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• W1969074879 abstract "Iron and copper are redox active metals essential for life. In the budding yeast Saccharomyces cerevisiae, expression of iron and copper genes involved in metal acquisition and utilization is tightly regulated at the transcriptional level. In addition iron and copper metabolism are inextricably linked because of the dependence on copper as a co-factor for iron uptake or mobilization. To further identify genes that function in iron and copper homeostasis, we screened for novel yeast mutants defective for iron limiting growth and thereby identified the CTI6 gene. Cti6 is a PHD finger-containing protein that has been shown to participate in the interaction of the Ssn6-Tup1 co-repressor with the Gcn5-containing SAGA chromatin-remodeling complex. In this report we show that CTI6 mRNA levels are increased under iron-limiting conditions, and that cti6 mutants display a growth defect under conditions of iron deprivation. Furthermore, we demonstrate that Cti6 is a nuclear protein that functionally associates with the Rpd3-Sin3 histone deacetylase complex involved in transcriptional repression. Cti6 demonstrates Rpd3-dependent transcriptional repression, and cti6 mutants exhibit an enhanced silencing of telomeric, rDNA and HMR loci, similar to mutants in genes encoding other Rpd3-Sin3-associated proteins. Microarray experiments with cti6 mutants grown under iron-limiting conditions show a down-regulation of telomeric genes and an up-regulation of Aft1 and Tup1 target genes involved in iron and oxygen regulation. Taken together, these data suggest a specific role for Cti6 in the regulation of gene expression under conditions of iron limitation. Iron and copper are redox active metals essential for life. In the budding yeast Saccharomyces cerevisiae, expression of iron and copper genes involved in metal acquisition and utilization is tightly regulated at the transcriptional level. In addition iron and copper metabolism are inextricably linked because of the dependence on copper as a co-factor for iron uptake or mobilization. To further identify genes that function in iron and copper homeostasis, we screened for novel yeast mutants defective for iron limiting growth and thereby identified the CTI6 gene. Cti6 is a PHD finger-containing protein that has been shown to participate in the interaction of the Ssn6-Tup1 co-repressor with the Gcn5-containing SAGA chromatin-remodeling complex. In this report we show that CTI6 mRNA levels are increased under iron-limiting conditions, and that cti6 mutants display a growth defect under conditions of iron deprivation. Furthermore, we demonstrate that Cti6 is a nuclear protein that functionally associates with the Rpd3-Sin3 histone deacetylase complex involved in transcriptional repression. Cti6 demonstrates Rpd3-dependent transcriptional repression, and cti6 mutants exhibit an enhanced silencing of telomeric, rDNA and HMR loci, similar to mutants in genes encoding other Rpd3-Sin3-associated proteins. Microarray experiments with cti6 mutants grown under iron-limiting conditions show a down-regulation of telomeric genes and an up-regulation of Aft1 and Tup1 target genes involved in iron and oxygen regulation. Taken together, these data suggest a specific role for Cti6 in the regulation of gene expression under conditions of iron limitation. Iron and copper are redox active metals that are essential for life in virtually all organisms and serve as catalytic co-factors for a wide variety of key cellular enzymes. Furthermore, much experimental evidence has established biochemical links between the ability of organisms to acquire copper and their ability to import iron into cells or distribute iron within cells or to peripheral tissues. Recent work on a high affinity iron uptake system in Saccharomyces cerevisiae, and on the multicopper oxidase ceruloplasmin in mammalian cells, firmly established the mechanistic requirement for copper as a redox cofactor that is essential for iron mobilization (1Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar, 2Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (561) Google Scholar). In Baker's yeast the Fet3 multicopper ferroxidase functions together with an iron permease (Ftr1) and ferric reductases (Fre1 and Fre2) at the plasma membrane in high affinity iron uptake. This reductive system of iron uptake is fully dependent on copper, a cofactor for the Fet3 multicopper oxidase (2Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (561) Google Scholar). The ability of yeast cells to incorporate copper into Fet3 within a late secretory compartment requires copper uptake by the Ctr1 high affinity copper transporter and delivery of the copper by a cytosolic metallochaperone Atx1 to the copper-transporting ATPase Ccc2, localized in the trans Golgi network membrane. Ccc2 delivers copper into the lumen of the secretory pathway where it is loaded into Fet3 (for review see Ref. 3O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2000; 275: 25057-25060Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). Yeast mutants defective for copper uptake through CTR1, or delivery to the lumen of the secretory compartment via mutations in ATX1 or CCC2, are unable to incorporate copper into the active sites of Fet3, resulting in defective high affinity iron uptake and a failure to grow under iron-limiting conditions. Consistent with this mechanism, the growth defect in limited iron and the absence of Fet3-dependent multicopper ferroxidase activity in ctr1, atx1, or ccc2 mutants can be reversed by the addition of copper to the growth medium.A key aspect to iron and copper homeostasis in S. cerevisiae is the regulation of transcription of genes encoding iron or copper homeostasis proteins in response to fluctuations in the availability of these two metals. Under conditions of copper limitation, the Mac1 copper-metalloregulatory transcription factor activates the expression of genes encoding components of the copper acquisition machinery that include the CTR1 and CTR3 high affinity plasma membrane copper transporters, the FRE1 and FRE7 metalloreductases, and other genes with as yet undescribed roles in copper homeostasis (2Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (561) Google Scholar, 4Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 5Yamaguchi-Iwai Y. Serpe M. Haile D. Yang W. Kosman D.J. Klausner R.D. Dancis A. J. Biol. Chem. 1997; 272: 17711-17718Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 6Gross C. Kelleher M. Iyer V.R. Brown P.O. Winge D.R. J. Biol. Chem. 2000; 275: 32310-32316Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Under conditions of copper adequacy, copper acquisition genes are not expressed. In response to iron deprivation S. cerevisiae cells use two iron-responsive transcription factors, Aft1 and Aft2, to stimulate the expression of genes involved in iron acquisition, the so-called iron regulon (7Rutherford J.C. Jaron S. Ray E. Brown P.O. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14322-14327Crossref PubMed Scopus (127) Google Scholar). Targets for Aft1/Aft2 regulation include (i) genes which protein products are involved in high affinity reductive iron uptake such as the plasma membrane metalloreductases FRE1–6 (8Georgatsou E. Alexandraki D. Yeast. 1999; 15: 573-584Crossref PubMed Scopus (61) Google Scholar), the high affinity iron transport complex composed of the iron permease FTR1 (9Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar) and the multicopper oxidase FET3 (10Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (582) Google Scholar) and the ATX1 copper chaperone and CCC2 copper-transporting ATPase, (ii) genes encoding components of siderophore iron uptake systems, which include the transporters ARN1–4 (11Yun C.W. Tiedeman J.S. Moore R.E. Philpott C.C. J. Biol. Chem. 2000; 275: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and the cell wall manoproteins FIT1–3 (12Protchenko O. Ferea T. Rashford J. Tiedeman J. Brown P.O. Botstein D. Philpott C.C. J. Biol. Chem. 2001; 276: 