Polymerase-Usage Sequencing Identifies Initiation Zones With Less Bias Throughout S phase in Mouse Embryonic Stem Cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE282548 | GEO | 2024/12/31
REPOSITORIES: GEO
Similar Datasets
Project description:The organization of mammalian DNA replication is poorly understood. We have produced genome-wide high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal and embryonic stem cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy number variations during S phase obtained using either high-density oligonucleotide tiling arrays or massively-parallel sequencing to produce replication timing profiles. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by SMARD analysis. Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarter of S phase and that ES cells replicate a larger proportion of their genome in early S phase than erythroid cells. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid firing origins are located near moderately expressed genes and that late firing origins are located far from genes.
2009-11-04 | GSE18679 | GEO
Project description:DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.
2011-07-06 | GSE30433 | GEO
Project description:DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins. Replicating DNA in cultured Arabidopsis cells was labeled with BrdU for 1 hour. Cells were harvested, nuclei extracted, and the nuclei were sorted by FACS as Early, Mid or Late S phase based on DNA content (DAPI signal). Newly replicated DNA was isolated by immunoprecipitation to BrdU, amplified, and labeled with Cy5/Cy3. Input DNA served as a reference and was labeled with Cy3/Cy5. Equal amounts of DNA was hybridized to a spotted cDNA that covers Arabidopsis chr4. Each S phase sample has 3 biological replicates with a dye swap serving as a technical replicate for a total of 18 arrays.
2011-07-06 | E-GEOD-30433 | biostudies-arrayexpress
Project description:Polymerase-Usage Sequencing Identifies Initiation Zones With Less Bias Throughout S phase in Mouse Embryonic Stem Cells
| PRJNA1189117 | ENA
Project description:Duplication of eukaryotic genomes during S phase is coordinated in space and time. In order to identify zones of initiation and cell-type as well as gender-specific plasticity of DNA replication, we profiled replication timing, histone acetylation and transcription throughout the Drosophila genome. We observed two waves of replication initiation with many distinct zones firing in early and multiple, less defined peaks at the end of S phase, suggesting that initiation becomes more promiscuous at the end of S phase. A comparison of different cell types revealed widespread plasticity of replication timing on autosomes. Most occur in large regions but only half coincide with local differences in transcription. In contrast to confined autosomal differences, a global shift in replication timing occurs throughout the single male X chromosome. Unlike in females, the dosage compensated X chromosome replicates almost exclusively early. This difference occurs at sites which are not transcriptionally hyperactivated, but show increased acetylation of lysine 16 of histone H4. This suggests a transcription-independent, yet chromosome-wide process related to chromatin. Importantly, H4K16ac is also enriched at initiation zones as well as early replicating regions on autosomes during S phase. Together, our data reveal novel organizational principles of DNA replication of the Drosophila genome and imply chromatin structure as a determinant of replication timing locally and chromosome-wide. Keywords: cell type comparison, chip-chip, replication timing
2009-02-10 | GSE13328 | GEO
Project description:Transcription Initiation Activity Sets Replication Origin Efficiency in Mammalian Cells
| PRJNA114781 | ENA
Project description:The organization of mammalian DNA replication is poorly understood. We have produced genome-wide high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal and embryonic stem cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy number variations during S phase obtained using either high-density oligonucleotide tiling arrays or massively-parallel sequencing to produce replication timing profiles. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by SMARD analysis. Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarter of S phase and that ES cells replicate a larger proportion of their genome in early S phase than erythroid cells. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid firing origins are located near moderately expressed genes and that late firing origins are located far from genes. Comparison of S/G1 from ES cells, MSCs and erythroid cells The purpose of the study was to measure replication timing by taking advantage of the fact that copy number of DNA increases from 2 two 4 during replication. Specifically, the study was designed to compare the number of copies of DNA of cells in the G1 phase of the cell cycle with the number of copies of DNA of cells in the S phase of the cell cycle. The cells in G1 and S of the cell cycle were obtained by sorting cells based on DNA content after staining with propidium iodide, a fluorescent compound that binds to DNA. We first tested the strategy with Nimblegen tiling arrays representing about 3% of the genome and found that it was indeed possible to measure such a small difference of copy number if a high-level of redundancy was used. We then extended the study genome-wide using massively parallel sequencing on either the SolID or the Illumina platform. Both platforms were tested and we found that for that application, both platforms were equivalent and provided sufficient number of reads to measure timing of replication genome-wide. We then attempted to better understand the correlation between timing of replication and transcription. We therefore obtained RNA from hESC and from erythroblast and hybridized them to standard Affymetrix U133plus2 arrays. This analysis revealed that it was possible to predict the replication timing patterns with relatively good accuracy (r=0.8) using expression data and distance of every genomic windows to express genes.
2009-11-04 | E-GEOD-18679 | biostudies-arrayexpress
Project description:Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS) suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation unveiling a new hallmark in ORI efficiency regulation in mammalian cells.
2009-03-05 | GSE15082 | GEO
Project description:The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.
2015-12-18 | GSE71051 | GEO
Project description:The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.
2015-12-18 | GSE71050 | GEO