Differential Subnuclear Localization and Replication Timing of Histone H3 Lysine 9 Methylation States

Mono-, di-, and trimethylation of specific histone residues adds an additional level of complexity to the range of histone modifications that may contribute to a histone code. However, it has not been clear whether different methylated states reside stably at different chromatin sites or whether they represent dynamic intermediates at the same chromatin sites. Here, we have used recently developed antibodies that are highly specific for mono-, di-, and trimethylated lysine 9 of histone H3 (MeK9H3) to examine the subnuclear localization and replication timing of chromatin containing these epigenetic marks in mammalian cells. Me1K9H3 was largely restricted to early replicating, small punctate domains in the nuclear interior. Me2K9H3 was the predominant MeK9 epitope at the nuclear and nucleolar periphery and colocalized with sites of DNA synthesis primarily in mid-S phase. Me3K9H3 decorated late-replicating pericentric heterochromatin in mouse cells and sites of DAPI-dense intranuclear heterochromatin in human and hamster cells that replicated throughout S phase. Disruption of the Suv39h1,2 or G9a methyltransferases in murine embryonic stem cells resulted in a redistribution of methyl epitopes, but did not alter the overall spatiotemporal replication program. These results demonstrate that mono-, di-, and trimethylated states of K9H3 largely occupy distinct chromosome domains.

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

10.1101/755629 ◽

2019 ◽

Cited By ~ 1

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.

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

Genes ◽

10.3390/genes10030196 ◽

2019 ◽

Vol 10 (3) ◽

pp. 196 ◽

Cited By ~ 6

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.

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Faculty Opinions recommendation of Differential subnuclear localization and replication timing of histone H3 lysine 9 methylation states.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1027229.326678 ◽

2005 ◽

Author(s):

J Julian Blow

Keyword(s):

Histone H3 ◽

Replication Timing ◽

Subnuclear Localization

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Histone H3 lysine 56 acetylation by Rtt109 is crucial for chromosome positioning

The Journal of Cell Biology ◽

10.1083/jcb.200806065 ◽

2008 ◽

Vol 183 (4) ◽

pp. 641-651 ◽

Cited By ~ 26

Author(s):

Shin-ichiro Hiraga ◽

Sotirios Botsios ◽

Anne D. Donaldson

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Histone Acetyltransferase ◽

Nuclear Periphery ◽

Histone H3 ◽

Epigenetic Inheritance ◽

S Phase ◽

Chromosome Localization ◽

Chromosome Domains ◽

H3k56 Acetylation

Correct intranuclear organization of chromosomes is crucial for many genome functions, but the mechanisms that position chromatin are not well understood. We used a layered screen to identify Saccharomyces cerevisiae mutants defective in telomere localization to the nuclear periphery. We find that events in S phase are crucial for correct telomere localization. In particular, the histone chaperone Asf1 functions in telomere peripheral positioning. Asf1 stimulates acetylation of histone H3 lysine 56 (H3K56) by the histone acetyltransferase Rtt109. Analysis of rtt109Δ and H3K56 mutants suggests that the acetylation/deacetylation cycle of the H3K56 residue is required for proper telomere localization. The function of H3K56 acetylation in localizing chromosome domains is not confined to telomeres because deletion of RTT109 also prevents the correct peripheral localization of a newly identified S. cerevisiae “chromosome-organizing clamp” locus. Because chromosome positioning is subject to epigenetic inheritance, H3K56 acetylation may mediate correct chromosome localization by facilitating accurate transmission of chromatin status during DNA replication.

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Processing of DNA prior to illegitimate recombination in mouse cells.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3779 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3779-3785 ◽

Cited By ~ 14

Author(s):

G Henderson ◽

J P Simons

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Embryonic Stem ◽

Illegitimate Recombination ◽

Strand Bias ◽

Dna Breaks ◽

Exogenous Dna ◽

Double Strand Dna Breaks ◽

Mouse Cells ◽

Exonuclease Digestion

In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 5'-->3' exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 3'-->5' exonuclease processing also occurs.