49244-49250Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), (iii) genes involved in the mobilization of iron from vacuolar stores, which include the Fet3-Ftr1 homologue complex formed by FET5 and FTH1 (13Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and the Nramp family member coded by SMF3 (14Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (179) Google Scholar, 15Portnoy M.E. Jensen L.T. Culotta V.C. Biochem. J. 2002; 362: 119-124Crossref PubMed Scopus (49) Google Scholar), (iv) the heme oxygenase homologue gene HMX1 involved in regulation of intracellular heme levels (16Protchenko O. Philpott C.C. J. Biol. Chem. 2003; 278: 36582-36587Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and other genes known or predicted to function in iron homeostasis are also transcriptionally activated by Aft1/Aft2 under iron deprivation. Therefore, copper and iron acquisition and distribution are regulated by metalloregulatory transcription factors that both activate or extinguish transcription in accordance with metal availability.To explore additional aspects of the regulation of copper and iron acquisition, we screened a yeast haploid knock out library to identify genes that, when deleted, give rise to a growth defect under conditions of iron limitation that is rescued by exogenous iron and copper. One such mutant was identified in which the CTI6 gene is insertionally deleted. Recent results show that Cti6 associates with the Ssn6(Cyc8)-Tup1 co-repressor (17Papamichos-Chronakis M. Petrakis T. Ktistaki E. Topalidou I. Tzamarias D. Mol. Cell. 2002; 9: 1297-1305Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The Ssn6-Tup1 complex is recruited to target promoters by different DNA-binding repressors including Mig1, Crt1, Rox1, and Sko1, and mediates repression of genes specifically required for growth under adverse conditions such as glucose starvation, DNA damage, hypoxia, or osmotic stress, respectively (for a review, see Ref. 18Smith R.L. Johnson A.D. Trends Biochem. Sci. 2000; 25: 325-330Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The mechanism of transcriptional repression includes, in addition to the interaction with the general transcription machinery, the specific interaction and recruitment of the Hda1, Rpd3, and Hos2 histone deacetylases (HDAC) 1The abbreviations used are: HDAC, histone deacetylase; BCS, bathocuproine disulfonic acid; BPS, bathophenantholine disulfonic acid; FOA, 5-fluoroorotic acid; GFP, green fluorescent protein; PHD, plant homeodomain; TBP, TATA-binding protein; SC, synthetic media.1The abbreviations used are: HDAC, histone deacetylase; BCS, bathocuproine disulfonic acid; BPS, bathophenantholine disulfonic acid; FOA, 5-fluoroorotic acid; GFP, green fluorescent protein; PHD, plant homeodomain; TBP, TATA-binding protein; SC, synthetic media. (19Watson A.D. Edmondson D.G. Bone J.R. Mukai Y. Yu Y. Du W. Stillman D.J. Roth S.Y. Genes Dev. 2000; 14: 2737-2744Crossref PubMed Scopus (129) Google Scholar, 20Chen G. Fernandez J. Mische S. Courey A.J. Genes Dev. 1999; 13: 2218-2230Crossref PubMed Scopus (352) Google Scholar, 21Wu J. Suka N. Carlson M. Grunstein M. Mol. Cell. 2001; 7: 117-126Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 22Robyr D. Suka Y. Xenarios I. Kurdistani S.K. Wang A. Suka N. Grunstein M. Cell. 2002; 109: 437-446Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 23Davie J.K. Edmondson D.G. Coco C.B. Dent S.Y. J. Biol. Chem. 2003; 278: 50158-50162Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Surprisingly, it has been shown that the Ssn6-Tup1 complex can also activate transcription of specific target promoters (GAL1 and ABN1) by recruiting the Gcn5 HAT-containing SAGA complex (17Papamichos-Chronakis M. Petrakis T. Ktistaki E. Topalidou I. Tzamarias D. Mol. Cell. 2002; 9: 1297-1305Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 24Proft M. Struhl K. Mol. Cell. 2002; 9: 1307-1317Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). This is possible because the Cti6 protein interacts simultaneously with the Ssn6-Tup1 and SAGA complexes, and