Nickel Resistance and Chromatin Condensation inSaccharomyces cerevisiae Expressing a Maize High Mobility Group I/Y Protein (2001)

Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions. HMG I/Y proteins may act as architectural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei. Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast Δrpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA. Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions. HMG I/Y proteins may act as architectural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei. Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast Δrpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA. high mobility group kilobase pair(s) untranslated region green fluorescent protein phosphate-buffered saline 5-fluoroorotic acid high pressure liquid chromatography Numerous metals are essential for living organisms, but they can be toxic when present in excess. A variety of mechanisms, including promotion of oxidative stress and genotoxicity, have been implicated in this toxicity process, and various defense mechanisms have evolved to protect cellular integrity (1Briat J.-F. Lebrun M. C. R. Acad. Sci. III. 1999; 322: 43-54Crossref PubMed Scopus (191) Google Scholar). Nickel has been reported to be an essential cofactor for urease in plants (2Eskew A.W. Welch R.M. Carey E.E. Science. 1983; 222: 621-623Crossref PubMed Scopus (245) Google Scholar). Other nickel-binding proteins required for urease activity have recently been reported in soybean (3Freyermuth S.K. Bacanamwo M. Polacco J.C. Plant J. 2000; 21: 53-60Crossref PubMed Google Scholar). However, at high concentrations, nickel ions are very toxic to many organisms, including plants, microorganisms, and mammals. Toxicity due to excess of nickel has been studied in animal systems. Nickel has been described as an allergen and a potent human and rodent carcinogen that induces human respiratory cancers (4Sunderman F.W.J. IARC Monogr. Eval. Carcinog. Risks Hum. 1984; 53: 127-142Google Scholar). This metal can induce oxidative damages through the production of reactive oxygen species in animal cells, leading to lipid peroxidation, chromosomal deletions, and cross-linking of proteins to DNA (5Costa M. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 321-337Crossref PubMed Google Scholar). However, nickel compounds selectively damage heterochromatic regions, and the major cause of nickel-induced carcinogenicity has been explained by an epigenetic mechanism (6Costa M. Am. J. Clin. Nutr. 1995; 61: 666S-669SCrossref PubMed Scopus (32) Google Scholar). Nickel compounds would induce an increase in chromatin condensation and methylation, causing neighboring genes, including potential antioncogenes, to be silenced. In Chinese hamster cells, nickel was shown to silence a gpt reporter gene inserted near a dense heterochromatic region susceptible to heterochromatin spreading and silencing (7Lee Y.W. Klein C.B. Kargacin B. Salnikow K. Kitahara J. Dowjat K. Zhitkovich A. Christie N.T. Costa M. Mol. Cell. Biol. 1995; 15: 2547-2557Crossref PubMed Scopus (334) Google Scholar). This mechanism seems to be conserved in Saccharomyces cerevisiae, in which Brodayet al. (8Broday L. Cai J. Costa M. Mutat. Res. 1999; 440: 121-130Crossref PubMed Scopus (39) Google Scholar) have shown that nickel can induce silencing of a reporter gene inserted in a telomeric region, involving DNA condensation, independent of methylation. During the last century, soils became enriched with heavy metals as a result of increasing industrial activities (9Daily G.C. Science. 1995; 269: 350-353Crossref PubMed Scopus (182) Google Scholar). Transfer of heavy metals from the soil to the plant may cause both phytotoxic symptoms and the potential accumulation of toxic metals in the edible part of crops. Nickel contamination arose from mining, metal refineries, smelting, sewage sludge, combustion of fuel fossils, and agricultural activities (10Barceloux D.G. J. Toxicol. Clin. Toxicol. 1999; 37: 239-258Crossref PubMed Scopus (257) Google Scholar). In plants, nickel ions may compete with the uptake of other cations such as Ca2+, Mg2+, Fe2+, and Zn2+ and induce Zn2+ or Fe2+ deficiencies that lead to characteristic plant chlorosis symptoms affecting the photosynthetic activity (11Marschner H. Mineral Nutrition of Higher Plants. 