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.

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Replication Timing and Transcription Identifies a Novel Fragility Signature Under Replication Stress

10.1101/716951 ◽

2019 ◽

Cited By ~ 1

Author(s):

Dan Sarni ◽

Takayo Sasaki ◽

Karin Miron ◽

Michal Irony Tur-Sinai ◽

Juan Carlos Rivera-Mulia ◽

...

Keyword(s):

Dna Replication ◽

Chromosomal Instability ◽

Temporal Order ◽

Replication Timing ◽

Replication Stress ◽

Fragile Sites ◽

Precise Mapping ◽

Chromosomal Fragility ◽

Transcriptional Changes ◽

Large Genes

AbstractCommon fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

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Delayed DNA replication in haploid human embryonic stem cells

10.1101/2021.05.11.443666 ◽

2021 ◽

Author(s):

Matthew Micheal Edwards ◽

Michael V. Zuccaro ◽

Ido Sagi ◽

Qiliang Ding ◽

Dan Vershkov ◽

...

Keyword(s):

Stem Cells ◽

Dna Replication ◽

Embryonic Stem Cells ◽

X Chromosome ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Replication Timing ◽

Genetic System ◽

X Chromosomes ◽

Genomic Regions

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X chromosome in haploids, consistent with the lack of X chromosome inactivation. Surprisingly, we also identified 21 autosomal regions that had dramatically delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also under-replicated in polyploid placental cells. The same delays were observed in female ESCs with two active X chromosomes, suggesting that increased X chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

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Delayed DNA replication in haploid human embryonic stem cells

Genome Research ◽

10.1101/gr.275953.121 ◽

2021 ◽

Author(s):

Matthew M. Edwards ◽

Michael V. Zuccaro ◽

Ido Sagi ◽

Qiliang Ding ◽

Dan Vershkov ◽

...

Keyword(s):

Stem Cells ◽

Dna Replication ◽

Embryonic Stem Cells ◽

X Chromosome ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Replication Timing ◽

Genetic System ◽

X Chromosomes ◽

Genomic Regions

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

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The Protective Role of Dormant Origins in Response to Replicative Stress

International Journal of Molecular Sciences ◽

10.3390/ijms19113569 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3569 ◽

Cited By ~ 11

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

10.1101/237628 ◽

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.

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Replication timing maintains the global epigenetic state in human cells

10.1101/2019.12.28.890020 ◽

2019 ◽

Cited By ~ 2

Author(s):

Kyle N. Klein ◽

Peiyao A. Zhao ◽

Xiaowen Lyu ◽

Daniel A. Bartlett ◽

Amar Singh ◽

...

Keyword(s):

Embryonic Stem ◽

Cancer Cell Line ◽

Replication Timing ◽

Cell Types ◽

Control Factor ◽

Epigenetic State ◽

Genome Wide ◽

A Genome ◽

A Cell ◽

Early Replication

AbstractDNA is replicated in a defined temporal order termed the replication timing (RT) program. RT is spatially segregated in the nucleus with early/late replication corresponding to Hi-C A/B chromatin compartments, respectively. Early replication is also associated with active histone modifications and transcriptional permissiveness. However, the mechanistic interplay between RT, chromatin state, and genome compartmentalization is largely unknown. Here we report that RT is central to epigenome maintenance and compartmentalization in both human embryonic stem cells (hESCs) and cancer cell line HCT116. Knockout (KO) of the conserved RT control factor RIF1, rather than causing discrete RT switches as previously suspected, lead to dramatically increased cell to cell heterogeneity of RT genome wide, despite RIF1’s enrichment in late replicating chromatin. RIF1 KO hESCs have a nearly random RT program, unlike all prior RIF1 KO cells, including HCT116, which show localized alterations. Regions that retain RT, which are prevalent in HCT116 but rare in hESCs, consist of large H3K9me3 domains revealing two independent mechanisms of RT regulation that are used to different extents in different cell types. RIF1 KO results in a striking genome wide downregulation of H3K27ac peaks and enrichment of H3K9me3 at large domains that remain late replicating, while H3K27me3 and H3K4me3 are re-distributed genome wide in a cell type specific manner. These histone modification changes coincided with global reorganization of genome compartments, transcription changes and a genome wide strengthening of TAD structures. Inducible degradation of RIF1 revealed that disruption of RT is upstream of genome compartmentalization changes. Our findings demonstrate that disruption of RT leads to widespread epigenetic mis-regulation, supporting previously speculative models in which the timing of chromatin assembly at the replication fork plays a key role in maintaining the global epigenetic state, which in turn drives genome architecture.