mediates SAGA and TBP recruitment, histone acetylation, and transcriptional activation (17Papamichos-Chronakis M. Petrakis T. Ktistaki E. Topalidou I. Tzamarias D. Mol. Cell. 2002; 9: 1297-1305Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Interestingly, recent studies in S. cerevisiae and Schizosaccharomyces pombe have also shown that the Tup1 complex (Tup11/Tup12 in S. pombe) modulates the expression of iron-regulated genes by associating with the Aft1 (Fep1 in S. pombe) transcription factor (25Znaidi S. Pelletier B. Mukai Y. Labbe S. J. Biol. Chem. 2004; 279: 9462-9474Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 26Fragiadakis G.S. Tzamarias D. Alexandraki D. EMBO J. 2004; 23: 333-342Crossref PubMed Scopus (34) Google Scholar).Here we demonstrate that the CTI6 gene is required for growth under iron-limiting conditions and for normal regulation of silencing. We demonstrate that CTI6 is localized to the nucleus, associated with the Rpd3-Sin3 HDAC complex, and exhibits Rpd3 histone deacetylase-dependent transcriptional repression. Cti6 protein contains a PHD finger domain that is essential for growth under low iron and regulation of telomeric silencing, but not for the transcriptional repression activity. Finally, microarray experiments suggest that Cti6 may act as a repressor under low iron conditions in concert with the Ssn6-Tup1 corepressor. We discuss a potential role for Cti6, the Rpd3-Sin3 HDAC and the Ssn6-Tup1 complexes in cellular adaptive responses for growth under iron-limiting conditions.EXPERIMENTAL PROCEDURESYeast Strains and Growth Conditions—Genotypes for the yeast strains used in this work are listed in Supplemental Materials Table S1. To test growth on low iron, cells were grown in synthetic media (SC) to exponential phase (A600 = 1.0) and spotted in 10-fold serial dilutions starting at A600 = 0.1 onto SC alone (complete) or SC containing 75–100 μm BPS and 75–100 μm BCS (low iron/copper), or 1 mm ferrozine (not shown). For silencing assays, cells were spotted in 5-fold serial dilutions starting at A600 = 1.0 onto SC (complete), SC lacking specific requirements (SC minus), or SC containing 1 g/liter 5-fluoroorotic acid (FOA).Plasmids—The wild type CTI6 gene and C95A, H100A, and C95A,H100A mutant alleles were amplified by PCR with Pfu Turbo DNA polymerase (Stratagene), cloned into pRS416 (CEN, URA3) and pRS415 (CEN, LEU2) vectors using SmaI and XhoI restriction sites, and sequenced. Wild type and mutant alleles of the CTI6 gene were cloned in phase into pBTM116 (pADH-LexA) plasmid, a gift from Ann Vojtek (University of Michigan), using SmaI and PstI restriction sites. The HDA1 gene was cloned into the pBTM116 plasmid using SmaI and SalI restriction sites. Site-directed mutagenesis of the CTI6 gene was performed by the overlap extension method (27Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). M1835 (pADH-lexA) and M1836 (pADH-SIN3-LexA) plasmids were gifts from David Stillman (University of Utah), pJH330 (INO1-LacZ) from John Lopes (Wayne State University), and p6HA (STE6-CYC1-LacZ) from Ira Herskowitz (University of California, San Francisco).DNA Microarray and RNA Blot Analysis—For microarray experiments wild type BY4741 and cti6 mutant cells were grown to exponential phase in liquid SC medium containing 150 μm BPS and 150 μm BCS. Total RNA was isolated with a modified hot phenol method (28Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3699) Google Scholar). RNA was further purified with a Qiagen RNeasy kit according to the manufacturer's instructions. The quality of the RNA samples was evaluated with an Agilent Bioanalyzer. Approximately 10 μg of total RNA from wild type and cti6 cells was labeled with Cy3 and Cy5 fluorescent dyes, respectively. Cy-labeled RNA samples were hybridized with an Operon oligonucleotide yeast array. Data acquisition was performed using a GenePix Pro 4000A laser scanner (Axon Instruments). Only spots that had median values 2 times background were considered. For further information about preparation of the slides for microarrays, synthesis of fluorescent-labeled cDNA, hybridization, scanning and data acquisition, and quality control steps visit the Duke Microarray Core Facility web site. For RNA blot analysis, PCR-amplified fragments were radiolabeled with 32P and used as probes.