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The yeast and human FACT complexes resolve R-loop-mediated transcription-replication conflicts

ABSTRACT: Transcription is a major obstacle for replication fork progression and transcription-replication collisions constitute a main cause of genome instability. At a genome-wide scale these obstacles can be detected by the accumulation of the replicative Rrm3 helicase required for RF progression through protein obstacles. Here we show that FACT, a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and that also has a role in replication, is needed to resolve transcription-replication conflicts in Saccharomyces cerevisiae. Importantly, ChIP-chip analyses of Rrm3 reveal that replication progression impairment in FACT mutants occur genome-wide, but preferentially at highly transcribed regions.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE51653 | GEO | 2014/03/17

SECONDARY ACCESSION(S): PRJNA224239

REPOSITORIES: GEO

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The yeast and human FACT complexes resolve R-loop-mediated transcription-replication conflicts

Project description:Transcription is a major obstacle for replication fork progression and transcription-replication collisions constitute a main cause of genome instability. At a genome-wide scale these obstacles can be detected by the accumulation of the replicative Rrm3 helicase required for RF progression through protein obstacles. Here we show that FACT, a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and that also has a role in replication, is needed to resolve transcription-replication conflicts in Saccharomyces cerevisiae. Importantly, ChIP-chip analyses of Rrm3 reveal that replication progression impairment in FACT mutants occur genome-wide, but preferentially at highly transcribed regions. ChIP-chip studies were perfomed with antibodies against Rrm3-FLAG in the yeast S. cerevisiae.

2014-03-17 | E-GEOD-51653 | biostudies-arrayexpress

ChIP-chip data of Npl3-Myc and Rrm3-Flag in wild-type and npl3M-bM-^HM-^F cells

Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Such instability increases in mutants with a suboptimal assembly of the nascent messenger ribonucleo-protein particle (mRNP), as THO/TREX and some heterogeneous nuclear ribonucleoproteins (hnRNPs) mutants. Here we show that yeast npl3M-bM-^HM-^F cells show genome-wide replication obstacles as determined by accumulation of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Npl3 is preferentially bound in wild-type cells. ChIP-chip studies were perfomed with antibodies against Myc-tagged Npl3 protein in wild-type cells of the yeast S. Cerevisiae, as well as Flag-tagged Rrm3 protein in both wild-type and npl3M-bM-^HM-^F cells.

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ChIP-chip data of Thp1-Flag and Sac3-Flag in wild-type cells, and Rrm3-Flag in sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells

Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Such instability increases in mutants with a suboptimal assembly of the nascent messenger ribonucleo-protein particle (mRNP), as THO/TREX and the NPC-associated THSC/TREX-2 complex. Here we show that yeast sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells accumulate genome-wide replication obstacles as determined by the distribution of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Sac3 and its interacting partner Thp1 are preferentially bound in wild-type cells. ChIP-chip studies were perfomed with antibodies against Flag-tagged Thp1 and Sac3 proteins in wild-type cells of the yeast S. Cerevisiae, as well as Flag-tagged Rrm3 protein in sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells that were compared with Rrm3 in wild-type cells from Santos-Pereira et al., 2013 (accession number GSE50185).

2014-10-08 | E-GEOD-56700 | biostudies-arrayexpress

ChIP-chip data of Npl3-Myc and Rrm3-Flag in wild-type and npl3∆ cells

2013-11-15 | GSE50185 | GEO

ChIP-chip data of Thp1-Flag and Sac3-Flag in wild-type cells, and Rrm3-Flag in sac3∆ and thp1∆ cells

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Mapping Replication Fork Pause Sites in S. cerevisiae

2009-06-15 | GSE16218 | GEO

Mapping Replication Fork Pause Sites in S. cerevisiae

Project description:Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. We alse mapped pause sites using a second replication fork component, Rrm3-13MYC and got similar results. Genomic input (labelled with Cy3) and IP'ed DNA (labelled with Cy5) using a MYC Ab of either DNA Pol2-13MYC or Rrm3-MYC from asynchronously grown S. cerevisiae cells in rich media were hybridized to whole-genome PCR-based arrays containing ORF and intergenic regions of the entire genome (Ivery et al 2001). At least three biological replication and one technical replicate (dye swap) were performed. Log2 transformed median normalized ratios (IP/IN) were averaged for each experiment and significant peaks of either DNA Pol2 or Rrm3 association were identified

2009-06-15 | E-GEOD-16218 | biostudies-arrayexpress

Genome-wide binding of THO and Sub2 in Saccharomyces cerevisiae

Project description:THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is an open question. ChIP-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome wide-distributions at active ORFs in yeast. In contrast to RNAPII, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a sharp 5M-bM-^@M-^YM-bM-^FM-^R3M-bM-^@M-^Y gradient. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by overexpression of RNase H1. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops. ChIP-chip studies were perfomed with tagged forms of the Hpr1 component of THO (Hpr1-FLAG), the Sub2 RNA-dependent ATPase of TREX (Sub2-FLAG), the Rpb3 subunit of RNA polymerase II (Rpb3-PK) and the Rrm3 protein (Rrm3-FLAG) in the yeast S. cerevisiae.

2011-06-28 | E-GEOD-24960 | biostudies-arrayexpress

The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription-replication conflicts

Project description:ATP-dependent chromatin remodelers are commonly mutated in human cancer. Mammalian SWI/SNF complexes comprise three conserved multi-subunit chromatin remodelers (cBAF, ncBAF and PBAF) that share the BRG1 (also known as SMARCA4) subunit responsible for the main ATPase activity. BRG1 is the most frequently mutated Snf2-like ATPase in cancer. Here we have investigated the role of SWI/SNF in genome instability, a hallmark of cancer cells, given its role in transcription, DNA replication and DNA damage repair. We show that depletion of BRG1 increases R-loops and R-loop-dependent DNA breaks, as well as transcription-replication conflicts. BRG1 colocalizes with R-loops and replication fork blocks, as determined by FANCD2 foci, with BRG1 depletion being epistatic to FANCD2 silencing. Our study, extended to other components of SWI/SNF, uncovers a key role of the SWI/SNF complex, in particular cBAF, in helping resolve R-loop-mediated transcription-replication conflicts; thus, unveiling a novel mechanism by which chromatin remodeling protects genome integrity.

2021-05-10 | GSE154631 | GEO

Rad53-mediated regulation of Rrm3 and Pif1 DNA helicases contributes to prevention of aberrant fork transitions under replication stress.

Project description:Replication stress activates the Mec1ATR and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1.

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