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Functional Genomics Analysis of the Yeast Iron Responsive Transcription Factor Aft1 Reveals Iron-Independent Functions

ABSTRACT: The Saccharomyces cerevisiae transcription factor Aft1 is activated in iron-deficient cells to induce the expression of iron regulon genes, which coordinate the increase of iron uptake and remodel cellular metabolism to survive low iron conditions. In addition, Aft1 has been implicated in numerous cellular processes including cell cycle progression and chromosome stability; however it is unclear if all cellular effects of Aft1 are mediated through iron homeostasis. To further investigate the cellular processes impacted by Aft1 using genome-wide synthetic lethal and synthetic dosage lethal screens, we identified greater than 70 deletion mutants that are sensitive to perturbations in AFT1 levels. Our genetic network reveals that Aft1 impacts a diverse range of cellular processes, including the RIM101 pH pathway, cell wall stability, DNA damage, protein transport, chromosome stability and mitochondrial function. Surprisingly, only a subset of mutants identified are sensitive to iron fluctuations or display genetic interactions with mutants of iron regulon genes AFT2 or FET3. We demonstrate that Aft1 works in parallel with the RIM101 pH pathway and the role of Aft1 in cell wall structure and DNA damage repair is mediated by iron. In contrast, we show that the role of Aft1 in chromosome maintenance is independent of its iron regulatory role and that alterations in iron levels do not impact chromosome loss rates. Our study clearly demonstrates a novel iron-independent role for Aft1.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE20531 | GEO | 2010/05/01

SECONDARY ACCESSION(S): PRJNA125145

REPOSITORIES: GEO

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Functional Genomics Analysis of the Yeast Iron Responsive Transcription Factor Aft1 Reveals Iron-Independent Functions

Project description:The Saccharomyces cerevisiae transcription factor Aft1 is activated in iron-deficient cells to induce the expression of iron regulon genes, which coordinate the increase of iron uptake and remodel cellular metabolism to survive low iron conditions. In addition, Aft1 has been implicated in numerous cellular processes including cell cycle progression and chromosome stability; however it is unclear if all cellular effects of Aft1 are mediated through iron homeostasis. To further investigate the cellular processes impacted by Aft1 using genome-wide synthetic lethal and synthetic dosage lethal screens, we identified greater than 70 deletion mutants that are sensitive to perturbations in AFT1 levels. Our genetic network reveals that Aft1 impacts a diverse range of cellular processes, including the RIM101 pH pathway, cell wall stability, DNA damage, protein transport, chromosome stability and mitochondrial function. Surprisingly, only a subset of mutants identified are sensitive to iron fluctuations or display genetic interactions with mutants of iron regulon genes AFT2 or FET3. We demonstrate that Aft1 works in parallel with the RIM101 pH pathway and the role of Aft1 in cell wall structure and DNA damage repair is mediated by iron. In contrast, we show that the role of Aft1 in chromosome maintenance is independent of its iron regulatory role and that alterations in iron levels do not impact chromosome loss rates. Our study clearly demonstrates a novel iron-independent role for Aft1. For this experiment 4 samples (2 reference and 2 test samples), consisting of 3 biological reps were hybridized. Cy3 one-color labelling was used for each sample.

2010-05-01 | E-GEOD-20531 | biostudies-arrayexpress

Aft2 in yeast iron regulation

Project description:Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1aft2 deletion was generated. Growth assays established that the single aft2 strain exhibited no iron-dependent phenotype. However, the double-mutant aft1aft2 strain was more sensitive to low-iron growth conditions than the single-mutant aft1 strain. A mutant allele of AFT2 (AFT2-1up), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory- deficient phenotype of the aft1 strain. The AFT2-1up allele also increased the uptake of 59Fe in an aft1 strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1up-dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1up allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed

2005-08-13 | E-GEOD-3122 | biostudies-arrayexpress

Candida glabrata WT and mutants under iron starvation

Project description:Transcriptional time series of Candida glabrata under iron starvation (SD medium without Fe). Wild type and deletion mutants of the iron-related transcription factors Aft1 and Sef1, as well as of the iron uptake transporter Ftr1 as a positive control.

2016-10-05 | GSE84816 | GEO

Aft2 in yeast iron regulation

2005-08-13 | GSE3122 | GEO

Iron-responsive genes in Lachancea kluyveri

Project description:To understand the iron-responsive gene expression in Lachancea kluyveri under high and low iron conditions, we perfomred the genome-wide gene expression profiling for the log-phase cells of Lachancea kluyveri wild type, sef1 deletion, aft1 deletion and sef1aft1 double deletion mutants under the YPD+400mM ferrous iron and YPD+ 200 mM BPS conditions.

