Role of the iron mobilization and oxidative stress regulons in the genomic response of yeast to hydroxyurea
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- Dubacq, C., Chevalier, A., Courbeyrette, R. et al. Mol Genet Genomics (2006) 275: 114. doi:10.1007/s00438-005-0077-5
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Hydroxyurea (HU) is a specific inhibitor of ribonucleotide reductase and thus impairs dNTP synthesis and DNA replication. The long-term transcriptional response of yeast cells to hydroxyurea was investigated using DNA microarrays containing all yeast coding sequences. We show that the redox-responsive Yap regulon and the iron-mobilization Aft regulon are activated in yeast cells treated with HU. Yap1 accumulates in the nucleus in response to HU, but HU activation of the Yap regulon was only partially dependent on Yap1 and yap1Δ mutants were not hypersensitive to HU. In contrast, deletion of the AFT1 and AFT2 transcription factor genes blocked the HU activation of a subset of the Aft regulon and the aft1Δ aft2Δ double mutant was hypersensitive to HU in an iron-suppressible manner. These results highlight the importance of the redox and iron mobilization regulons in the cellular response to HU.
KeywordsDNA damage responseHydroxyureaAft1 and Yap1 transcription factorsRibonucleotide reductaseYeast
DNA Damage Response
Environmental stress response
Reverse transcriptase polymerase chain reaction
Ribonucleotide reductase (RNR) furnishes precursors for DNA synthesis by catalyzing the reduction of ribonucleoside diphosphates into deoxyribonucleoside diphosphates (Eklund et al. 2001). Eukaryotic RNRs are hetero-tetrameric enzymes containing two large subunits and two small subunits. The large subunit (R1) contains both catalytic and allosteric sites, whereas the small subunit (R2) contains an oxo-diferric iron center that is required for the formation of a catalytically essential tyrosyl-free radical (Eklund et al. 2001). Given its essential role in DNA synthesis and repair, small molecule inhibitors of RNR might be effective drugs to treat cancers. Several types of inhibitor are being actively developed. One type involves iron chelators that may inhibit the formation of the oxo-diferric center on the small subunit (Le and Richardson 2002). These chelators are expected to have pleiotropic effects on cells since many enzymes use iron as cofactors, but some studies suggest that RNR activity is preferentially sensitive to their action (Furukawa et al. 1992; Nyholm et al. 1993a). Another type is radical scavengers that quench the tyrosyl radical of R2. Hydroxyurea (HU) is the best-characterized RNR inhibitor of this class (Eklund et al. 2001). Although high concentrations of HU are required for RNR inhibition, its only essential target in the cell appears to be RNR because overexpression or mutation of R2 confers increased cellular resistance to HU (Sneeden and Loeb 2004).
The cellular response to HU has been most extensively studied in Saccharomyces cerevisiae. Inhibition of RNR and dNTP synthesis by HU triggers an S-phase checkpoint response that leads to transcriptional activation of RNR genes, the stabilization of stalled replication forks, and the inhibition of cell-cycle progression (Osborn et al. 2002). The screening of deletion mutants of all non-essential genes has also identified numerous genes that contribute to cellular tolerance to HU in as yet unknown ways (Parsons et al. 2004). SNF1 is an example of a non-essential gene that is required for wild-type levels of resistance to HU (Dubacq et al. 2004). The Snf1 kinase is a member of the AMP-activated kinase family that responds to various stresses, but its exact role in promoting HU tolerance has not been determined. Ctk1, a non-essential kinase that phosphorylates the C-terminal heptapeptide repeats of the large subunit of RNA polymerase II, is also required for cellular resistance to HU (Ostapenko and Solomon 2003). A recent analysis of the transcriptome of yeast cells treated for short periods of time with HU uncovered a role for the Ctk1 kinase in the transcriptional regulation of a subset of HU-induced genes (Ostapenko and Solomon 2003). We were interested in identifying genes that contribute to the cellular resistance to HU during long-term exposure to the drug, as might occur during cancer chemotherapy. We thus carried out transcriptional profiling of yeast cells treated for up to 6 h with high levels of HU.
