Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

Epigenetic plasticity safeguards heterochromatin configuration in mammals.

Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.

SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication.

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold-attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein U (HNRNPU), contributes to the formation of open chromatin structure. Here, we demonstrate that SAF-A promotes the normal progression of DNA replication and enables resumption of replication after inhibition. We report that cells depleted of SAF-A show reduced origin licensing in G1 phase and, consequently, reduced origin activation frequency in S phase. Replication forks also progress less consistently in cells depleted of SAF-A, contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed two distinct effects of SAF-A depletion: first, the boundaries between early- and late-replicating domains become more blurred; and second, SAF-A depletion causes replication timing changes that tend to bring regions of discordant domain compartmentalisation and replication timing into concordance. Associated with these defects, SAF-A-depleted cells show elevated formation of phosphorylated histone H2AX (γ-H2AX) and tend to enter quiescence. Overall, we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

Formation of a multi-layered 3-dimensional structure of the heterochromatin compartment during early mammalian development.

During embryogenesis in mammals, the 3-dimensional (3D) genome organization changes globally in parallel with transcription changes in a cell-type specific manner. This involves the progressive formation of heterochromatin, the best example of which is the inactive X chromosome (Xi) in females, originally discovered as a compact 3D structure at the nuclear periphery known as the Barr body. The heterochromatin formation on the autosomes and the Xi is tightly associated with the differentiation state and the developmental potential of cells, making it an ideal readout of the cellular epigenetic state. At a glance, the heterochromatin appears to be uniform. However, recent studies are beginning to reveal a more complex picture, with multiple hierarchical levels co-existing within the heterochromatin compartment. Such hierarchical levels appear to exist in the heterochromatin compartment on autosomes as well as on the Xi. Here, we review recent progress in our understanding of the 3D genome organization changes during the period of differentiation surrounding pluripotency in vivo and in vitro, with a focus on the heterochromatin compartment. We first look at the whole genome, then focus on the Xi, and discuss their differences and similarities. Finally, we present a unified view of how the heterochromatin compartment is formed and regulated during early development. In particular, we emphasize that there are multiple layers within the heterochromatic compartment on both the autosomes and the Xi, with regulatory mechanisms common and specific to each layer.

Microrheology for Hi-C Data Reveals the Spectrum of the Dynamic 3D Genome Organization.

The one-dimensional information of genomic DNA is hierarchically packed inside the eukaryotic cell nucleus and organized in a three-dimensional (3D) space. Genome-wide chromosome conformation capture (Hi-C) methods have uncovered the 3D genome organization and revealed multiscale chromatin domains of compartments and topologically associating domains (TADs). Moreover, single-nucleosome live-cell imaging experiments have revealed the dynamic organization of chromatin domains caused by stochastic thermal fluctuations. However, the mechanism underlying the dynamic regulation of such hierarchical and structural chromatin units within the microscale thermal medium remains unclear. Microrheology is a way to measure dynamic viscoelastic properties coupling between thermal microenvironment and mechanical response. Here, we propose a new, to our knowledge, microrheology for Hi-C data to analyze the dynamic compliance property as a measure of rigidness and flexibility of genomic regions along with the time evolution. Our method allows the conversion of an Hi-C matrix into the spectrum of the dynamic rheological property along the genomic coordinate of a single chromosome. To demonstrate the power of the technique, we analyzed Hi-C data during the neural differentiation of mouse embryonic stem cells. We found that TAD boundaries behave as more rigid nodes than the intra-TAD regions. The spectrum clearly shows the dynamic viscoelasticity of chromatin domain formation at different timescales. Furthermore, we characterized the appearance of synchronous and liquid-like intercompartment interactions in differentiated cells. Together, our microrheology data derived from Hi-C data provide physical insights into the dynamics of the 3D genome organization.

Epigenetic differences between naïve and primed pluripotent stem cells.

It has been 8 years since the concept of naïve and primed pluripotent stem cell states was first proposed. Both are states of pluripotency, but exhibit slightly different properties. The naïve state represents the cellular state of the preimplantation mouse blastocyst inner cell mass, while the primed state is representative of the post-implantation epiblast cells. These two cell types exhibit clearly distinct developmental potential, as evidenced by the fact that naïve cells are able to contribute to blastocyst chimeras, while primed cells cannot. However, the epigenetic differences that underlie the distinct developmental potential of these cell types remain unclear, which is rather surprising given the large amount of active investigation over the years. Elucidating such epigenetic differences should lead to a better understanding of the fundamental properties of these states of pluripotency and the means by which the naïve-to-primed transition occurs, which may provide insights into the essence of stem cell commitment.

A feedback loop that drives cell death and proliferation and its defect in intestinal stem cells.