2019-04-26 | GSE130296 | GEO

Chemical potentiator of copper-accumulation and -toxicity: Probing iron- regulons of Saccharomyces cerevisiae

Project description:RNA-seq was used to assess mRNA transcript abundance in wild type and fra2Δ S. cerevisiae (BY4741) cells treated with 2-(6-benzyl-2-pyridyl)quinazoline (BPQ) and CuSO4. BPQ potentiates copper toxicity and in yeast, in common with other organisms, a major cause of copper toxicity is damage of iron-sulphur clusters. Iron sensing within yeast relies on mitochondrial iron-sulphur cluster biosynthesis and therefore treatment with BPQ and copper can be used to mimic iron deficiency. Fra2 is known to be a key component of the iron sensing mechanism; however, this mechanism can operate, to an extent, independently of Fra2. BPQ (+CuSO4) treatment was used with the aim of probing the regulation of the iron regulon of S. cerevisiae and the role of Fra2 in the suppression of the low iron response. This study has uncovered nine new Cth2 target-transcripts, plus a new Aft1 target-gene and paralogous non-target. Fra2 dominates basal repression of the iron regulon in iron-replete cultures, however, Fra2-independent control of the iron regulon is also observed with CTH2 appearing to be atypically Fra2-dependent. Transcripts from untreated and CuSO4 treated cells were included as controls.

2014-06-04 | GSE54045 | GEO

Endoplasmic reticulum–mitochondria junction is required for iron homeostasis

Project description:The Endoplasmic Reticulum–Mitochondria Encounter Structure (ERMES) is a protein complex that tethers the two organelles and creates the physical basis for communication between them. ERMES functions in lipid and calcium exchange between the ER and mitochondria, mitochondrial protein import and maintenance of mitochondrial morphology and genome. Here we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of ERMES known roles in calcium regulation, phospholipid biosynthesis or mitochondrial biology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our study reveals that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels.

2017-06-20 | GSE99499 | GEO

Chemical potentiator of copper-accumulation and -toxicity: Probing iron- regulons of Saccharomyces cerevisiae

Project description:RNA-seq was used to assess mRNA transcript abundance in wild type and fra2M-NM-^T S. cerevisiae (BY4741) cells treated with 2-(6-benzyl-2-pyridyl)quinazoline (BPQ) and CuSO4. BPQ potentiates copper toxicity and in yeast, in common with other organisms, a major cause of copper toxicity is damage of iron-sulphur clusters. Iron sensing within yeast relies on mitochondrial iron-sulphur cluster biosynthesis and therefore treatment with BPQ and copper can be used to mimic iron deficiency. Fra2 is known to be a key component of the iron sensing mechanism; however, this mechanism can operate, to an extent, independently of Fra2. BPQ (+CuSO4) treatment was used with the aim of probing the regulation of the iron regulon of S. cerevisiae and the role of Fra2 in the suppression of the low iron response. This study has uncovered nine new Cth2 target-transcripts, plus a new Aft1 target-gene and paralogous non-target. Fra2 dominates basal repression of the iron regulon in iron-replete cultures, however, Fra2-independent control of the iron regulon is also observed with CTH2 appearing to be atypically Fra2-dependent. Transcripts from untreated and CuSO4 treated cells were included as controls. Three independent biological replicates were analysed for each condition (BPQ and CuSO4 treated wild type and fra2M-NM-^T cells, CuSO4 treated wild type and fra2M-NM-^T cells and untreated wild type and fra2M-NM-^T cells)

2014-06-04 | E-GEOD-54045 | biostudies-arrayexpress

Transcription profiling of yeast double aft1aft2 deletion mutant in growth assays

2005-06-29 | E-SMDB-1517 | biostudies-arrayexpress

Iron uptake in yeast

Project description:Analysis of iron-regulated gene expression in Saccharomyces cerevisiae using cDNA microarrays has identified three putative cell wall proteins that are directly regulated by Aft1p, the major iron-dependent transcription factor in yeast. FIT1, FIT2, and FIT3 (for facilitator of iron transport) were more highly expressed in strains grown in low concentrations of iron and in strains in which AFT1-1(up), a constitutively active allele of AFT1, was expressed. Northern blot analysis confirmed that FIT1, FIT2, and FIT3 mRNA transcript levels were increased 60-230-fold in response to iron deprivation in an Aft1p-dependent manner. Fit1p was localized exclusively to the cell wall by indirect immunofluorescence. Deletion of the FIT genes, individually or in combination, resulted in diminished uptake of iron bound to the siderophores ferrioxamine B and ferrichrome, without diminishing the uptake of ferric iron salts, or the siderophores triacetylfusarinine C and enterobactin. FIT-deletion strains exhibited increased expression of Aft1p target genes as measured by a FET3-lacZ reporter gene or by Arn1p Western blotting, indicating that cells respond to the absence of FIT genes by up-regulating systems of iron uptake. Aft1p activation in FIT-deleted strains occurred when either ferrichrome or ferric salts were used as sources of iron during growth, suggesting that the FIT genes enhance uptake of iron from both sources. Enzymatic digestion of the cell wall resulted in the release of significant amounts of iron from cells, and the relative quantity of iron released was reduced in FIT-deletion strains. Fit1p, Fit2p, and Fit3p may function by increasing the amount of iron associated with the cell wall and periplasmic space. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set

2005-10-22 | GSE3496 | GEO

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