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.

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.

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.

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.

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.

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.

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.

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.

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.

Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing.

Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.

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.

Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding?

BACKGROUND: Hi-C is derived from chromosome conformation capture (3C) and targets chromatin contacts on a genomic scale. This method has also been used frequently in scaffolding nucleotide sequences obtained by de novo genome sequencing and assembly, in which the number of resultant sequences rarely converges to the chromosome number. Despite its prevalent use, the sample preparation methods for Hi-C have not been intensively discussed, especially from the standpoint of genome scaffolding. RESULTS: To gain insight into the best practice of Hi-C scaffolding, we performed a multifaceted methodological comparison using vertebrate samples and optimized various factors during sample preparation, sequencing, and computation. As a result, we identified several key factors that helped improve Hi-C scaffolding, including the choice and preparation of tissues, library preparation conditions, the choice of restriction enzyme(s), and the choice of scaffolding program and its usage. CONCLUSIONS: This study provides the first comparison of multiple sample preparation kits/protocols and computational programs for Hi-C scaffolding by an academic third party. We introduce a customized protocol designated "inexpensive and controllable Hi-C (iconHi-C) protocol," which incorporates the optimal conditions identified in this study, and demonstrate this technique on chromosome-scale genome sequences of the Chinese softshell turtle Pelodiscus sinensis.

The Eleanor ncRNAs activate the topological domain of the ESR1 locus to balance against apoptosis.

MCF7 cells acquire estrogen-independent proliferation after long-term estrogen deprivation (LTED), which recapitulates endocrine therapy resistance. LTED cells can become primed for apoptosis, but the underlying mechanism is largely unknown. We previously reported that Eleanor non-coding RNAs (ncRNAs) upregulate the ESR1 gene in LTED cells. Here, we show that Eleanors delineate the topologically associating domain (TAD) of the ESR1 locus in the active nuclear compartment of LTED cells. The TAD interacts with another transcriptionally active TAD, which is 42.9 Mb away from ESR1 and contains a gene encoding the apoptotic transcription factor FOXO3. Inhibition of a promoter-associated Eleanor suppresses all genes inside the Eleanor TAD and the long-range interaction between the two TADs, but keeps FOXO3 active to facilitate apoptosis in LTED cells. These data indicate a role of ncRNAs in chromatin domain regulation, which may underlie the apoptosis-prone nature of therapy-resistant breast cancer cells and could be good therapeutic targets.

Single-cell DNA replication profiling identifies spatiotemporal developmental dynamics of chromosome organization.

In mammalian cells, chromosomes are partitioned into megabase-sized topologically associating domains (TADs). TADs can be in either A (active) or B (inactive) subnuclear compartments, which exhibit early and late replication timing (RT), respectively. Here, we show that A/B compartments change coordinately with RT changes genome wide during mouse embryonic stem cell (mESC) differentiation. While A to B compartment changes and early to late RT changes were temporally inseparable, B to A changes clearly preceded late to early RT changes and transcriptional activation. Compartments changed primarily by boundary shifting, altering the compartmentalization of TADs facing the A/B compartment interface, which was conserved during reprogramming and confirmed in individual cells by single-cell Repli-seq. Differentiating mESCs altered single-cell Repli-seq profiles gradually but uniformly, transiently resembling RT profiles of epiblast-derived stem cells (EpiSCs), suggesting that A/B compartments might also change gradually but uniformly toward a primed pluripotent state. These results provide insights into how megabase-scale chromosome organization changes in individual cells during differentiation.

DNA Replication Timing Enters the Single-Cell Era.

In mammalian cells, DNA replication timing is controlled at the level of megabase (Mb)-sized chromosomal domains and correlates well with transcription, chromatin structure, and three-dimensional (3D) genome organization. Because of these properties, DNA replication timing is an excellent entry point to explore genome regulation at various levels and a variety of studies have been carried out over the years. However, DNA replication timing studies traditionally required at least tens of thousands of cells, and it was unclear whether the replication domains detected by cell population analyses were preserved at the single-cell level. Recently, single-cell DNA replication profiling methods became available, which revealed that the Mb-sized replication domains detected by cell population analyses were actually well preserved in individual cells. In this article, we provide a brief overview of our current knowledge on DNA replication timing regulation in mammals based on cell population studies, outline the findings from single-cell DNA replication profiling, and discuss future directions and challenges.

Genome-wide stability of the DNA replication program in single mammalian cells.

Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.

Practical Analysis of Hi-C Data: Generating A/B Compartment Profiles.

Recent advances in next-generation sequencing (NGS) and chromosome conformation capture (3C) analysis have led to the development of Hi-C, a genome-wide version of the 3C method. Hi-C has identified new levels of chromosome organization such as A/B compartments, topologically associating domains (TADs) as well as large megadomains on the inactive X chromosome, while allowing the identification of chromatin loops at the genome scale. Despite its powerfulness, Hi-C data analysis is much more involved compared to conventional NGS applications such as RNA-seq or ChIP-seq and requires many more steps. This presents a significant hurdle for those who wish to implement Hi-C technology into their laboratory. On the other hand, genomics data repository sites sometimes contain processed Hi-C data sets, allowing researchers to perform further analysis without the need for high-spec workstations and servers. In this chapter, we provide a detailed description on how to calculate A/B compartment profiles from processed Hi-C data on the autosomes and the active/inactive X chromosomes.

Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs.

Multicellular organisms consist of multiple cell types. The identity of these cells is primarily maintained by cell-type-specific gene expression programs; however, mechanisms that suppress these programs are poorly defined. Here we show that serum response factor (Srf), a transcription factor that is activated by various extracellular stimuli, can repress cell-type-specific genes and promote cellular reprogramming to pluripotency. Manipulations that decrease ß-actin monomer quantity result in the nuclear accumulation of Mkl1 and the activation of Srf, which downregulate cell-type-specific genes and alter the epigenetics of regulatory regions and chromatin organization. Mice overexpressing Srf exhibit various pathologies including an ulcerative colitis-like symptom and a metaplasia-like phenotype in the pancreas. Our results demonstrate an unexpected function of Srf via a mechanism by which extracellular stimuli actively destabilize cell identity and suggest Srf involvement in a wide range of diseases.