2nd Ed. Academic Press Ltd., London1995Google Scholar). Mechanisms of nickel resistance in the plant kingdom have been particularly studied using nickel hyperaccumulators, which are plants adapted to nickel-rich soils that accumulate high concentrations of nickel in the shoots without showing any toxicity symptoms. InAlyssum bertolonii, Krämer et al. (12Krämer U. Cotter-Howells J.D. Charnock J.M. Baker A.J.M. Smith J.A. Nature. 1996; 379: 635-638Crossref Scopus (705) Google Scholar) have shown that increasing free histidine in the xylem sap enhances translocation of nickel to the shoots, which could explain the metal hyperaccumulating phenotype of this plant. These authors suggested that free histidine may be involved in chelating nickel during the xylem transport. In the aerial parts of plants, vacuolar sequestration of nickel is thought to be one of the major mechanism of resistance, although the precise mechanism of metal uptake in the vacuoles has not yet been described in detail. However, very little is known concerning the molecular mechanisms that are involved in the resistance of plants to increasing concentrations of nickel. In a search for plant genes conferring resistance to nickel toxicity, we expressed a maize root cDNA library in S. cerevisiaeand screened for yeast transformants resistant to nickel toxicity. One of the isolated maize cDNAs encoded an HMG1 I/Y protein whose expression in yeast conferred a specific resistance to nickel, likely by interfering with the nickel-induced DNA condensation. The S. cerevisiae strains used in this study were as follows: F113 (Mat a, can1, inol-13, ura3-52); UCC1001, a derivative of the YPH250 strain (Mat a,ura3-52, ade2-101ochre , trp1-Δ1, his3-Δ200, leu2-Δ1, lys2-801ochre ,TELadh4::URA3) with the URA3gene inserted 1.3 kbp from the telomere on chromosome VII (13Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1124) Google Scholar); YDS21U, a derivative of the YDS2 strain (Mat a , ade2-1, can1-100, his3-11,15 leu2-3,112 trp1-1, URA3::TELVR) with the URA3 gene inserted 2.1 kbp from the telomere on chromosome V (14Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (514) Google Scholar); and SRG39 (Δrpd3), isogenic to YDS21U, but withrpd3::LEU2. Yeast strains were grown at 30 °C in 1% yeast extract, 2% peptone, and 2% dextrose or in synthetic medium with dextrose (SD medium; 0.17% yeast nitrogen base without amino acid and ammonium sulfate (Difco), 2% dextrose, and 0.5% ammonium sulfate) supplemented with the required amino acids. To assay metal resistance, cultures were grown in liquid SD medium for 24 h to saturation, and serial dilutions were then spotted onto agar minimum medium (15Campbell I. Campbell I. Duffus J.H. Yeast: A practical approach. IRL Press, Oxford, England1988: 277-279Google Scholar) with some modifications (0.5% ammonium sulfate as nitrogen source and Eagle basel medium vitamins from Life Technologies, Inc.) and containing 0.5 mm NiSO4 or different metals at concentrations inhibiting yeast cell growth. The F113 yeast strain was transformed by the lithium acetate method (16Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (361) Google Scholar) with a cDNA library from maize roots (17Loulergue C. Lebrun M. Briat J.-F. Gene ( Amst. PubMed Scopus Google Scholar) in the high expression vector Yeast. PubMed Scopus Google Scholar). transformants isolated on medium were and at °C in transformants were then screened for nickel resistance on agar minimum medium containing 0.5 mm of growth at 30 the to on this toxic medium were and were isolated from independent resistant yeast transformants and strain J. A 2nd Ed. Scholar). conferred nickel resistance to The were by the was with DNA and by to the at the were by The ZmHMG I/Y2cDNA was the vector M. L. Yeast. 1991; 7: PubMed Scopus Google Scholar) and the vector M. Plant J. Google in which we the URA3 gene with the to this in Yeast was from cell cultures grown in SD medium to using as described by et al. J. A 2nd Ed. Scholar). was as described by and Briat S. Briat J.