Materials and methods
Strains and media
Strains used in this study
MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
Giaever et al. (2002)
MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
Giaever et al. (2002)
MATα his3Δ200 leu2-3,112 ura3-52
Vincent et al. (2001)
MATα his3 leu2 lys2 ura3 snf1Δ10
Vyas et al. (2001)
MATa can1-100 ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1
Allen et al. (1994)
MATα trp1-63 leu2-3,112 gcn4-101 his3-609
Blaiseau et al. (2001)
MATα trp1-63 leu2-3,112 gcn4-101 his3-609 aft2::KanMX4
Blaiseau et al. (2001)
MATα trp1-63 leu2-3,112 gcn4-101 his3-609 aft1::TRP1
Blaiseau et al. (2001)
MATα trp1-63 leu2-3,112 gcn4-101 his3-609 aft1::TRP1 aft2::KanMX4
Blaiseau et al. (2001)
Quantitative reverse transcriptase-polymerase chain reactions (RT-PCR)
RNA was prepared from cells with an RNeasy extraction kit (Qiagen). RNA was converted to cDNA by reverse transcription with random hexanucleotide primers and 1 μg of total RNA, 1 mM dNTPs, and 1 μl RNasin for 1.5 h at 42°C, followed by a 5 min heat inactivation at 95°C. For each gene, real-time quantitative RT-PCR amplification was performed with an ABI Prism 7,000 according to the manufacturer’s directions.
Strains were grown to mid-log phase in rich medium and submitted to the indicated treatments. Total RNA was isolated using RNeasy extraction kit (Qiagen) and cDNA was synthesized and labeled using the protocol previously described (Fauchon et al. 2002) for the W303 strain with or without HU, or using an indirect protocol with incorporation of aminoallyl-dUTP during cDNA synthesis followed by coupling to CyDye mono-reactive Cy5 or Cy3 (Amersham; see GEO accession numbers GSE1915, GSE1941, GSE1942, and GSE1943 for details). DNA microarrays were prepared by the Service de Génomique Fonctionnelle, CEA/Evry, and data capture and analysis were performed with GenePix and GeneSpring software as described (Fauchon et al. 2002). The cut off for significant spots required that 70% of the pixels be more intense than the background plus twice the standard deviation in at least one channel. Each set of RNA samples was hybridized to two different microarrays performed with an inversion of the fluorochromes to avoid incorporation bias. With these criteria, 4,000–5,000 coding sequences of the approximately 6,200 sequences on the microarrays yielded significant signals depending on the particular experiment and hybridization condition. The complete data set for the arrays was deposited in the GEO database (http://www.ncbi.nlm.nih.gov/projects/geo/; accession numbers GSE1915, GSE1941, GSE1942, and GSE1943).
Cells transformed with a YAP1–GFP plasmid (2 μ, LEU2, generously provided by Agnès Delaunay) were cultivated to O.D.600=0.5–0.6 in a synthetic medium lacking leucine, centrifuged and resuspended in PBS prior to observation using a Leica DMRXA fluorescence microscope.