Cell death and proliferation are at a glance dichotomic events, but occasionally coupled. Caspases, traditionally known to execute apoptosis, play non-apoptotic roles, but their exact mechanism remains elusive. Here, using Drosophila intestinal stem cells (ISCs), we discovered that activation of caspases induces massive cell proliferation rather than cell death. We elucidate that a positive feedback circuit exists between caspases and JNK, which can simultaneously drive cell proliferation and cell death. In ISCs, signalling from JNK to caspases is defective, which skews the balance towards proliferation. Mechanistically, two-tiered regulation of the DIAP1 inhibitor rpr, through its transcription and its protein localization, exists. This work provides a conceptual framework that explains how caspases perform apoptotic and non-apoptotic functions in vivo and how ISCs accomplish their resistance to cell death.

Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types.

To identify evolutionarily conserved features of replication timing and their relationship to epigenetic properties, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two human induced pluripotent stem cell lines (hiPSCs), and compared them with related mouse cell types. Results confirm the conservation of coordinately replicated megabase-sized "replication domains" punctuated by origin-suppressed regions. Differentiation-induced replication timing changes in both species occur in 400- to 800-kb units and are similarly coordinated with transcription changes. A surprising degree of cell-type-specific conservation in replication timing was observed across regions of conserved synteny, despite considerable species variation in the alignment of replication timing to isochore GC/LINE-1 content. Notably, hESC replication timing profiles were significantly more aligned to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Comparison with epigenetic marks revealed a signature of chromatin modifications at the boundaries of early replicating domains and a remarkably strong link between replication timing and spatial proximity of chromatin as measured by Hi-C analysis. Thus, early and late initiation of replication occurs in spatially separate nuclear compartments, but rarely within the intervening chromatin. Moreover, cell-type-specific conservation of the replication program implies conserved developmental changes in spatial organization of chromatin. Together, our results reveal evolutionarily conserved aspects of developmentally regulated replication programs in mammals, demonstrate the power of replication profiling to distinguish closely related cell types, and strongly support the hypothesis that replication timing domains are spatially compartmentalized structural and functional units of three-dimensional chromosomal architecture.

Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.

Differentiation of mouse embryonic stem cells (mESCs) is accompanied by changes in replication timing. To explore the relationship between replication timing and cell fate transitions, we constructed genome-wide replication-timing profiles of 22 independent mouse cell lines representing 10 stages of early mouse development, and transcription profiles for seven of these stages. Replication profiles were cell-type specific, with 45% of the genome exhibiting significant changes at some point during development that were generally coordinated with changes in transcription. Comparison of early and late epiblast cell culture models revealed a set of early-to-late replication switches completed at a stage equivalent to the post-implantation epiblast, prior to germ layer specification and down-regulation of key pluripotency transcription factors [POU5F1 (also known as OCT4)/NANOG/SOX2] and coinciding with the emergence of compact chromatin near the nuclear periphery. These changes were maintained in all subsequent lineages (lineage-independent) and involved a group of irreversibly down-regulated genes, at least some of which were repositioned closer to the nuclear periphery. Importantly, many genomic regions of partially reprogrammed induced pluripotent stem cells (piPSCs) failed to re-establish ESC-specific replication-timing and transcription programs. These regions were enriched for lineage-independent early-to-late changes, which in female cells included the inactive X chromosome. Together, these results constitute a comprehensive "fate map" of replication-timing changes during early mouse development. Moreover, they support a model in which a distinct set of replication domains undergoes a form of "autosomal Lyonization" in the epiblast that is difficult to reprogram and coincides with an epigenetic commitment to differentiation prior to germ layer specification.

Replication dynamics identifies the folding principles of the inactive X chromosome.

Chromosome-wide late replication is an enigmatic hallmark of the inactive X chromosome (Xi). How it is established and what it represents remains obscure. By single-cell DNA replication sequencing, here we show that the entire Xi is reorganized to replicate rapidly and uniformly in late S-phase during X-chromosome inactivation (XCI), reflecting its relatively uniform structure revealed by 4C-seq. Despite this uniformity, only a subset of the Xi became earlier replicating in SmcHD1-mutant cells. In the mutant, these domains protruded out of the Xi core, contacted each other and became transcriptionally reactivated. 4C-seq suggested that they constituted the outermost layer of the Xi even before XCI and were rich in escape genes. We propose that this default positioning forms the basis for their inherent heterochromatin instability in cells lacking the Xi-binding protein SmcHD1 or exhibiting XCI escape. These observations underscore the importance of 3D genome organization for heterochromatin stability and gene regulation.

Replication timing: a fingerprint for cell identity and pluripotency.

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify "replication timing fingerprints" unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization.

G9a selectively represses a class of late-replicating genes at the nuclear periphery.

We have investigated the role of the histone methyltransferase G9a in the establishment of silent nuclear compartments. Following conditional knockout of the G9a methyltransferase in mouse ESCs, 167 genes were significantly up-regulated, and no genes were strongly down-regulated. A partially overlapping set of 119 genes were up-regulated after differentiation of G9a-depleted cells to neural precursors. Promoters of these G9a-repressed genes were AT rich and H3K9me2 enriched but H3K4me3 depleted and were not highly DNA methylated. Representative genes were found to be close to the nuclear periphery, which was significantly enriched for G9a-dependent H3K9me2. Strikingly, although 73% of total genes were early replicating, more than 71% of G9a-repressed genes were late replicating, and a strong correlation was found between H3K9me2 and late replication. However, G9a loss did not significantly affect subnuclear position or replication timing of any non-pericentric regions of the genome, nor did it affect programmed changes in replication timing that accompany differentiation. We conclude that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery.