-F. Plant 1999; 37: Scopus Google Scholar). of were were with I/Y2 of the cDNA and a by B. to was at °C in and were with and at The was then at °C on were from yeast cultures grown to in SD Yeast cells were in acid and then with The was in liquid in of and by the of and for The was in Nature. PubMed Scopus Google and for The were and the was were on a and onto The ZmHMG I/Y2 protein was using a by H. of Plant and of J. A. Plant Cell. Google Scholar) at a of and the was using to at a of was with the A ZmHMG I/Y2 protein was by the DNA in with the region of the ZmHMG I/Y2 cDNA at the in the The DNA was by using and from the by B. a the DNA was of the the at Yeast cells transformed with the were grown in SD medium to and then by Yeast were with mm and and then with for at Yeast cells were with and then with at a of for Yeast cells were and in of were with a with a and to a was using a and a was using a and a and the cells were by were using were grown for 24 h in SD medium containing various NiSO4 concentrations. Yeast cells were by and with mm and then were in of acid at °C for the were with and in a Yeast cells were with an The was by Yeast cells were grown to and in for were with of mm and with A for h at °C and for h at Yeast cells were with and were then and in of containing was using a To the of histidine to the leading to nickel resistance of transformed yeast cells, the UCC1001, YDS21U, and SRG39 yeast strains were transformed with a The gene was by using and from the to strains for The and strains were grown in medium for silencing of the URA3 The cells were then at in medium containing various nickel concentrations and grown for h at 30 of serial dilutions were spotted onto SD with or without was of at 30 To the of cells were onto SD medium with or without and of growth at 30 To plant genes conferring nickel resistance to we transformed the F113 yeast strain with a maize root cDNA The resistant transformants were on a minimum medium containing 0.5 an for yeast growth and I/Y in A. the cDNAs isolated from this conferred nickel resistance to yeast of the cDNAs with cDNAs encoding HMG proteins One of was ZmHMG I/Y2 and was of the wild-type yeast strain F113 with the ZmHMG I/Y2 cDNA a high as as a conferred growth on 0.5 mm In when in a high of the the maize cDNA not nickel resistance to the transformed yeast cells, that of the cDNA was required for resistance I/Y in Nickel resistance of yeast cells transformed with the ZmHMG I/Y2 cDNA was shown to be and was observed with to mm NiSO4 in the medium of the phenotype was by yeast cells transformed with the ZmHMG I/Y2 cDNA on medium supplemented with concentrations of various A resistance to and resistance to and were observed of I/Y2 cDNA an of amino encoding a protein with a molecular of This is by a of base and by a of base The amino acid with plant proteins of the HMG I/Y The region is to the of and four AT hook motifs were observed the motifs the protein to with plant containing AT-rich Plant J. 1995; 7: PubMed Scopus Google Scholar). The ZmHMG I/Y2 protein with a reported maize HMG I/Y Gene ( Amst. 1999; PubMed Scopus Google Scholar). However, the proteins are likely to be encoded by different genes the and of the ZmHMG I/Y2 cDNA are not conserved and in the of the reported maize HMG I/Y cDNA. A high of of the ZmHMG I/Y2 protein was observed with other plant HMG I/Y proteins to from J. A. Plant Cell. Google to from J. A. Res. PubMed Scopus Google and HMG I/Y R. J.C. Plant Mol. Biol. PubMed Scopus Google Scholar). and of or protein the presence of the ZmHMG I/Y2 and protein in cells transformed with the ZmHMG I/Y2 but not in cells transformed with the empty vector or with the ZmHMG I/Y2 cDNA in the vector and of in were by the with a The of the and of the were with the of the isolated cDNA and of the HMG I/Y proteins are to the they to DNA and may act as M. R. Res. Mol. Biol. 1996; PubMed Google Scholar). the mechanisms by which ZmHMG I/Y2 is to nickel resistance to yeast cells, was to The part of the ZmHMG I/Y2 protein was in to the and expression of the protein was in living yeast Expression of the ZmHMG in yeast conferred nickel resistance to the yeast cells not The activity of the fusion protein with a the vector containing the the of the without any ZmHMG I/Y2 cDNA was expressed in Expression