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High resolution mapping of DNA replication origins in human genome

ABSTRACT: Because of the lack of information, regulation of DNA replication initiation in mammals is still poorly understood. In order to identify general rules, we have mapped replication origins along 1% of the human genome in HeLa cells. We found large gene-poor regions lacking origin and G+C rich regions containing clusters of closely spaced origins. Half of the 283 origins mapped are within or near CpG islands. The connection with gene expression is further reinforced by the observation that most origins overlap with DNAseI hypersensitive sites found at transcriptional regulatory elements. We show, however, that this association is independent of chromatin structure and transcriptional activity. Replication timing analyses coupled to our origin mapping demonstrate that origin dense regions and isolated origins are replicated at every moment in S phase. All together, our data suggest that a relatively strict origin-timing programme regulates DNA replication of the human genome. Keywords: Nascent strands, ENCODE project, HeLAS3 cells, SNS-Chip

ORGANISM(S): Homo sapiens

PROVIDER: GSE10217 | GEO | 2008/10/22

SECONDARY ACCESSION(S): PRJNA108275

REPOSITORIES: GEO

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High resolution mapping of DNA replication origins in human genome

Project description:Because of the lack of information, regulation of DNA replication initiation in mammals is still poorly understood. In order to identify general rules, we have mapped replication origins along 1% of the human genome in HeLa cells. We found large gene-poor regions lacking origin and G+C rich regions containing clusters of closely spaced origins. Half of the 283 origins mapped are within or near CpG islands. The connection with gene expression is further reinforced by the observation that most origins overlap with DNAseI hypersensitive sites found at transcriptional regulatory elements. We show, however, that this association is independent of chromatin structure and transcriptional activity. Replication timing analyses coupled to our origin mapping demonstrate that origin dense regions and isolated origins are replicated at every moment in S phase. All together, our data suggest that a relatively strict origin-timing programme regulates DNA replication of the human genome. Keywords: Nascent strands, ENCODE project, HeLAS3 cells, SNS-Chip Four independent preparations of Short Nascent Strands (SNS) were performed. In order to have enough material for microarray hybridisation, we coupled the stringent preparation of SNS with the TLAD method, a technique of linear amplification that can generate several Âµg of amplified material from 10-20 ng of DNA (Liu et al., 2003).Two were amplified by TLAD (experiments A and B) and hybridized on DNA microarrays, and the other two (experiments C and D) were used for the validation by real-time quantitative PCR (qPCR) of results obtained on micro-arrays. We performed also a gDNA/gDNA hybridization where gDNA are also amplified by TLAD to order to do a control.

2010-05-18 | E-GEOD-10217 | biostudies-arrayexpress

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

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

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

Project description:The chromatin at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how the chromatin composition controls the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we used site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae . Using mass spectrometry, we define the histone modification landscape and identify the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to the known origin interactors, we find novel origin-associated factors, such as the kinetochore-associated Ask1/DASH complex. Strikingly, we show that Ask1 regulates the replication timing control of specific origins in yeast. Thus, our unbiased approach identifies functionally-relevant proteomes at single-copy loci and would be widely applicable to provide an in-depth quantitative characterization of histone modification and protein interaction networks of chromatin at any genomic locus of interest.

2023-02-16 | PXD032930 | Pride

Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase (BrdU)

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. 5 total experiments with replicates

2015-12-18 | E-GEOD-71050 | biostudies-arrayexpress

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.

2023-05-10 | PXD031984 | Pride

LSD1 Facilitates Euchromatic Origin Firing and Regulates Replication Timing [Repli-seq]

Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.

2022-03-07 | GSE157470 | GEO

LSD1 Facilitates Euchromatic Origin Firing and Regulates Replication Timing [ChIP-seq]

2022-03-07 | GSE157469 | GEO

LSD1 Facilitates Euchromatic Origin Firing and Regulates Replication Timing [RNA-seq]

2022-03-07 | GSE157468 | GEO

Forkhead transcription factors establish origin timing and long-range clustering in S. cerevisiae [Array Data]

Project description:The replication of eukaryotic chromosomes is organized temporally and spatially within the nucleus through epigenetic regulation of replication origin function. The characteristic initiation timing of specific origins is thought to reflect their chromatin environment or sub-nuclear positioning, however the mechanism remains obscure. Here we show that the yeast Forkhead transcription factors, Fkh1 and Fkh2, are global determinants of replication origin timing. Forkhead regulation of origin timing is independent of local levels or changes of transcription. Instead, we show that Fkh1 and Fkh2 are required for the clustering of early origins and their association with the key initiation factor Cdc45 in G1-phase, suggesting that Fkh1 and Fkh2 selectively recruit origins to emergent replication factories. Fkh1 and Fkh2 bind Fkh-activated origins, and interact physically with ORC, providing a plausible mechanism to cluster origins. These findings add a new dimension to our understanding of the epigenetic basis for differential origin regulation and its connection to chromosomal domain organization.

2012-01-20 | GSE33702 | GEO

Forkhead transcription factors establish origin timing and long-range clustering in S. cerevisiae [Sequence Data]

2012-01-20 | GSE33677 | GEO

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