Cell cycle dynamics and developmental dynamics of the 3D genome: toward linking the two timescales.

In the mammalian cell nucleus, chromosomes are folded differently in interphase and mitosis. Interphase chromosomes are relatively decondensed and display at least two unique layers of higher-order organization: topologically associating domains (TADs) and cell-type-specific A/B compartments, which correlate well with early/late DNA replication timing (RT). In mitosis, these structures rapidly disappear but are gradually reconstructed during G1 phase, coincident with the establishment of the RT program. However, these structures also change dynamically during cell differentiation and reprogramming, and yet we are surprisingly ignorant about the relationship between their cell cycle dynamics and developmental dynamics. In this review, we summarize the recent findings on this topic, discuss how these two processes might be coordinated with each other and its potential significance.

Large-Scale Chromatin Rearrangements in Cancer.

Epigenetic abnormalities are extremely widespread in cancer. Some of them are mere consequences of transformation, but some actively contribute to cancer initiation and progression; they provide powerful new biological markers, as well as new targets for therapies. In this review, we examine the recent literature and focus on one particular aspect of epigenome deregulation: large-scale chromatin changes, causing global changes of DNA methylation or histone modifications. After a brief overview of the one-dimension (1D) and three-dimension (3D) epigenome in healthy cells and of its homeostasis mechanisms, we use selected examples to describe how many different events (mutations, changes in metabolism, and infections) can cause profound changes to the epigenome and fuel cancer. We then present the consequences for therapies and briefly discuss the role of single-cell approaches for the future progress of the field.

Global reorganization of replication domains during embryonic stem cell differentiation.

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these "replicon clusters" coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call "replication domains," separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.

Domain-wide regulation of DNA replication timing during mammalian development.

Studies of replication timing provide a handle into previously impenetrable higher-order levels of chromosome organization and their plasticity during development. Although mechanisms regulating replication timing are not clear, novel genome-wide studies provide a thorough survey of the extent to which replication timing is regulated during most of the early cell fate transitions in mammals, revealing coordinated changes of a defined set of 400-800 kb chromosomal segments that involve at least half the genome. Furthermore, changes in replication time are linked to changes in sub-nuclear organization and domain-wide transcriptional potential, and tissue-specific replication timing profiles are conserved from mouse to human, suggesting that the program has developmental significance. Hence, these studies have provided a solid foundation for linking megabase level chromosome structure to function, and suggest a central role for replication in domain-level genome organization.

Regulation of mammalian 3D genome organization and histone H3K9 dimethylation by H3K9 methyltransferases.

Histone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and overlaps well with lamina-associated domains and the B compartment defined by Hi-C. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. Here, we investigated genome-wide H3K9me2 distribution, transcriptome, and 3D genome organization in mouse embryonic stem cells following the inhibition or depletion of H3K9 methyltransferases (MTases): G9a, GLP, SETDB1, SUV39H1, and SUV39H2. We show that H3K9me2 is regulated by all five MTases; however, H3K9me2 and transcription in the A and B compartments are regulated by different MTases. H3K9me2 in the A compartments is primarily regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments is regulated by all five MTases. Furthermore, decreased H3K9me2 correlates with changes to more active compartmental state that accompanied transcriptional activation. Thus, H3K9me2 contributes to inactive compartment setting.

Dynamics of transcription-mediated conversion from euchromatin to facultative heterochromatin at the Xist promoter by Tsix.

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment.

The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

Autosomal lyonization of replication domains during early Mammalian development.

It has been exactly 50 years since it was discovered that duplication of the eukaryotic genome follows a defined temporal order as cells progress through S-phase. While the mechanism of this replication-timing program still remains a mystery, various correlations of this program with both static and dynamic properties of chromatin render it an attractive forum to explore previously impenetrable higher-order organization of chromosomes. Indeed, studies of DNA replication have provided a simple and straightforward approach to address physical organization of the genome, both along the length of the chromosome as well as in the context of the 3-dimensional space in the cell nucleus. In this chapter, we summarize the 50-years history of the pursuit for understanding the replication-timing program and its developmental regulation, primarily in mammalian cells. We begin with the discovery of the replication-timing program, discuss developmental regulation of this program during X-inactivation in females as well as on autosomes and then describe the recent findings from genome-wide dissection of this program, with special reference to what takes place during mouse embryonic stem cell differentiation. We make an attempt to interpret what these findings might represent and discuss their potential relevance to embryonic development. In doing so, we revive an old concept of "autosomal Lyonization" to describe "facultative heterochromatinization" and irreversible silencing of individual replication domains on autosomes reminiscent of the stable silencing of the inactive X chromosome, which takes place at a stage equivalent to the postimplantation epiblast in mice.