of this in yeast cells a spreading of the the cell To ZmHMG I/Y2 could nickel resistance to yeast cells, we that this protein could the nickel of cells either by or nickel accumulation by sequestration of nickel on a acid of a yeast protein expressing the ZmHMG I/Y2 protein in the of the protein in the a between the ZmHMG I/Y2 protein and nickel not Furthermore, expression of I/Y2 cDNA in yeast cells grown at various nickel concentrations not lead to a in nickel concentrations with cells transformed with a empty These that ZmHMG nickel resistance of yeast cells is not related to in nickel mechanisms have been suggested for resistance to nickel toxicity and an increase in free which is an of nickel, and nickel in the K. K. J. PubMed Google J. 1999; PubMed Google Scholar). in not the that the ZmHMG I/Y2 protein could histidine nickel to the without affecting the nickel content of the To this we measured the of ZmHMG I/Y2 on free histidine and on the expression of various genes involved in histidine or and in nickel the a free histidine in ZmHMG yeast cells with cells not that of gene encoding a for histidine gene encoding a of a vacuolar to a nickel and gene encoding a high histidine K. K. J. PubMed Google J. 1999; PubMed Google Scholar) was in yeast cells expressing ZmHMG I/Y2 with cells transformed with the empty not In the of toxicity, et J. A. R.D. J. PubMed Scopus Google Scholar) have shown that yeast cells grown toxic have cell in To nickel could the cell which could be expression of I/Y2 yeast cells were grown in various nickel concentrations and by cell major in the through the cell were observed in to nickel a in the of yeast cells to was at or in the Expression of the ZmHMG I/Y2 cDNA in yeast cells restored the of nickel. However, this of nickel on the through the cell is to explain the of the Nickel toxicity has been reported to be to chromatin condensation in leading to increased gene silencing at the telomere (8Broday L. Cai J. Costa M. Mutat. Res. 1999; 440: 121-130Crossref PubMed Scopus (39) Google Scholar). expressed in the ZmHMG I/Y2 cDNA in the this of protein is known to AT-rich with M. R. Res. Mol. Biol. 1996; PubMed Google Scholar). that the ZmHMG I/Y2 protein could nickel resistance to yeast cells by interfering with nickel-induced chromatin condensation. this by using yeast strains in which gene was inserted to the telomere on chromosome VII in or on chromosome V in Expression of gene cell growth on a medium containing the This enables the due to telomeric silencing on chromatin structure (13Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1124) Google Scholar). then silencing at could be by ZmHMG I/Y2 in either the or presence of various nickel concentrations Expression of the ZmHMG I/Y2 cDNA to and in resistance in the of nickel when URA3 was inserted on chromosome VII or on chromosome These that the ZmHMG I/Y2 protein chromatin condensation in leading to telomeric silencing. with either or NiSO4 of yeast cells expressing the empty with URA3 inserted on chromosome to a increase in resistance, but nickel silencing in yeast cells expressing the ZmHMG I/Y2 cDNA. URA3 was inserted on chromosome increase in resistance to nickel was observed in cells expressing the empty However, with and NiSO4 of yeast cells expressing the ZmHMG I/Y2 cDNA to a in resistance from to and is to that cells from the and strains transformed with the ZmHMG I/Y2 cDNA were resistant to 0.5 mm NiSO4 not can be from that gene silencing at the telomere can be increased by ZmHMG I/Y2 and that nickel, at in some this silencing. Therefore, can be that nickel resistance by the ZmHMG I/Y2 protein could be to chromatin structure modification, interfering with the of nickel on DNA condensation. The method used the of silencing to a specific region of the the To the between chromatin condensation and nickel resistance in yeast cells expressing the ZmHMG I/Y2 we used a yeast strain a of the gene that was reported to chromatin in an structure (14Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (514) Google Scholar). The S. was to nickel with the and growth was by mm NiSO4 Expression of I/Y2 cDNA in the yeast Δrpd3 restored nickel in to with the empty vector The level of nickel