β-Galactosidase Assays—Cells were grown in selective media to exponential phase. For INO1-LacZ assays 1 mm choline and 0.75 mm inositol were added to the medium. Cells were harvested and β-galactosidase activity was measured in permeabilized cells as previously described (29Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).Fluorescence Microscopy—For Cti6 subcellular localization, wild type and mutant proteins were tagged with green fluorescent protein (GFP) at the carboxyl terminus as previously described (30Pena M.M. Puig S. Thiele D.J. J. Biol. Chem. 2000; 275: 33244-33251Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Cells were grown in selective media to exponential phase, and fluorescence visualized, photographed, and image processed as previously described (27Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). For staining of yeast nuclei cells were incubated for 15 min with 10 μg/ml 4′,6-diamidino-2-phenylindole.RESULTScti6 Mutants Exhibit a Growth Defect under Iron-limiting Conditions—The acquisition of iron through the Fet3 multicopper ferroxidase is a copper-dependent process, and mutations in the copper uptake pathway (ctr1) or in the delivery of copper to the secretory compartment (atx1 or ccc2) render cells defective in high affinity iron uptake, which can be corrected by increased exogenous copper levels. We screened a haploid yeast deletion library to ascertain if mutations in other loci conferred growth defects under iron-limited conditions that are rescued by extracellular iron and copper addition. One mutant, cti6, showed a dramatic growth defect under iron scarcity achieved by addition of the extracellular Fe(II) chelator BPS plus the Cu(I) chelator BCS (low iron/copper, Fig. 1, A and B). cti6 growth defects were also observed by using the intracellular Fe(II) chelator ferrozine or BPS alone (data not shown), but because addition of BCS further decreases the availability of iron, BPS and BCS were used for further studies. Under these conditions cti6 mutants show a growth defect similar to ctr1, atx1, and ccc2 mutants (Fig. 1A). The growth defect of the cti6 mutant was fully complemented by the addition of low concentrations of iron. Only 10 μm iron suppressed the growth defect of the atx1 and cti6 mutants, and up to 25 μm iron was required for growth recovery of ctr1 and ccc2 cells (Fig. 1A). Addition of copper also complemented the cti6 growth defect (Fig. 1A). Addition of 5 μm copper stimulated growth of the atx1 and cti6 mutants. Higher copper concentrations robustly suppressed the growth defect of cti6 and ctr1 (25 μm), and ccc2 (50 μm) mutants (Fig. 1A). cti6 growth defect in low iron was also rescued by reintroducing the wild type or an allele in which the Cti6 carboxyl terminus was tagged with GFP in a centromeric plasmid under the control of CTI6 wild type promoter (Fig. 1B). Taken together, these results demonstrate that cti6 mutants are defective for growth under conditions of iron scarcity and are rescued by addition of extracellular iron and copper.CTI6 mRNA Levels Are Increased under Low Iron Conditions—Because the CTI6 gene is required for growth under iron-limited conditions, we ascertained whether CTI6 mRNA levels are regulated by iron availability. RNA blotting was performed using wild type cells, cells defective in the iron-sensing transcription factor Aft1 (aft1), and cells lacking both Aft1 and Aft2 (aft1 aft2). Cells were grown to exponential phase under iron-replete (+100 μm iron) and iron-limited (+100 μm BPS) conditions. The multicopper ferroxidase gene FET3, whose expression is induced under low iron conditions by the Aft1 transcription factor, was used as a control for iron-regulated gene expression. As previously reported (31Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (313) Google