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Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Genetics ◽

10.1093/genetics/iyab147 ◽

2021 ◽

Author(s):

Souradip Das ◽

Madison Caballero ◽

Tatyana Kolesnikova ◽

Igor Zhimulev ◽

Amnon Koren ◽

...

Keyword(s):

Dna Replication ◽

Copy Number ◽

Genome Stability ◽

Timing Analysis ◽

Replication Timing ◽

Critical Function ◽

Replication Fork Progression ◽

Polyploid Cells ◽

Genomic Regions ◽

Insight Into

Abstract Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

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Replication timing maintains the global epigenetic state in human cells

Science ◽

10.1126/science.aba5545 ◽

2021 ◽

Vol 372 (6540) ◽

pp. 371-378

Author(s):

Kyle N. Klein ◽

Peiyao A. Zhao ◽

Xiaowen Lyu ◽

Takayo Sasaki ◽

Daniel A. Bartlett ◽

...

Keyword(s):

Dna Replication ◽

Histone Modifications ◽

Temporal Order ◽

Three Dimensional ◽

Replication Timing ◽

Human Cells ◽

Genome Architecture ◽

Chromatin Modifications ◽

Complete Elimination ◽

Epigenetic State

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

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DNA methylation is required to maintain DNA replication timing precision and 3D genome integrity

10.1101/2020.10.15.338855 ◽

2020 ◽

Author(s):

Qian Du ◽

Grady C. Smith ◽

Phuc Loi Luu ◽

James M. Ferguson ◽

Nicola J. Armstrong ◽

...

Keyword(s):

Dna Methylation ◽

Dna Replication ◽

Replication Timing ◽

Genome Architecture ◽

Genome Organisation ◽

Dna Hypomethylation ◽

3D Genome ◽

Timing Precision ◽

The Impact ◽

Dna Replication Timing

AbstractDNA replication timing and three-dimensional (3D) genome organisation occur across large domains associated with distinct epigenome patterns to functionally compartmentalise genome regulation. However, it is still unclear if alternations in the epigenome, in particular cancer-related DNA hypomethylation, can directly result in alterations to cancer higher order genome architecture. Here, we use Hi-C and single cell Repli-Seq, in the colorectal cancer DNMT1 and DNMT3B DNA methyltransferases double knockout model, to determine the impact of DNA hypomethylation on replication timing and 3D genome organisation. First, we find that the hypomethylated cells show a striking loss of replication timing precision with gain of cell-to-cell replication timing heterogeneity and loss of 3D genome compartmentalisation. Second, hypomethylated regions that undergo a large change in replication timing also show loss of allelic replication timing, including at cancer-related genes. Finally, we observe the formation of broad ectopic H3K4me3-H3K9me3 domains across hypomethylated regions where late replication is maintained, that potentially prevent aberrant transcription and loss of genome organisation after DNA demethylation. Together, our results highlight a previously underappreciated role for DNA methylation in maintenance of 3D genome architecture.

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Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

10.1101/2021.04.19.440528 ◽

2021 ◽

Author(s):

Souradip Das ◽

Madison Caballero ◽

Tatyana Kolesnikova ◽

Igor Zhimulev ◽

Amnon Koren ◽

...

Keyword(s):

Dna Replication ◽

Copy Number ◽

Genome Stability ◽

Timing Analysis ◽

Replication Timing ◽

Critical Function ◽

Replication Fork Progression ◽

Polyploid Cells ◽

Genomic Regions ◽

Insight Into

Regulation of DNA replication and copy number are necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rif1 is key regulator of RT and has a critical function in copy number control in polyploid cells. In a previous study (Munden et al., 2018), we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 functions both upstream and downstream of SUUR to promote UR. Our work provides new mechanistic insight into the process of UR and its links to RT.

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