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Study of RepID/PHIP in DNA Replication inhitiation in Mammalian Cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE80298 | GEO | 2016/06/02

SECONDARY ACCESSION(S): PRJNA318517

REPOSITORIES: GEO

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Study of RepID/PHIP in DNA Replication inhitiation in Mammalian Cells

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

Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

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Single-copy locus isolation identifies distinct proteomes of early- and late-firing DNA replication origin chromatin

Project description:The chromatin at origins of replication is the template for DNA replication initiation in eukaryotic genomes. However, whether the chromatin composition instructs individual origins to fire early-efficiently (EE) or late-inefficiently (LI) remains to be investigated. Here, we used site-specific recombination and a single-locus chromatin isolation approach to purify selected EE and LI replication origins from chromosome III of Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of defined ~1kb native chromatin regions surrounding the EE and LI replication start sites. We confirm expected origin interactors but also find unexpected interactions, such as the Ask1 subunit of the kinetochore-associated DASH complex. Strikingly, we show that Ask1 is a critical factor in the replication timing control of specific origins in yeast. These results highlight that our unbiased approach can specifically identify the functionally-relevant proteome at a single-copy locus of interest and provide a detailed view of histone modification and protein interaction networks on replication origin chromatin. The current dataset contains proteomic data used for the histone PTM analysis.

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High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

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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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