Pervasive transcription-dependent chromatin remodeling influences the replication initiation program
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE111058 | GEO | 2018/02/24
REPOSITORIES: GEO
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Project description:Pervasive transcription-dependent chromatin remodeling influences the replication initiation program
| PRJNA435729 | ENA
Project description:In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3 cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors. BrdU-IP-seq analysis of origin activity in wt, sir2 and rpd3 cells, aligned against genomic DNA (sacCer3) and rDNA sequences Please note that the wt WCE #1 and #2 samples are whole-cell extracts from wild-type cells arrested in HU that were used to calculate the log ratio of the BrdU IP #1 and #2 batches, respectively.
2014-05-14 | E-GEOD-57617 | biostudies-arrayexpress
Project description:In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3 cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors. BrdU-IP-chip analysis of origin usage in different yeast HDAC mutants
2014-03-26 | E-GEOD-56171 | biostudies-arrayexpress
Project description:Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unkown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that thsese deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism. In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated. To this end, we systematically deleted seven well-characterized origins on the left arm of S. cerevisiae chromosome VI and analyzed (1) Orc2 localization during G2/M arrest and (2) BrdU incorporation during synchronous release from G1 arrest into S-phase, and compared the results to wild-type cell signals. For Orc2 ChIP-Chip experiments, Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested in G2/M using antibodies against Flag. For BrdU ChIP-Chip experiments, actively replicating DNA was isolated from cells harboring a single integrated BrdU incorporation vector released synchronously into 200mM HU using antibodies against BrdU. Immunoprecipitated and input (Orc2) or G1 (BrdU) DNA was then amplified and competitively hybridized to high-resolution strand-specific microarrays covering chromosomes III, VI, and XII.
2014-09-26 | E-GEOD-61743 | biostudies-arrayexpress
Project description:In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3 cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors. MNase-seq analysis of nucleosome position in wt, sir2 and rpd3 cells, aligned against genomic DNA (sacCer3; *sorted_s3.bed) and rDNA sequences (*rdna_nucleosomes.bed)
2014-05-14 | E-GEOD-57618 | biostudies-arrayexpress
Project description:Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4∆ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1∆ cells, and checkpoint-defective mec1∆ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres. 30 total samples: (6 samples - BrdU- HU arrest 45min with 2 replicates, strains: WT, rif1 delta, pfa4 delta) (12 samples -S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: WT, rif1 delta, mec1_100) (12 samples - S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: sml1 delta, sml1 delta rif1 delta, sml1 delta mec1 delta)
2014-06-01 | E-GEOD-55130 | biostudies-arrayexpress
Project description:Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4∆ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1∆ cells, and checkpoint-defective mec1∆ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres. 4 samples BrdU-IP-seq in HU, 2 strains with 2-replicates each (strains:WT and rif1 delta)
2014-06-01 | E-GEOD-55155 | biostudies-arrayexpress
Project description:We describe a modification of initiation site sequencing (ini-seq, Langley et al., NAR 2016) in which density gradient centrifugation separates ‘heavy’ replicated DNA containing BrdU from DNA containing only ‘light’ dTTP before sequencing. This approach allows us to assign a replication initiation efficiency score to each of the 23905 origins identified. We also performed Short Nascent Strand sequencing (SNS-seq), an established method to map replication origins, in the same cell line as in Ini-seq2 for comparison.
2022-06-15 | GSE186675 | GEO
Project description:modENCODE_submission_710 This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: Early origins of replication were identified by treating cells with hydroxyurea (HU), a potent inhibitor of nucleotide synthesis, in the presence of the nucleotide analogue BrdU. Treatment of synchronized S2-DRSC cells with HU stalls replication forks and activates the intra S-phase checkpoint, thereby limiting BrdU incorporation to those sequences immediately adjacent to early activating replication origins. BrdU enriched sequences surrounding early origins of replication are then enriched by immunoprecipitation with an anti-BrdU antibody. Early origins are then detected by hybridization to Agilent genomic tiling arrays. Peaks are called using MA2C (http://liulab.dfci.harvard.edu/MA2C/MA2C.htm) Keywords: CHIP-chip For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2009-07-29 | GSE17286 | GEO
Project description:modENCODE_submission_709 This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: Early origins of replication were identified by treating cells with hydroxyurea (HU), a potent inhibitor of nucleotide synthesis, in the presence of the nucleotide analogue BrdU. Treatment of synchronized Kc167 cells with HU stalls replication forks and activates the intra S-phase checkpoint, thereby limiting BrdU incorporation to those sequences immediately adjacent to early activating replication origins. BrdU enriched sequences surrounding early origins of replication are then enriched by immunoprecipitation with an anti-BrdU antibody. Early origins are then detected by hybridization to Agilent genomic tiling arrays. Peaks are called using MA2C (http://liulab.dfci.harvard.edu/MA2C/MA2C.htm) Keywords: CHIP-chip For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2009-07-29 | GSE17285 | GEO