Scholar), FET3 mRNA levels increased under low iron conditions, and this induction was dependent on the Aft1 transcription factor (Fig. 1, C and D). However, the levels of CTI6 mRNA were only slightly increased in wild type cells grown under iron limitation (Fig. 1, C and D). This CTI6 basal expression was absent in a cti6 deletion mutant (data not shown). CTI6 mRNA levels under low iron conditions were considerably increased in both the aft1 and aft1 aft2 mutants, which sense a much more severe iron starvation because of the loss of expression of genes involved in iron uptake (Fig. 1, C and D). Unlike FET3, the CTI6 promoter lacks Aft1-Aft2-binding sites, consistent with our observation that Aft1 and Aft2 do not mediate the elevation in CTI6 steady state mRNA levels in response to iron deprivation. Interestingly, increased CTI6 (YPL181w) mRNA levels have also been reported previously in mutants defective in frataxin (Yfh1), a mitochondrial matrix protein required for iron-sulfur synthesis and export from mitochondria (32Foury F. Talibi D. J. Biol. Chem. 2001; 276: 7762-7768Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Both aft1 and yfh1 mutant cells grown under low iron conditions show an increase in iron deficiency compared with wild type cells because of a defect in iron uptake and mobilization. Taken together, these results indicate that CTI6 mRNA steady state levels increase during conditions of iron scarcity in a manner that is independent of the Aft1/2 iron-responsive transcription factors.The Cti6 PHD Finger Is Important for Growth under Iron-limiting Conditions—Cti6 is a PHD finger-containing protein. PHD fingers are zinc finger-like motifs of ∼60 amino acids defined by seven cysteines and one histidine arranged as C4HC3, with non-conserved intervening sequences. The PHD finger has been found in many eukaryotic proteins including transcription factors and proteins involved in chromatin-mediated transcriptional regulation (reviewed in Ref. 33Aasland R. Gibson T.J. Stewart A.F. Trends Biochem. Sci. 1995; 20: 56-59Abstract Full Text PDF PubMed Scopus (747) Google Scholar). The structure of the PHD finger of the KAP-1 corepressor has been solved recently showing that it binds two zinc atoms in a cross-brace RING-like arrangement (34Capili A.D. Schultz D.C. Rauscher I.F. Borden K.L. EMBO J. 2001; 20: 165-177Crossref PubMed Scopus (162) Google Scholar). According to this model, Cti6 protein could coordinate one zinc atom to cysteine residues 75, 77, and 103, and histidine residue 100 (site I), whereas the second zinc atom would coordinate to cysteine residues 92, 95, 117, and 120 (site II). To ascertain whether the PHD finger in the Cti6 protein is important for growth under conditions of iron deprivation, cysteine residue 95, a putative metal ligand for site II, and histidine residue 100, a predicted zinc ligand for site I, were converted to alanine by site-directed mutagenesis (Fig. 2A). Both single mutants were unable to complement the cti6 growth defect under iron-limiting conditions (Fig. 2B). The double mutant C95A,H100A did not show any additional growth defect (Fig. 2B). Cti6 PHD finger mutants epitope-tagged with GFP were expressed at similar levels as wild type Cti6 and were properly localized (Fig. 3 and data not shown). These results strongly suggest that the integrity of the Cti6 PHD finger is important for growth under conditions of iron scarcity.Fig. 2The Cti6 PHD finger is required for growth under iron-limiting conditions. A, schematic representation of the PHD finger in Cti6 protein. Cysteine 95 and histidine 100 residues (white characters) where mutagenized to alanine. B, BY4741 wild type (WT), and cti6 cells transformed with pRS416 (vector), pRS416-CTI6, pRS416-CTI6-C95A, pRS416-CTI6-H100A, and pRS416-CTI6-C95A,H100A were assayed for growth onto SC (complete) and SC containing 100 μm BPS and 100 μm BCS media (Low Fe/Cu).View Large Image Figure ViewerDownload (PPT)Fig. 3Cti6 is a nuclear protein. cti6 cells expressing pRS416-Cti6-GFP or pRS416-Cti6-C95A,H100A-GFP