Genome-wide stability of DNA replication program in single mammalian cells
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ABSTRACT:
PROVIDER: PRJNA427668 | ENA |
REPOSITORIES: ENA
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Project description: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.
2018-12-13 | GSE108556 | GEO
Project description:Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program
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Project description:Faithful DNA replication is essential for genome integrity. Under-replicated DNA leads to chromosome segregation defects, which are reportedly common during embryogenesis. However, DNA replication regulation remains poorly understood in early mammalian embryos. Here, we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and discovered an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing (RT) program, and the entire genome replicated gradually and uniformly using extremely slow-moving forks. In 4-cell embryos, a somatic-cell-like RT program commenced abruptly. However, the fork speed was still slow, S-phase was extended, and SLX4 DNA repair foci increased during G2/M, which was followed by a transient increase in chromosome segregation errors. Importantly, live imaging captured longer S-phase of error cells, and the breakpoints identified by single-cell genome sequencing were enriched in late-replicating regions. By the 8-cell stage, forks gained speed, S-phase was no longer extended, and chromosome aberrations disappeared. Thus, a transient period of genomic instability exists during normal mouse development, which is preceded by a fragile S-phase lacking the coordination between replisome-level regulation and megabase-scale RT regulation, implicating the importance of their coordination for genome integrity.
2024-06-24 | GSE226309 | GEO
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of DNA replication origins in K562 mammalian cells. We have optimized a specific method of peak detection adapted to the signal produced by the sequencing of short nascent strands (SNS) that are specific of replication initiation, with the aim to have both a good sensitivity and specificity. We demonstrated the existence of the spatiao-temporal program of DNA replication driven by a specific epigenetic signature. K562 human cells; five samples subjected to SNS-Seq.
2014-05-01 | E-GEOD-46189 | biostudies-arrayexpress
Project description:Germline DNA replication timing shapes mammalian genome composition
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Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of DNA replication origins in K562 mammalian cells. We have optimized a specific method of peak detection adapted to the signal produced by the sequencing of short nascent strands (SNS) that are specific of replication initiation, with the aim to have both a good sensitivity and specificity. We demonstrated the existence of the spatiao-temporal program of DNA replication driven by a specific epigenetic signature.
2014-05-01 | GSE46189 | GEO
Project description:Mammalian chromosome replication starts from distinct sites, but the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. We identified a replication initiation determinant (RepID) protein that binds a subset of replication initiation sites. A large fraction of RepID binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from Rep-ID bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication initiation events. These observations are consistent with a model suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. Nascent strands were purified with the lambda exonuclease methods from HCT116 cells and sequenced. Chromatin from unsyncrhonized untreated cultures of U2OS cells was subjected to ChIP-Seq with antibody directed against RepID/PHIP
2016-06-02 | E-GEOD-80298 | biostudies-arrayexpress
Project description:Histone chaperones are an important class of factors that regulate chromatin accessibility for DNA-templated processes. Spt6 is a conserved histone chaperone and key regulator of transcription and chromatin structure. However, its functions outside of these roles have been little explored. In this work, we demonstrate a role for Spt6 in DNA replication and more broadly as a regulator of genome stability. Spt6 binds the replication machinery and mutations or loss of Spt6 impair DNA replication in vivo. Additionally, spt6 mutants are sensitive to DNA replication stress inducing agents, with increased sensitivity when combined with loss of DNA replication associated factors. Furthermore, spt6 mutants have elevated levels of DNA double strand breaks and are hyper-recombinogenic. R-loops do not appear to be elevated in spt6 mutants. In total, we identify Spt6 as a regulator of genome stability, at least in part through a role for Spt6 in DNA replication.
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Project description:DNA Replication Timing (RT) has been suggested to play an important role in shaping the mammalian genome by affecting mutation rates. Previous analyses were limited in that they relied on somatic DNA RT profiles, while to fully understand the influences of RT on the mammalian genome, germ cell RT information is necessary, as only germline mutations are passed to offspring and thus affect genomic composition. Using an improved RT mapping technique that allows mapping the RT from limited amounts of cells, we measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs).
2018-06-26 | GSE109804 | GEO
Project description:Mammalian chromosome replication starts from distinct sites, but the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. We identified a replication initiation determinant (RepID) protein that binds a subset of replication initiation sites. A large fraction of RepID binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from Rep-ID bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication initiation events. These observations are consistent with a model suggesting that RepID facilitates replication initiation at a distinct group of human replication origins.
2016-06-02 | GSE80298 | GEO