Project description:Developing stickleback embryos were dissociated and sorted for S-phase and G0/G1-phase cell populations. DNA was extracted from each population and sequenced. In a mixed S-phase population, regions that replicate earlier are at higher copy number (up to 2x) than regions that replicate later. The read depth in S-phase, normalized to the read depth in G-phase, thus represents replication timing.
Project description:Replication timing is cell type specific, is tightly linked to the 3D nuclear organisation of the genome and is considered an epigenetic fingerprint. In spite of its importance in maintaining the epigenome the developmental regulation of replication timing in mammals in vivo has not been explored. Here, using single cell Repli-seq, we generated the genome-wide replication timing maps of mouse embryos from the zygote until the blastocyst stage.
Project description:DNA replication is initiated at multiple sites or origins enriched with AT-rich sequences at various times during the S-phase. While current studies of genome-wide DNA replication profiles have focused on the timing of replication and the location of origins, the efficiency of replication/firing at various origins remains unclear. In this study, we show different efficiencies of DNA replication at various loci by using ORF-specific DNA microarrays. DNA copy-number increases as a function of time at individual loci are approximated to near-sigmoidal models for estimation of replication initiation and completion timings in HU-challenged cells. Duplicating times (from initiation to completion) vary from loci to loci, partly contributing to various firing efficiencies at origins. DNA replication timing profiles are strikingly similar to the reported patterns of enriched ssDNA, suggesting that majority stalled forks are restored for resumption of DNA replication. Although the DNA replication timing profiles are disrupted in HU-challenged cds1? cells, ~85% of potential origins overlapped with those found in wild type cells, significantly, most of which represents inefficiently fired origins in wild type cells. Together, our result indicates that replication checkpoint plays a role in monitoring efficient origins and thus maintaining global DNA replication patterns in HU-challenged cells. Keywords: WT or Cds1 HU synchronized cells released in HU free media and harvested at different time points vs WT or Cds1 synchronized with HU for 3 hrs. We analyzed 32 arrays for WT and 38 arrays for Cds1 cells which were synchronized with HU and released in HU free media and harvested at different time points. At least two biological repeats were done for each time points.
Project description:This SuperSeries is composed of the following subset Series: GSE17980: Genome-Wide Dynamics of Replication Timing Revealed by In Vitro Models of Mouse Embryogenesis (Expression) GSE17983: Genome-Wide Dynamics of Replication Timing Revealed by In Vitro Models of Mouse Embryogenesis (WG_CGH; Replication Timing) GSE17980 (Expression): 8 cell lines, with a total of 14 individual replicates (i.e. 6 in duplicates, 2 in single replicates) GSE17983 (WG_CGH; Replication Timing): 22 cell lines, with a total of 36 individual replicates (i.e. 14 in duplicates, 8 in single replicates)
Project description:DNA replication is initiated at multiple sites or origins enriched with AT-rich sequences at various times during the S-phase. While current studies of genome-wide DNA replication profiles have focused on the timing of replication and the location of origins, the efficiency of replication/firing at various origins remains unclear. In this study, we show different efficiencies of DNA replication at various loci by using ORF-specific DNA microarrays. DNA copy-number increases as a function of time at individual loci are approximated to near-sigmoidal models for estimation of replication initiation and completion timings in HU-challenged cells. Duplicating times (from initiation to completion) vary from loci to loci, partly contributing to various firing efficiencies at origins. DNA replication timing profiles are strikingly similar to the reported patterns of enriched ssDNA, suggesting that majority stalled forks are restored for resumption of DNA replication. Although the DNA replication timing profiles are disrupted in HU-challenged cds1? cells, ~85% of potential origins overlapped with those found in wild type cells, significantly, most of which represents inefficiently fired origins in wild type cells. Together, our result indicates that replication checkpoint plays a role in monitoring efficient origins and thus maintaining global DNA replication patterns in HU-challenged cells. Keywords: WT or Cds1 HU synchronized cells released in HU free media and harvested at different time points vs WT or Cds1 synchronized with HU for 3 hrs.
