Reorganization of the DNA replication landscape during adipogenesis is closely linked with adipogenic gene expression.

The temporal order of DNA replication along the chromosomes is thought to reflect the transcriptional competence of the genome. During differentiation of mouse 3T3-L1 cells into adipocytes, cells undergo one or two rounds of cell division called mitotic clonal expansion (MCE). MCE is an essential step for adipogenesis; however, little is known about the regulation of DNA replication during this period. Here, we performed genome-wide mapping of replication timing (RT) in mouse 3T3-L1 cells before and during MCE, and identified a number of chromosomal regions shifting toward either earlier or later replication through two rounds of replication. These RT changes were confirmed in individual cells by single-cell DNA-replication sequencing. Coordinate changes between a shift toward earlier replication and transcriptional activation of adipogenesis-associated genes were observed. RT changes occurred before the full expression of these genes, indicating that RT reorganization might contribute to the mature adipocyte phenotype. To support this, cells undergoing two rounds of DNA replication during MCE had a higher potential to differentiate into lipid droplet-accumulating adipocytes, compared with cells undergoing a single round of DNA replication and non-replicating cells.

scRepli-Seq: A Powerful Tool to Study Replication Timing and Genome Instability.

Advances in "omics" technology have made it possible to study a wide range of cellular phenomena at the single-cell level. Recently, we developed single-cell DNA replication sequencing (scRepli-seq) that measures replication timing (RT) by copy number differences between replicated and unreplicated genomic DNA in replicating single mammalian cells. This method has been used to reveal previously unrecognized static and dynamic natures of several hundred kilobases to a few megabases-scale chromosomal units called RT domains. Because RT domains are highly correlated to A/B compartments detected by Hi-C, scRepli-seq data can be used to predict the 3D organization of the genome in the nuclear space. scRepli-seq, which essentially measures the copy number, can also be applied to study genome instability.

Camptothecin-Induced Replication Stress Affects DNA Replication Profiling by E/L Repli-Seq.

E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.

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.

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.

The NSD2/WHSC1/MMSET methyltransferase prevents cellular senescence-associated epigenomic remodeling.

Senescent cells may possess the intrinsic programs of metabolic and epigenomic remodeling, but the molecular mechanism remains to be clarified. Using an RNAi-based screen of chromatin regulators, we found that knockdown of the NSD2/WHSC1/MMSET methyltransferase induced cellular senescence that augmented mitochondrial mass and oxidative phosphorylation in primary human fibroblasts. Transcriptome analysis showed that loss of NSD2 downregulated the expression of cell cycle-related genes in a retinoblastoma protein (RB)-mediated manner. Chromatin immunoprecipitation analyses further revealed that NSD2 was enriched at the gene bodies of actively transcribed genes, including cell cycle-related genes, and that loss of NSD2 decreased the levels of histone H3 lysine 36 trimethylation (H3K36me3) at these gene loci. Consistent with these findings, oncogene-induced or replicative senescent cells showed reduced NSD2 expression together with lower H3K36me3 levels at NSD2-enriched genes. In addition, we found that NSD2 gene was upregulated by serum stimulation and required for the induction of cell cycle-related genes. Indeed, in both mouse and human tissues and human cancer cell lines, the expression levels of NSD2 were positively correlated with those of cell cycle-related genes. These data reveal that NSD2 plays a pivotal role in epigenomic maintenance and cell cycle control to prevent cellular senescence.

Genome-wide analysis of DNA methylation profile identifies differentially methylated loci associated with human intervertebral disc degeneration.

BACKGROUND: Environmental and endogenous factors under genetic predisposition are considered to initiate the human intervertebral disc (IVD) degeneration. DNA methylation is an essential mechanism to ensure cell-specific gene expression for normal development and tissue stability. Aberrant epigenetic alterations play a pivotal role in several diseases, including osteoarthritis. However, epigenetic alternations, including DNA methylation, in IVD degeneration have not been evaluated. The purpose of this study was to comprehensively compare the genome-wide DNA methylation profiles of human IVD tissues, specifically nucleus pulpous (NP) tissues, with early and advanced stages of disc degeneration. METHODS: Human NP tissues were used in this study. The samples were divided into two groups: early stage degeneration (n = 8, Pfirrmann's MRI grade: I-III) and advanced stage degeneration (n = 8, grade: IV). Genomic DNA was processed for genome-wide DNA methylation profiling using the Infinium MethylationEPIC BeadChip array. Extraction of raw methylation data, clustering and scatter plot of each group values of each sample were performed using a methylation module in GenomeStudio software. The identification of differentially methylated loci (DMLs) and the Gene Ontology (GO) analysis were performed using R software with the ChAMP package. RESULTS: Unsupervised hierarchical clustering revealed that early and advanced stage degenerated IVD samples segregated into two main clusters by their DNA methylome. A total of 220 DMLs were identified between early and advanced disc degeneration stages. Among these, four loci were hypomethylated and 216 loci were hypermethylated in the advanced disc degeneration stage. The GO enrichment analysis of genes containing DMLs identified two significant GO terms for biological processes, hemophilic cell adhesion and cell-cell adhesion. CONCLUSIONS: We conducted a genome-wide DNA methylation profile comparative study and observed significant differences in DNA methylation profiles between early and advanced stages of human IVD degeneration. These results implicate DNA methylation in the process of human IVD degeneration.

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.

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.

Mapping mammalian replication domains using the ion torrent semiconductor sequencing platform.

