Transcriptional regulators compete with nucleosomes post-replication
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE76120 | GEO | 2016/04/07
SECONDARY ACCESSION(S): PRJNA306358
REPOSITORIES: GEO
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Project description:The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression. S.cerevisiae oligonucleotide microarrays were provided by Affymetrix (S.cerevisiae Tiling 1.0R, P/N 900645). BrdU and proteins ChIP-chip analyses were carried out as described (Fachinetti et al., M Cell, 2010).
2014-09-12 | E-GEOD-61030 | biostudies-arrayexpress
Project description:The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.
2014-09-12 | GSE61030 | GEO
Project description:Upon replication stress, the Mec1ATR kinase triggers the downregulation of transcription, reducing the level of RNA polymerase on chromatin to facilitate replication fork progression. We identify a hydroxyurea-induced phosphorylation site at Mec1-S1991 that contributes to the eviction of RNAPII and RNAPIII during replication stress. The non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII with a Rpb3-TAP fusion, suggesting that lethality arises from replication-transcription conflicts. Coincident with a failure to repress gene expression, highly transcribed genes like GAL1 persist at nuclear pores in mec1-S1991A cells. Consistently, we find pore proteins and several components controlling RNAPII and RNAPIII transcription are phosphorylated in a Mec1-dependent manner, suggesting that Mec1-S1991 phosphorylation limits conflicts between replication and either RNA polymerase complex. We further show that Mec1 contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase.
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Project description:Upon replication stress, the Mec1ATR kinase triggers the downregulation of transcription, reducing the level of RNA polymerase on chromatin to facilitate replication fork progression. We identify a hydroxyurea-induced phosphorylation site at Mec1-S1991 that contributes to the eviction of RNAPII and RNAPIII during replication stress. The non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII with a Rpb3-TAP fusion, suggesting that lethality arises from replication-transcription conflicts. Coincident with a failure to repress gene expression, highly transcribed genes like GAL1 persist at nuclear pores in mec1-S1991A cells. Consistently, we find pore proteins and several components controlling RNAPII and RNAPIII transcription are phosphorylated in a Mec1-dependent manner, suggesting that Mec1-S1991 phosphorylation limits conflicts between replication and either RNA polymerase complex. We further show that Mec1 contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase.
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Project description:Upon replication stress, the Mec1ATR kinase triggers the downregulation of transcription, reducing the level of RNA polymerase on chromatin to facilitate replication fork progression. We identify a hydroxyurea-induced phosphorylation site at Mec1-S1991 that contributes to the eviction of RNAPII and RNAPIII during replication stress. The non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII with a Rpb3-TAP fusion, suggesting that lethality arises from replication-transcription conflicts. Coincident with a failure to repress gene expression, highly transcribed genes like GAL1 persist at nuclear pores in mec1-S1991A cells. Consistently, we find pore proteins and several components controlling RNAPII and RNAPIII transcription are phosphorylated in a Mec1-dependent manner, suggesting that Mec1-S1991 phosphorylation limits conflicts between replication and either RNA polymerase complex. We further show that Mec1 contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase.
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Project description:Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we have developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease that is normalized for nucleosome density, NCAM (Normalized Chromatin Accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress. In this study we sought to analyze the chromatin structural changes that take place at sites of DNA replication. To this end, we obtained yeast cell populations synchronously undergoing DNA replication by M-NM-1-factor G1 arrest and release. In order to analyze chromatin structure at sites of DNA replication we first mapped the genomic locations undergoing DNA replication to high-resolution, strand-specific microarrays tiling chromosomes III, VI, and XII, covering ~14% of the genome. Two complementary approaches were taken: (1) we mapped fork positions by chromatin immunoprecipitation (ChIP) of a FLAG-tagged DNA polymerase 1 (Pol1), a replication fork component and (2) we mapped the sites of active DNA synthesis by generating DNA copy number profiles. We then analyzed chromatin structure at the sites of DNA replication by Micrococcal nuclease mononucleosome mapping and by generating histone H3 density maps. We further generated a Normalized Chromatin Accessibility to MNase (NCAM) signal by normalizing MNase mononucleosome signal for histone H3 density. NCAM signal represents a measure of nucleosome accessibility to MNase that is normalized for nucleosome content. These three approaches allowed us to assess potential changes in nucleosome positioning, nucleosome density, and nucleosome accessibility during DNA replication. We searched for changes in chromatin structure by comparing, during S phase, regions undergoing DNA replication to those not yet replicated, and also by comparing the same region before replication (not replicated, G1 arrested control) and during DNA replication in S phase. We typically harvested S phase cells at 30 or 60 minutes after release from G1 arrest. Experimental conditions included releasing cells into rich media at 24M-BM-0C or into rich media containing 200 mM Hydroxyurea (HU). Both conditions slowed down replication fork rate and made these experiments feasible. For each strain, samples for the different experiments (Chromatin for Pol1 and H3 ChIP, in vivo MNase digestion, and DNA for DNA copy number profiles) were harvested simultaneously for each time point. Therefore, comparisons between time points for each strain must be made using samples from the same replicate experiment. Our analysis included WT cells, as well as S phase checkpoint mutants (M-NM-^Tmec1 M-NM-^Tsml1 and mec1-100 M-NM-^Tsml1), as well as control strains (M-NM-^Tsml1).
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