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Lack of the pif1 helicase enhances zinc tolerance in S. cerevisiae

ABSTRACT: The Saccharomyces cerevisiae gene PIF1 encodes a conserved eukaryotic DNA helicase required for both mitochondrial and nuclear DNA integrity. Our previous work revealed that a pif1D strain is tolerant to zinc overload. We demonstrate here that this effect is independent of the Pif1 helicase activity and is only observed when the protein is absent from the mitochondria. pif1 cells accumulate abnormal amounts of mitochondrial zinc and iron. Transcriptional profiling reveals that pif1 cells under standard growth conditions overexpress aconitase-related genes. When exposed to zinc, pif1 cells show lower induction of genes encoding iron (siderophores) transporters and higher expression of genes related to oxidative stress responses than wild type cells. Coincidently, pif1 mutants are less prone to zinc-induced oxidative stress and display a higher reduced/oxidized glutathione ratio. Strikingly, although pif1 cells contain normal amounts of the Aco1 protein, they completely lack aconitase activity. Loss of Aco1 activity is also observed when the cell expresses a non-mitochondrially targeted form of Pif1. We postulate that lack of Pif1 forces aconitase to play its DNA protective role as nucleoid protein and that this would trigger a domino effect on iron homeostasis resulting in increased zinc tolerance.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE18411 | GEO | 2010/06/01

SECONDARY ACCESSION(S): PRJNA118263

REPOSITORIES: GEO

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Lack of the pif1 helicase enhances zinc tolerance in S. cerevisiae

Project description:The Saccharomyces cerevisiae gene PIF1 encodes a conserved eukaryotic DNA helicase required for both mitochondrial and nuclear DNA integrity. Our previous work revealed that a pif1D strain is tolerant to zinc overload. We demonstrate here that this effect is independent of the Pif1 helicase activity and is only observed when the protein is absent from the mitochondria. pif1 cells accumulate abnormal amounts of mitochondrial zinc and iron. Transcriptional profiling reveals that pif1 cells under standard growth conditions overexpress aconitase-related genes. When exposed to zinc, pif1 cells show lower induction of genes encoding iron (siderophores) transporters and higher expression of genes related to oxidative stress responses than wild type cells. Coincidently, pif1 mutants are less prone to zinc-induced oxidative stress and display a higher reduced/oxidized glutathione ratio. Strikingly, although pif1 cells contain normal amounts of the Aco1 protein, they completely lack aconitase activity. Loss of Aco1 activity is also observed when the cell expresses a non-mitochondrially targeted form of Pif1. We postulate that lack of Pif1 forces aconitase to play its DNA protective role as nucleoid protein and that this would trigger a domino effect on iron homeostasis resulting in increased zinc tolerance. Four samples were analyzed: WT and pif1 mutant both, in the presence and in the absence of 5 mM ZnCl2 for 1 h. We compared the expression profile of: 1) WT+Zn vs WT-Zn, 2) pif1 mutant+Zn vs pif1 mutant-Zn, 3) pif1 mutant vs WT, and 4) pif1 mutant+Zn vs WT+Zn. A Dye-swap was carried out for each comparison of samples. Total number of chips analyzed: 8.

2010-05-31 | E-GEOD-18411 | biostudies-arrayexpress

The Pif1 helicase is actively inhibited during meiotic recombination to prevent excess length of gene conversions

Project description:Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversion events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, formed of the Mer3 helicase and the MutL heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops in vitro, allowing long synthesis required for break-induced replication. We show that Pif1 is recruited genome wide to meiotic double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutL recruitment to recombination sites. in vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutL ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutL may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity to promote meiotic recombination-related DNA synthesis is restrained by the Mer3-MutL ensemble to prevent long gene conversion events and possibly associated mutagenesis.

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Expression changes by Endodermal PIF1: SCR:PIF1/pifQ transgenic plant vs. pif1pif3pif4pif5 (pifQ) mutant

Project description:Transcriptional profiling of dark-grown Arabidopsis seedlings comparing SCR:PIF1/pifQ transgenic plant with pif1pif3pif4pif5 quadruple mutant (pifQ). Seedlings were grown under dark condition for 2.5 days. Goal was to determine the effects of endodermal PIF1 in dark-grown seedlings. Two-condition experiment, SCR:PIF1/pifQ vs. pifQ Biological replicates: 3 pifQ replicates, 3 SCR:PIF1 replicates.

2016-08-01 | E-GEOD-71918 | biostudies-arrayexpress

Expression changes by Endodermal PIF1: SCR:PIF1/pifQ transgenic plant vs. pif1pif3pif4pif5 (pifQ) mutant

2016-08-01 | GSE71918 | GEO

Genome-wide identification of PIF1-binding sites and direct-target genes of PIF1 transcriptional regulation in skotomorphogenesis

Project description:Dark-grown seedlings exhibit skotomorphogenic development. Genetic and molecular evidence indicates that a quartet of Arabidopsis Phytochrome (phy)-Interacting bHLH Factors (PIF1, 3, 4 and 5) are critically necessary to maintaining this developmental state, and that light activation of phy induces a switch to photomorphogenic development by inducing rapid degradation of the PIFs. Here, using combined ChIP-seq and RNA-seq analyses, we have identified genes that are direct targets of PIF1 transcriptional regulation, and we provide evidence that the quartet collectively regulate these genes by shared, direct binding to the target promoters in promoting skotomorphogenesis. Three biological replicates data of PIF1-binding sites were collected by comparing the parallel ChIP samples from Myc-epitope-tagged-PIF1 (P1M) overexpressing transgenic seedlings and the wild-type (WT) control.

2014-10-02 | E-GEOD-43283 | biostudies-arrayexpress

Transcription profiling by array of Arabidopsis seedlings mutant for pif1

Project description:Arabidopsis bHLH transcription factor PIF1 has been shown function as a negative regulator of Chlorophyll biosynthesis. Pif1-2 mutant accumulate more protochlorophylide during prolonged dark growth condations, therefore the comparation between Wt and pif1-2 mutant will help us find the PIF1 target genes in Chlorophyll biosynthesis pathway at genome wide level under the dark grown conditions. We used microarrays to detail the global gene expression under the regulation of PIF1. Experiment Overall Design: 4 days dark grown Wt and pif1-2 seedlings were collected for isolation RNA. Standard Affymetrix protocal and 25K Affymetrix chip (ATH1) were used for Microarry hybridition.

2008-06-18 | E-GEOD-11594 | biostudies-arrayexpress

Pif1-family helicases cooperate to suppress widespread replication fork arrest at tRNA genes

Project description:Saccharomyces cerevisiae encodes two distinct Pif1-family helicases – Pif1 and Rrm3 – which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging- strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes; consistent with this observation, we find that head-on collisions between tRNA transcription and replisome progression are under-represented in the S. cerevisiae genome. Further, we demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable.

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Genome-wide identification of PIF1-binding sites and direct-target genes of PIF1 transcriptional regulation in skotomorphogenesis

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Expression data from dark grown Arabidopsis Wild type (Wt, col-o) and pif1-2 (At2g20810, Salk_072677) mutant seedlings

2008-05-30 | GSE11594 | GEO