Transcriptional profiling of yeast cells treated with HU
The genomic response to hydroxyurea is partly a non-specific stress response
Genes repressed during long-term exposure to hydroxyurea
Cell wall biogenesis
DSE1, DSE2, CWP1, PST1
EGT2, CTS1, SCW11, DSE4
Bud site selection, cellpolarity
MSB2, BUD9, AXL2
Regulation of mitosis
HTA2, HTB1, HTA1, SFH1, IOC4, NET1, RAP1, HST3, SGF73
SWI5, TUP1, REB1, SET2, SPT6, LEO1
Drug response: PDR5; DNA topology: TOF2; RNA helicase: DBP2; Meiotic chromosome segregation: CSM4; rRNA/ribosome processing: UTP10, RRP8, MAK16, NOP15; mRNA degradation: PUF3; Protein modification: PSA1, CDC5, UBP12; Protein synthesis: CBP6, RML2; Protein nucleus import: MLP1; Pseudo-hyphal growth: TEC1, STE12, ASH1; Purine base metabolism: ADE13, MIS1; Conjugation: YNL174W; SPB duplication: KAR1; Thiamin transport: PHO3; translation: TIF4631; vacuole fusion: VTC2; aromatic compound catabolism: PAD1
YOR263C, YGL101W, DSE3, YMR193C-A, YDR428C, YIL130W, YIL158W, YOR315W, YJL051W, YNL057W, YNL058C, YNL087W, YNR066C, YER184C
Identification of novel regulons induced by HU
Activation of the Yap regulon by HU
To address the involvement of the Yap1 transcription factor in the HU-induced expression of these genes, a global analysis of gene expression was performed with yap1Δ and wild-type strains during HU exposure (Fig. 3a, yap1Δ). We found that 10/41 of the HU-induced genes belonging to the Yap regulon were repressed twofold or more in the yap1Δ mutant treated with HU (Fig. 4b, bold). Thus, the HU induction of these genes is probably under the direct control of the Yap1 transcription factor. However, many of the HU-induced genes of the Yap regulon were repressed less than twofold following YAP1 deletion (Fig. 3a). Moreover, the yap1Δ null mutant was not hypersensitive to HU (data not shown). We suggest that another functionally redundant transcription factor may participate in the activation of the Yap regulon in response to HU in yap1Δ mutants.
HSP31 is a gene whose induction by HU is dependent on Yap1 (Fig. 4b), although it is not a known target gene of Yap1 in response to oxidative stress. We also identified three genes (CYC1, TRR1, and ROX1) whose expression was repressed in yap1Δ compared to the wild type during HU exposure, but whose expression was not modified in wild-type cells treated with HU. The basal expression of these three genes during normal growth may depend in part on Yap1.
Yap1 is mainly cytosolic in yeast cells under normal growth conditions, but it accumulates in the nucleus and activates the Yap1 regulon in response to oxidative stress, or when cells are treated with electrophilic compounds or with some drugs (Azevedo et al. 2003). We monitored the subcellular localization of a Yap1–GFP fusion protein after treating cells with 200 mM HU in comparison with the same cells treated with 0.3 mM H2O2 (Fig. 4c). As previously reported, Yap1–GFP rapidly and transiently accumulated in the nucleus after treating cells with these low levels of H2O2 (Delaunay et al. 2000). The transient nature of this response is due to the rapid detoxification of H2O2 within cells. Yap1–GFP also accumulated in the nucleus in response to HU, but with slower kinetics relative to H2O2. Nuclear accumulation of Yap1–GFP was observed in 50% of the cells after 1 h of incubation with 200 mM HU and was sustained for the 3 h duration of the experiment in the presence of the drug. The induced nuclear accumulation of Yap1 further confirms a role for Yap1 in the cellular response to HU.
Aft1 and Aft2 are required for tolerance to hydroxyurea
A limited role for the Snf1 kinase in the transcriptional response to HU
We used microarrays containing all yeast coding sequences to study the long-term transcriptional response of yeast cells to treatment with the ribonucleotide reductase inhibitor hydroxyurea. In addition to the expected activation of the DNA damage response (DDR) and environmental stress response (ESR) regulons, we observed activation of the Yap1 and Aft regulons. The Yap1 regulon is activated upon treatment of yeast cells with oxidative agents or electrophiles (Azevedo et al. 2003). Two distinct pathways lead to Yap1 nuclear accumulation. H2O2 induces a specific disulfide bond between Cys-303 and Cys-589 that is catalyzed by the hydrogen peroxide sensor Gpx3 (Delaunay et al. 2002). In contrast, thiol-reactive electrophiles, such as alkylating agents and some heavy metals, are believed to directly modify C-terminal cysteines of Yap1 independently of Gpx3 (Azevedo et al. 2003). In both cases, nuclear export of Yap1 is inhibited. Accumulation of Yap1 in the nucleus contributes to activation of the Yap regulon encoding proteins implicated in redox homeostasis, in drug and metal detoxification, and in multidrug efflux from the cell (Gounalaki and Thireos 1994; Toone et al. 2001). Hydroxyurea is not an electrophile and is unlikely to react directly with thiols. The slow kinetics of Yap1 nuclear accumulation in the presence of HU is also consistent with an indirect action of HU leading to Yap1 nuclear accumulation. HU blocks DNA synthesis by inhibiting ribonucleotide reductase through a single electron transfer reaction that quenches a tyrosyl radical contained within the small subunit of the enzyme (Eklund et al. 2001). This reaction would also generate a hydroperoxy radical form of HU that could diffuse away from the reduced RNR and act as an oxidizing agent. However, we found that Yap1–GFP still accumulated in the nucleus in a gpx3Δ mutant in response to HU, as did a Yap1–C598A–GFP mutant in a wild-type strain treated with HU (data not shown). Thus, Yap1 nuclear accumulation in response to HU does not occur through the activation pathway used by H2O2. It remains possible that hydroperoxy radical forms of HU directly or indirectly modify Yap1 cysteines.