Here, we show that semiconductor-based sequencing technology can be used to map mammalian replication domains, chromosomal units with similar DNA replication timing. Replicating DNA purified from mammalian cells was successfully sequenced by the Ion Torrent platform. The resultant replication domain map of mouse embryonic stem cells is comparable to those obtained by the conventional microarray-based method.

Correlation between histone acetylation and expression of Notch1 in human lung carcinoma and its possible role in combined small-cell lung carcinoma.

Combined small-cell lung carcinoma (cSCLC) is composed of small-cell lung carcinoma (SCLC) admixed with non-small-cell lung carcinoma (NSCLC). Evaluating the molecular differences between SCLC and NSCLC could lead to a better understanding of the pathogenesis of such neoplasms. Therefore, in this study, we investigated the correlation between histone acetylation and Notch1 expression in lung carcinoma. Using chromatin immunoprecipitation (ChIP) assay, we measured the level of acetylated histone H3 around the promoter region of Notch1 in SCLC and NSCLC cells. We then treated SCLC cells with trichostatin A (TSA) and characterized the level of histone H3 acetylation at Notch1. In addition, TSA-treated cells were injected into immune-compromised mice, for analysis of the ex vivo tumor xenograft phenotype. The level of acetylated histone H3 surrounding the Notch1 promoter was lower in lung cancer cells not expressing Notch1. Tumors originated from TSA-treated SCLC cells occasionally formed an epithelial-like glandular arrangement of cells; with Notch1 expression and decreased expression of neuroendocrine (NE) markers. Histone deacetylation around the promoter region of Notch1 inhibits Notch1 protein expression in SCLC and the restoration of Notch1 expression in SCLC leads to the concurrent appearance of epithelial-like areas within the SCLC, which could provide a possible mechanism for histogenesis of cSCLC.

The SETD8/PR-Set7 Methyltransferase Functions as a Barrier to Prevent Senescence-Associated Metabolic Remodeling.

Cellular senescence is an irreversible growth arrest that contributes to development, tumor suppression, and age-related conditions. Senescent cells show active metabolism compared with proliferating cells, but the underlying mechanisms remain unclear. Here we show that the SETD8/PR-Set7 methyltransferase, which catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), suppresses nucleolar and mitochondrial activities to prevent cellular senescence. SETD8 protein was selectively downregulated in both oncogene-induced and replicative senescence. Inhibition of SETD8 alone was sufficient to trigger senescence. Under these states, the expression of genes encoding ribosomal proteins (RPs) and ribosomal RNAs as well as the cyclin-dependent kinase (CDK) inhibitor p16INK4A was increased, with a corresponding reduction of H4K20me1 at each locus. As a result, the loss of SETD8 concurrently stimulated nucleolar function and retinoblastoma protein-mediated mitochondrial metabolism. In conclusion, our data demonstrate that SETD8 acts as a barrier to prevent cellular senescence through chromatin-mediated regulation of senescence-associated metabolic remodeling.

Anatomy of Mammalian Replication Domains.

Genetic information is faithfully copied by DNA replication through many rounds of cell division. In mammals, DNA is replicated in Mb-sized chromosomal units called "replication domains." While genome-wide maps in multiple cell types and disease states have uncovered both dynamic and static properties of replication domains, we are still in the process of understanding the mechanisms that give rise to these properties. A better understanding of the molecular basis of replication domain regulation will bring new insights into chromosome structure and function.

Direct visualization of replication dynamics in early zebrafish embryos.

We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

Retinoblastoma protein promotes oxidative phosphorylation through upregulation of glycolytic genes in oncogene-induced senescent cells.

Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene-induced senescence (OIS). A combination of real-time metabolic monitoring, and metabolome and gene expression analyses showed that OIS-induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB-depleted or downstream glycolytic enzyme-depleted OIS cells, suggesting that RB-mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells.

Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains.

BACKGROUND: Cellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains ('replication domains'), but few gene products have been identified whose disruption affects these properties. RESULTS: Here we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF. CONCLUSIONS: Loss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells.

Low-cost fabrication of centimetre-scale periodic arrays of single plasmid DNA molecules.

We report the development of a low-cost method to generate a centimetre-scale periodic array of single plasmid DNA molecules of 11 kilobase pairs. The arrayed DNA molecules are amenable to enzymatic and physical manipulations.

Development of a single-cell array for large-scale DNA fluorescence in situ hybridization.

DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitates analysis of the FISH results with the FISH-FINDER computer program.

Developmental control of replication timing defines a new breed of chromosomal domains with a novel mechanism of chromatin unfolding.

We recently identified a set of chromosome domains that are early replicating uniquely in pluripotent cells. Their switch from early to late replication occurs just prior to germ layer commitment, associated with a stable form of gene silencing that is difficult to reverse. Here, we discuss results demonstrating that these domains are among the least sensitive regions in the genome to global digestion by either MNase or restriction enzymes. This inaccessible chromatin state persists whether these regions are in their physically distended early replicating or compact late replicating configuration, despite dramatic changes in 3D chromatin folding and long-range chromatin interactions, and despite large changes in transcriptional activity. This contrasts with the strong correlation between early replication, accessibility, transcriptional activity and open chromatin configuration that is observed genome-wide. We put these results in context with findings from other studies indicating that many structural (DNA sequence) and functional (density and activity of replication origins) properties of developmentally regulated replication timing ("switching") domains resemble properties of constitutively late replicating domains. This suggests that switching domains are a type of late replicating domain within which both replication timing and transcription are subject to unique or additional layers of control not experienced by the bulk of the genome. We predict that understanding the unusual structure of these domains will reveal a novel principle of chromosome folding.