were grown in SC-ura to exponential phase, stained with 4′,6-diamidino-2-phenylindole (DAPI), and visualized by fluorescence microscopy. The same nuclear localization for both wild type and mutant was observed when cells were grown in 100 μm Fe(NH4)2(SO4)2, 100 μm CuSO4, 100 μm BPS or 100 μm BCS containing media (data not shown). DIC, differential interference microscopy.View Large Image Figure ViewerDownload (PPT)To ascertain the specificity of the iron limitation growth defect observed for cti6 mutants we analyzed the growth rates, under iron-limited conditions, for the 15 additional mutants corresponding to genes in the S. cerevisiae genome coding for proteins containing PHD fingers. Of these, only cells harboring an insertional inactivation of the PHO23 gene also showed a considerable growth defect under iron scarcity (Supplemental Materials Fig. S1). Interestingly, Pho23 has been recently shown to be a component of the Rpd3-Sin3 HDAC required for regulation of gene expression and silencing (35Loewith R. Smith J.S. Meijer M. Williams T.J. Bachman N. Boeke J.D. Young D. J. Biol. Chem. 2001; 276: 24068-24074Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).Cti6 Is a Nuclear Protein—The molecular function of the PHD finger motif has not been elucidated but, in analogy with LIM (C2HC5) and RING (C3HC4) domains, it has been suggested that PHD fingers could be involved in protein-protein interactions. Proteins containing PHD, LIM, or RING domains are often involved in transcriptional control either directly, or by recruiting co-repressors or co-activators (33Aasland R. Gibson T.J. Stewart A.F. Trends Biochem. Sci. 1995; 20: 56-59Abstract Full Text PDF PubMed Scopus (747) Google Scholar). To obtain further insight into the function of Cti6 we epitope-tagged the protein at the carboxyl terminus with the GFP to determine its subcellular localization. Expression of the Cti6-GFP fusion protein under the control of the CTI6 promoter fully rescued the growth defect observed for a cti6 mutant strain under low iron conditions (Fig. 1C). Fluorescence microcopy of these cells showed a Cti6-GFP fluorescence signal that overlapped with the nuclear stain 4′,6-diamidino-2-phenylindole (Fig. 3, top panels). Mutagenesis of amino acid residues 95 and 100 (C95A,H100A mutant, Fig. 3, bottom panels), or growth under different concentrations of iron or copper availability (not shown) did not affect Cti6-GFP nuclear localization or protein levels. These results demonstrate that Cti6 is a nuclear protein, and strongly suggest that the integrity of the PHD finger, whereas essential for its role in cell growth under conditions of iron deprivation, is not required for its targeting to the nucleus.A LexA-Cti6 Fusion Protein Mediates Rpd3-dependent Transcriptional Repression—Two independent reports have described large scale approaches to analyze multiprotein complexes in S. cerevisiae via the use of tandem affinity purification or FLAG epitopes (36Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3974) Google Scholar, 37Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. Donaldson I. Schandorff S. Shewnarane J. Vo M. Taggart J. Goudreault M. Muskat B. Alfarano C. Dewar D. Lin Z. Michalickova K. Willems A.R. Sassi H. Nielsen P.A. Rasmussen K.J. Andersen J.R. Johansen L.E. Hansen L.H. Jespersen H. Podtelejnikov A. Nielsen E. Crawford J. Poulsen V. Sorensen B.D. Matthiesen J. Hendrickson R.C. Gleeson F. Pawson T. Moran M.F. Durocher D. Mann M. Hogue C.W. Figeys D. Tyers M. Nature. 2002; 415: 180-183Crossref PubMed Scopus (3054)" @default.
• W1969074879 created "2016-06-24" @default.
• W1969074879 creator A5009177870 @default.
• W1969074879 creator A5047741580 @default.
• W1969074879 creator A5062686245 @default.
• W1969074879 date "2004-07-01" @default.
• W1969074879 modified "2023-10-15" @default.
• W1969074879 title "Cti6 Is an Rpd3-Sin3 Histone Deacetylase-associated Protein Required for Growth under Iron-limiting Conditions in Saccharomyces cerevisiae" @default.
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