The production of hydroperoxy radical forms of HU may constitute an oxidative stress that would be combated by activation of the Yap regulon. Unexpectedly, deletion of YAP1 only partially blocked HU activation of the Yap regulon, and yap1Δ mutants were not hypersensitive to HU. These results suggest that other transcription factors participate in this activation. Candidates include the seven other Yap1-like proteins encoded within the yeast genome, but further experimental work is necessary to test their possible involvement (Fernandes et al. 1997).
In addition to quenching its tyrosyl radical, treatment of mammalian RNR with HU also leads to reduction and loss of the oxo-diiron center from the small subunit (Nyholm et al. 1993b). This diferric iron center is required for regenerating a tyrosyl radical within R2. Cells have poorly studied pathways for assembling this oxo-diiron center, but it is known that two ferrous ions must first bind to apo R2. Active R2 is then formed by reaction with oxygen to give an oxo-diferric iron center and a tyrosyl radical (Eklund et al. 2001). We found that treatment of yeast cells with HU leads to activation of the Aft regulon that is implicated in iron mobilization in response to iron deprivation. Treatment of yeast cells with HU induces the transcription of the RNR2 and RNR4 genes encoding RNR small subunits. Rnr2 binds iron, but Rnr4 lacks three conserved iron-binding residues and does not itself bind iron. Instead, it forms heterodimers with Rnr2 and acts as a cofactor for Rnr2 assembly and activity (Chabes et al. 2000; Sommerhalter et al. 2004). Assembly of iron into newly synthesized Rnr2 subunits, and possibly the reassembly of diferric iron centers in old Rnr2 subunits attacked by HU, could lead to depletion of cytosolic iron and activation of the Aft regulon in order to mobilize iron from the vacuole (Rouault and Tong 2005). It is also possible that a specific chaperone involved in efficient assembly of the Rnr2 diferric iron center is expressed as a part of the Aft regulon. Strikingly, deletion of AFT1 and AFT2, encoding the transcriptional factors required for activation of the Aft regulon, sensitized yeast cells to HU in a manner that could be suppressed by the addition of excess iron to the growth medium. This result illustrates the physiological importance of the Aft regulon in the cellular response to HU.
Among the 126 genes induced in both S288C and W303 genetic contexts by a 2 h treatment with 200 mM HU, we identified 26 genes belonging to known regulons (Crt, Aft, Yap) and 32 additional genes induced during the environmental stress response. Thus, there remain 68 HU-induced genes that have not yet been assigned to known regulons and whose eventual roles in the cellular response to HU remain to be elucidated.
We thank P.-L. Blaiseau, A. Dancis, A. Delaunay, Y. Pereira, M. Toledano, and Y. Yamaguchi-Iwai for providing yeast strains, plasmids, and discussions. We are grateful to M. Werner for access to the microarray analysis platform and for general support. C.D. was supported by an Allocation Couplée de l’Ecole Normale Supérieure de Paris. This work was also supported by grants from the Association pour la Recherche sur le Cancer (ARC number 4470).