resistance was observed between the wild-type strain and the yeast Δrpd3 mutant. These the that expression of the ZmHMG I/Y2 cDNA in yeast DNA condensation and nickel resistance to yeast In this we isolated a maize cDNA encoding a ZmHMG I/Y2 protein that nickel resistance when expressed in This protein was by expressing a maize cDNA library cerevisiae and for growth of transformed cells on a medium containing 0.5 mm Expression of the ZmHMG I/Y2 cDNA in yeast cells to at to mm and the conferred resistance was specific to nickel. However, in the nickel content of transformed yeast cells were This to that the ZmHMG I/Y2 protein could nickel toxicity. One for such a resistance could be that the binding of the ZmHMG I/Y2 protein to chromatin would have specific on gene in on genes encoding proteins involved in histidine and in nickel the K. K. J. PubMed Google J. 1999; PubMed Google Scholar). However, in histidine content and in and were between ZmHMG cells and cells not is I/Y2 expression in yeast of genes involved in nickel through histidine nickel the However, at this be that expression of other genes could be through ZmHMG I/Y2 binding to the of which is for nickel Nickel compounds oxidative damage in cells in the heterochromatic of M. S. Klein C.B. Salnikow K. Sci. PubMed Scopus Google Scholar). This is achieved through the of nickel with cellular such as specific of 1999; PubMed Scopus Google Scholar) or L. Salnikow K. M. Costa M. Res. 2000; Google Scholar). In animal cells, nickel is a carcinogen gene expression through DNA and methylation, for to the silencing of genes (7Lee Y.W. Klein C.B. Kargacin B. Salnikow K. Kitahara J. Dowjat K. Zhitkovich A. Christie N.T. Costa M. Mol. Cell. Biol. 1995; 15: 2547-2557Crossref PubMed Scopus (334) Google Scholar). the other HMG I/Y proteins are to as architectural modifying chromatin structure M. R. Res. Mol. Biol. 1996; PubMed Google Scholar). These proteins four AT hook motifs that binding to the minor groove of AT-rich DNA with The maize HMG I/Y protein is in the of transformed yeast cells leading to that nickel resistance could be due to chromatin structure by this plant In S. cerevisiae, nickel compounds have been reported to induce URA3 gene silencing at the telomere and to result in a epigenetic (8Broday L. Cai J. Costa M. Mutat. Res. 1999; 440: 121-130Crossref PubMed Scopus (39) Google Scholar). The level of silencing was the of the reporter gene from the In with this we observed silencing at by nickel ions when the URA3 gene was inserted 1.3 kbp from the telomere on chromosome but not when the URA3 gene was 2.1 kbp on chromosome V Expression of I/Y2 cDNA in yeast increased silencing at the level as nickel but was not the of the URA3 gene from the telomere The ZmHMG I/Y2 protein URA3 gene silencing at the as nickel, but without to growth. This may a for this protein in the of nickel binding to the URA3 gene was inserted 2.1 kbp from the telomere on chromosome silencing in to expression of the ZmHMG I/Y2 cDNA was in the presence of nickel an of compounds in the of telomeric silencing. Furthermore, of the of nickel and ZmHMG I/Y2 was in a yeast strain a of the gene This strain is by increased at and of that with increased activity (14Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (514) Google but an increased silencing at telomeric In this the increased nickel sensitivity observed be with increased spreading of However, nickel of the Δrpd3 strain not increase silencing of the URA3 gene at the telomere not of the high nickel sensitivity of the could a chromatin structure to nickel ions due to increased Expression of I/Y2 cDNA in the yeast Δrpd3 restored a wild-type level of nickel sensitivity This result is in with a of ZmHMG I/Y2 in chromatin condensation restricting nickel accessibility to DNA. HMG I/Y as as a synthetic AT hook to PubMed Scopus Google Scholar). a proteins such as are in chromatin accessibility K. J. PubMed Scopus (257) Google R. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). plant HMG I/Y proteins an region to the Plant J. 1995; 7: PubMed Scopus Google Scholar). is to that this be involved in the of plant HMG I/Y proteins with chromatin J. J. 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