Project description:DNA replication is spatially and temporally regulated during S-phase. DNA replication timing is established in early G1-phase at a point referred to as TDP (timing decision point). We show that Rif1 (Rap1-interacting-factor1), originally identified as a telomere binding factor in yeast, is a critical determinant of the replication timing program in human cells. Depletion of Rif1 results in specific loss of mid-S replication foci profiles, stimulation of initiation events in early S-phase and changes in long-range replication timing domain structures. Overall replication timing is shifted toward mid-S in both directions, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear insoluble structures at late-M to early-G1 and regulates the chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early S-phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures through regulating higher-order chromatin architecture. HeLa cells (ATCC), with a total of 4 individual replicates
Project description:We have characterized allele-specific regulation of replication in human cultured primary basophilic erythroblasts using TimEX-seq. We show that in most of the genome the timing of replication of the two chromosome homologs is robustly and tightly regulated since the two alleles replicate almost at the same time. We also show that small genetic differences such as SNPs and indels do not affect replication timing. We identify two major causes of replication asynchrony: the presence of large structural variants and parental imprinting. Both are associated with the formation of asynchronously replicated domains that can reach several megabases in size. We also report that replication timing domains have a previously undetected fine structure. Compare DNA content in cells in S and G1 phase of cell cycle using TimEX-seq The goal of these experiments was to measure the timing of replication in human basophilic erythroblasts in an allele-specific manner by comparing DNA content in cells in S and G1 phase of cell cycle using TimEX-seq. Cells in S phase were obtained by sorting propidium iodide stained exponentially growing basophilic erythroblasts produce after 14 days of culture of circulating peripheral blood stem and progenitor cells. The cells in G1, which are used to normalize the results from the cells in S phase for mapability, were circulating mononuclear cells (WBCs) which are in the G1 cell for the cell cycle at 99.5%. The processed files represent S/G1 ratio values which are surrogate values for the timing of replication. Allele-specific TimEX-seq profiles and hi-resolution non-allele specific profiles are provided at different smoothing levels. The following processed files are derived from the multiple files as indicated below; >FNY01_3_2_Ery_MAT_S.bed is generated from FNY01_3_2_Ery_round *_S_Phase.bed >FNY01_3_2_Ery_PAT_S.bed is generated from FNY01_3_2_Ery_round *_S_Phase.bed >FNY01_3_2_Ery_MAT_G1.bed is generated from FNY01_3_2_WBC_round *_G1_600.bed FNY01_3_2_WBC_round *_G1_300.bed >FNY01_3_2_WBC_PAT_G1.bed is generated from FNY01_3_2_WBC_round *_G1_600.bed FNY01_3_2_WBC_round *_G1_300.bed >FNY01_3_2_Ery_S_G1 ratio_MAT_100kb_smooth.bedGraph is from FNY01_3_2_Ery_MAT_S.bed FNY01_3_2_Ery_MAT_G1.bed >FNY01_3_2_Ery_S_G1 ratio_PAT_100kb_smooth.bedGraph is from FNY01_3_2_Ery_PAT_S.bed FNY01_3_2_Ery_PAT_G1.bed >FNY01_3_2_Ery_S_G1 ratio_unsmooth.bedGraph, FNY01_3_2_Ery_S_G1 ratio_20Kb_smooth.bedGraph, and FNY01_3_2_Ery_S_G1 ratio_100Kb_smooth.bedGraph are from FNY01_3_2_Ery_round *_S_Phase.bed FNY01_3_2_WBC_round *_G1_600.bed FNY01_3_2_WBC_round *_G1_300.bed >FNY01_3_2&3_3_Ery* files are generated from 14 .bed files linked to the corresponding sample records. Please note that *3_3* files follow the same pattern as *3_2*
Project description:We have characterized allele-specific regulation of replication in human cultured primary basophilic erythroblasts using TimEX-seq. We show that in most of the genome the timing of replication of the two chromosome homologs is robustly and tightly regulated since the two alleles replicate almost at the same time. We also show that small genetic differences such as SNPs and indels do not affect replication timing. We identify two major causes of replication asynchrony: the presence of large structural variants and parental imprinting. Both are associated with the formation of asynchronously replicated domains that can reach several megabases in size. We also report that replication timing domains have a previously undetected fine structure.