliferating lymphoid cells for replication of constitutive heterochromatin.

Stem cells have the unique ability to self-renew and differentiate into specialized cell types. Epigenetic mechanisms, including histones and their post-translational modifications, play a crucial role in regulating programs integral to a cell’s identity, like gene expression and DNA replication. However, the transcriptional, chromatin, and replication timing profiles of adult stem cells in vivo remain poorly understood. Containing germline stem cells (GSCs) and somatic cyst stem cells (CySCs), the Drosophila testis provides an excellent in vivo model for studying adult stem cells. However, the small number of stem cells and cellular heterogeneity of this tissue have limited comprehensive genomic studies.In this study, we developed cell type-specific genomic techniques to analyze the transcriptome, histone modification patterns, and replication timing of GSC-like and CySC-like cells. Single cell RNA sequencing validated previous findings on GSC-CySC intercellular communication and revealed high expression of chromatin regulators in GSC-like cells. To characterize chromatin landscapes, we developed a cell-type-specific chromatin profiling assay to map H3K4me3-, H3K27me3-, and H3K9me3-enriched regions, corresponding to euchromatic, facultative heterochromatic, and constitutive heterochromatic domains, respectively. Finally, we determined cell type-specific replication timing profiles, integrating our in vivo datasets with published data using cultured cell lines. Our results reveal that GSC-like cells display a distinct replication program compared to somatic lineages, that aligns with chromatin state differences. Collectively, our integrated transcriptomic, chromatin, and replication datasets provide a comprehensive framework for understanding genome regulation differences between these in vivo stem cell populations, demonstrating the power of multi-omics in uncovering cell type-specific regulatory features.

Stem cells have the unique ability to self-renew and differentiate into specialized cell types. Epigenetic mechanisms, including histones and their post-translational modifications, play a crucial role in regulating programs integral to a cell's identity, like gene expression and DNA replication. However, the transcriptional, chromatin, and replication timing profiles of adult stem cells in vivo remain poorly understood. Containing germline stem cells (GSCs) and somatic cyst stem cells (CySCs), the Drosophila testis provides an excellent in vivo model for studying adult stem cells. However, the small number of stem cells and the cellular heterogeneity of this tissue have limited comprehensive genomic studies. In this study, we develop cell-type-specific genomic techniques to analyze the transcriptome, histone modification patterns, and replication timing of germline stem cell (GSC)–like and somatic cyst stem cell (CySC)–like cells. Single-cell RNA sequencing validates previous findings on GSC–CySC intercellular communication and reveals a high expression of chromatin regulators in GSC-like cells. To characterize chromatin landscapes, we develop a cell-type-specific chromatin profiling assay to map H3K4me3-, H3K27me3-, and H3K9me3-enriched regions, corresponding to the euchromatic, facultative heterochromatic, and constitutive heterochromatic domains, respectively. Finally, we determine cell-type-specific replication timing profiles, integrating our in vivo data sets with published data using cultured cell lines. Our results reveal that GSC-like cells display a distinct replication program, compared with somatic lineages, that aligns with chromatin state differences. Collectively, our integrated transcriptomic, chromatin, and replication data sets provide a comprehensive framework for understanding genome regulation differences between these in vivo stem-cell populations, demonstrating the power of multiomics in uncovering cell-type-specific regulatory features.

DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (>100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.

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.

Correct nuclear position is crucial for cellular function and tissue development. Depending on cell context, however, the cytoskeletal elements responsible for nuclear positioning vary. While these cytoskeletal mechanisms have been intensely studied in single cells, how nuclear positioning is linked to tissue morphology is less clear. Here, we compare apical nuclear positioning in zebrafish neuroepithelia. We find that kinetics and actin-dependent mechanisms of nuclear positioning vary in tissues of different morphology. In straight neuroepithelia, nuclear positioning is controlled by Rho-ROCK-dependent myosin contractility. In contrast, in basally constricted neuroepithelia, a novel formin-dependent pushing mechanism is found for which we propose a proof-of-principle force generation theory. Overall, our data suggest that correct nuclear positioning is ensured by the adaptability of the cytoskeleton to cell and tissue shape. This in turn leads to robust epithelial maturation across geometries. The conclusion that different nuclear positioning mechanisms are favored in tissues of different morphology highlights the importance of developmental context for the execution of intracellular processes.

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.

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

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.

Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers.

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.

BackgroundIn mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function.ResultsWe show here that the inactive X in ICF cells, which appears to be hypomethylated at all CpG islands, exhibits normal histone modification patterns. In addition, in Rett cells with no functional MeCP2 methyl-CpG binding protein, the inactive X also exhibits normal histone modification patterns.ConclusionsThese data suggest that DNA methylation and the associated methyl-DNA binding proteins may not play a critical role in determining histone modification patterns on the mammalian inactive X chromosome at the sites analyzed.