High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE65692 | GEO | 2015/04/23
SECONDARY ACCESSION(S): PRJNA274733
REPOSITORIES: GEO
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Project description:At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, which genetic and chromatin features define metazoan replication start sites remains largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and show their ability to transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence specificity, mark and predict replication origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship between DNA topology, chromatin, transcription and replication initiation across metazoa. Our dataset consists of SNS-Seq profiles in Drosophila S2 and Bg3 cells. Small nascent leading strands (SNS) have been purified with an enhanced sensitivity SNS purification protocol. For standard SNS-Seq experiments (from pooled gradient fractions, denoted as "pool"), SNS were purified from two biological replicates. For single-fraction SNS-Seq experiments, two libraries were prepared for each fraction. The genomic coordinates of S2 and Bg3 replication origins are also provided.
2015-04-23 | E-GEOD-65692 | biostudies-arrayexpress
Project description:Some features underlying replication origin activation in metazoan cells have been identified, but little is known about their regulation during metazoan development. Using the nascent strand purification method, we identified replication origins throughout Caenorhabditis elegans embryonic development and found that the origin repertoire is thoroughly reorganized after gastrulation onset. During the pluripotent embryonic stages (pre-gastrula), potential cruciform structures and open chromatin are determinant factors to establish replication origins. The enrichment of replication origins in transcription factor binding sites and their presence inside promoters of highly transcribed genes, particularly operons, argue that transcriptional activity contributes to replication initiation before gastrulation. After the gastrula transition, when differentiation programs are set in the embryos, origins are particularly selected at enhancers, in the vicinity of CGI-like sequences, and non-coding genes. Our findings suggest that origin selection coordinates replication initiation with transcriptional programs during metazoan development.
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Project description:To unveil the still-elusive nature of metazoan replication origins, we identified them genome-wide and at unprecedented high-resolution in mouse ES cells. This allowed initiation sites (IS) and initiation zones (IZ) to be differentiated. We then characterized their genetic signatures and organization and integrated these data with 43 chromatin marks and factors. Our results reveal that replication origins can be grouped into three main classes with distinct organization, chromatin environment and sequence motifs. Class 1 contains relatively isolated, low-efficiency origins that are poor in epigenetic marks and are enriched in an asymmetric AC repeat at the initiation site. Late origins are mainly found in this class. Class 2 origins are particularly rich in enhancer elements. Class 3 origins are the most efficient and are associated with open chromatin and polycomb protein-enriched regions. The presence of Origin G-rich Repeated elements (OGRE) potentially forming G-quadruplexes (G4) was confirmed at most origins. These coincide with nucleosome-depleted regions located upstream of the initiation sites, which are associated with a labile nucleosomes containing H3K64ac. These data demonstrate that specific chromatin landscapes and combinations of specific signatures regulate origin localization. They explain the frequently-observed links between DNA replication and transcription. They also emphasize the plasticity of metazoan replication origins, and suggest that in multicellular eukaryotes, the combination of distinct genetic features and chromatin configurations act in synergy to define and adapt the origin profile.
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Project description:Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal DNA, triggering a series of molecular events that culminate in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome requires multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern with relevance to differentiation, genome stability and evolution. It is unclear whether ORC-origin interactions are relevant to the regulation of origin activation time. Here we applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. The two classes of origins were distinct in terms of local nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Importantly, origins that fired in late S-phase, but not those that fired in early S-phase, showed a significant dependence on the ORC-origin DNA interaction for ORC binding in vivo. Therefore we demonstrate for the first time a connection between ORC-origin binding mechanisms and the regulation of origin activation time at a genome-wide level. Analysis of ORC genomic DNA binding across three ORC concentrations.
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Project description:In metazoans, thousands of DNA replication origins (Oris) are activated to replicate DNA at each cell cycle. Although their timing of activation is better understood, their genomic organization and their genetic nature remain elusive. Here, we identified Oris by nascent strand (NS) purification and characterized their common features by performing a genome-wide analysis in both Drosophila and mouse cell lines. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, suggesting that this epigenetic mark is not crucial for defining the initiation event. Moreover, nascent strands at the borders of CGIs show a striking bimodal distribution, suggestive of a dual initiation event. We also found that Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A over-representation at 5’ and 3’ of the Ori sites, respectively. GC-rich elements are detected, which are good predictors of Oris, in both mouse and Drosophila, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris are strongly correlated with HP1 binding sites. At the chromosome level, we show that Oris are organized in Ori-rich and -poor regions that co-localize with early and late replicating domains, respectively. Finally, the genome-wide analysis was coupled with a DNA combing analysis of the in-vivo spacing of replication origins. The results suggest that Oris are in large excess, and organized in groups of site-specific but flexible origins that define replicons, where a single origin is used in each group. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation of DNA replication to environmental cues and cell fates.
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Project description:Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal DNA, triggering a series of molecular events that culminate in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome requires multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern with relevance to differentiation, genome stability and evolution. It is unclear whether ORC-origin interactions are relevant to the regulation of origin activation time. Here we applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. The two classes of origins were distinct in terms of local nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Importantly, origins that fired in late S-phase, but not those that fired in early S-phase, showed a significant dependence on the ORC-origin DNA interaction for ORC binding in vivo. Therefore we demonstrate for the first time a connection between ORC-origin binding mechanisms and the regulation of origin activation time at a genome-wide level.
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