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

2017 ◽  
Author(s):  
Saori Takahashi ◽  
Hisashi Miura ◽  
Takahiro Shibata ◽  
Koji Nagao ◽  
Katsuzumi Okumura ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Developmental Plasticity ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Small Degree ◽  
3D Genome ◽  
Inactive X Chromosome ◽  
Genome Wide ◽  
Individual Mouse

ABSTRACTHere, we report the establishment of a single-cell DNA replication sequencing method, scRepli-seq, which is a simple 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 replication profiles, which were conserved from cell to cell. Haplotype-resolved scRepli-seq revealed similar replication timing 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, and we discovered that developmentally regulated domains are a source of such variability, suggesting a link between cell-to-cell heterogeneity and developmental plasticity. Together, our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into 3D genome organization.

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

2020 ◽  
Vol 15 (12) ◽  
pp. 4058-4100
Author(s):  
Hisashi Miura ◽  
Saori Takahashi ◽  
Takahiro Shibata ◽  
Koji Nagao ◽  
Chikashi Obuse ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Cell ◽  
Mammalian Cells ◽  
Replication Timing ◽  
Genome Wide

scholarly journals Functional correlation of H3K9me2 and nuclear compartment formation

2020 ◽  
Author(s):  
Kei Fukuda ◽  
Chikako Shimura ◽  
Hisashi Miura ◽  
Akie Tanigawa ◽  
Takehiro Suzuki ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Epigenetic Mark ◽  
3 Dimensional ◽  
3D Genome ◽  
Functional Correlation ◽  
Genome Wide

AbstractBackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear.ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation.ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

scholarly journals Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 196 ◽  
Author(s):  
Phoebe Oldach ◽  
Conrad A. Nieduszynski
Keyword(s):  
Dna Replication ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Replication Timing ◽  
S Phase ◽  
Genome Architecture ◽  
3D Genome ◽  
Synchronized Cells ◽  
Dna Replication Timing ◽  
Phase Entry

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

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

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Kei Fukuda ◽  
Chikako Shimura ◽  
Hisashi Miura ◽  
Akie Tanigawa ◽  
Takehiro Suzuki ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Epigenetic Mark ◽  
3 Dimensional ◽  
3D Genome ◽  
Genome Wide ◽  
H3k9 Dimethylation

AbstractHistone 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.

scholarly journals High Resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells

2019 ◽  
Author(s):  
Peiyao A. Zhao ◽  
Takayo Sasaki ◽  
David M. Gilbert
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Cell Types ◽  
S Phase ◽  
List Type ◽  
Cell Type ◽  
Histone Marks ◽  
Cell Type Specific

ABSTRACTDNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. RT is developmentally regulated and correlated with chromatin conformation and local transcriptional potential. Here we present RT profiles of unprecedented temporal resolution in two human embryonic stem cell lines, human colon carcinoma line HCT116 as well as F1 subspecies hybrid mouse embryonic stem cells and their neural progenitor derivatives. Strong enrichment of nascent DNA in fine temporal windows reveals a remarkable degree of cell to cell conservation in replication timing and patterns of replication genome-wide. We identify 5 patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation were found throughout S phase and resolved to ~50kb precision. Temporal transition regions were resolved into segments of uni-directional replication punctuated with small zones of inefficient initiation. Small and large valleys of convergent replication were consistent with either termination or broadly distributed initiation, respectively. RT correlated with chromatin compartment across all cell types but correlations of initiation time to chromatin domain boundaries and histone marks were cell type specific. Haplotype phasing revealed previously unappreciated regions of allele-specific and alleleindependent asynchronous replication. Allele-independent asynchrony was associated with large transcribed genes that resemble common fragile sites. Altogether, these data reveal a remarkably deterministic temporal choreography of DNA replication in mammalian cells.Highly homogeneous replication landscape between cells in a populationInitiation zones resolved within constant timing and timing transition regionsActive histone marks enriched within early initiation zones while enrichment of repressive marks is cell type specific.Transcribed long genes replicate asynchronously.

scholarly journals DNA Replication Timing Enters the Single-Cell Era

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 221 ◽  
Author(s):  
Ichiro Hiratani ◽  
Saori Takahashi
Keyword(s):  
Dna Replication ◽  
Single Cell ◽  
Cell Population ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Three Dimensional ◽  
Replication Timing ◽  
3D Genome ◽  
Population Analyses ◽  
Dna Replication Timing

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.

scholarly journals Cohesin-mediated genome architecture does not define DNA replication timing domains

2019 ◽  
Author(s):  
Phoebe Oldach ◽  
Conrad A Nieduszynski
Keyword(s):  
Dna Replication ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Replication Timing ◽  
S Phase ◽  
Genome Architecture ◽  
3D Genome ◽  
Synchronized Cells ◽  
Dna Replication Timing ◽  
Phase Entry

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

scholarly journals The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 266
Author(s):  
Shin-ichiro Takebayashi ◽  
Tyrone Ryba ◽  
Kelsey Wimbish ◽  
Takuya Hayakawa ◽  
Morito Sakaue ◽  
...  
Keyword(s):  
Dna Replication ◽  
Temporal Order ◽  
Expression System ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Dna Methyltransferases ◽  
Dna Hypomethylation ◽  
Transcriptional Changes ◽  
A Cell ◽  
Genomic Regions

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.

scholarly journals The Protective Role of Dormant Origins in Response to Replicative Stress

2018 ◽  
Vol 19 (11) ◽  
pp. 3569 ◽  
Author(s):  
Lilas Courtot ◽  
Jean-Sébastien Hoffmann ◽  
Valérie Bergoglio
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Replication Timing ◽  
Protective Role ◽  
Entire Genome ◽  
Replicative Stress ◽  
A Cell ◽  
The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

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