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Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin

The Journal of Cell Biology ◽

10.1083/jcb.200601113 ◽

2006 ◽

Vol 174 (2) ◽

pp. 185-194 ◽

Cited By ~ 40

Author(s):

Rong Wu ◽

Prim B. Singh ◽

David M. Gilbert

Keyword(s):

Replication Timing ◽

Pericentric Heterochromatin ◽

S Phase ◽

Fine Tuning ◽

G1 Phase ◽

Late Replication ◽

Free System ◽

Replication Time ◽

A Cell ◽

Early Replication

Mouse chromocenters are clusters of late-replicating pericentric heterochromatin containing HP1 bound to trimethylated lysine 9 of histone H3 (Me3K9H3). Using a cell-free system to initiate replication within G1-phase nuclei, we demonstrate that chromocenters acquire the property of late replication coincident with their reorganization after mitosis and the establishment of a global replication timing program. HP1 dissociated during mitosis but rebound before the establishment of late replication, and removing HP1 from chromocenters by competition with Me3K9H3 peptides did not result in early replication, demonstrating that this interaction is neither necessary nor sufficient for late replication. However, in cells lacking the Suv39h1,2 methyltransferases responsible for K9H3 trimethylation and HP1 binding at chromocenters, replication of chromocenter DNA was advanced by 10–15% of the length of S phase. Reintroduction of Suv39h1 activity restored the later replication time. We conclude that Suv39 activity is required for the fine-tuning of pericentric heterochromatin replication relative to other late-replicating domains, whereas separate factors establish a global replication timing program during early G1 phase.

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Selection for synchronized replication of genes encoding the same protein complex during tumorigenesis

10.1101/496059 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ying Chen ◽

Ke Li ◽

Xiao Chu ◽

Lucas B. Carey ◽

Wenfeng Qian

Keyword(s):

Cell Cycle ◽

Cancer Cells ◽

Protein Complex ◽

Embryonic Stem ◽

Replication Timing ◽

S Phase ◽

Proliferating Cells ◽

Genes Encoding ◽

Dosage Balance ◽

Selection For

ABSTRACTDNA replication alters the dosage balance among genes; at the mid-S phase, early-replicating genes have doubled their copies while late-replicating genes have not. Dosage imbalance among proteins, especially within members of a protein complex, is toxic to cells. Here, we propose the synchronized replication hypothesis: genes sensitive to stoichiometric relationships will be replicated simultaneously to maintain stoichiometry. In support of this hypothesis, we observe that genes encoding the same protein complex have similar replication timing, but surprisingly, only in fast-proliferating cells such as embryonic stem cells and cancer cells. The synchronized replication observed in cancer cells, but not in slow-proliferating differentiated cells, is due to convergent evolution during tumorigenesis that restores synchronized replication timing within protein complexes. Collectively, our study reveals that the selection for dosage balance during S phase plays an important role in the optimization of the replication-timing program; that this selection is relaxed during differentiation as the cell cycle is elongated, and restored as the cell cycle shortens during tumorigenesis.

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A non-transcriptional function of Yap orchestrates the DNA replication program

10.1101/2021.11.18.468628 ◽

2021 ◽

Author(s):

Rodrigo Meléndez García ◽

Olivier Haccard ◽

Albert Chesneau ◽

Hemalatha Narassimprakash ◽

Jerome E Roger ◽

...

Keyword(s):

Stem Cells ◽

Dna Replication ◽

Embryonic Stem ◽

Replication Timing ◽

S Phase ◽

Hippo Signaling ◽

Downstream Effector ◽

Egg Extracts ◽

Replication Foci ◽

Eukaryotic Organisms

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signaling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and that Yap depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. Furthermore, we identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that both Yap and Rif1 depletion trigger an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as breaks to control the DNA replication program in early embryos and post-embryonic stem cells.

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