Nickel Resistance and Chromatin Condensation in Saccharomyces cerevisiae Expressing a Maize High Mobility Group I/Y Protein*

Céline Forzani, Clarisse Loulergue, Stéphane Lobréaux, Jean-François Briat, and Michel LebrunDagger

From Biochimie et Physiologie Moléculaire des Plantes, CNRS Unité Mixte de Recherche 5004, Université Montpellier 2, Institut National de la Recherche Agronomique, Ecole Nationale Supérieure d'Agronomie, 2 place Viala, F-34060 Montpellier Cedex 1, France

Received for publication, August 16, 2000, and in revised form, January 22, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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/Y2 cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast Delta rpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1). Nickel has been reported to be an essential cofactor for urease in plants (2). Other nickel-binding proteins required for urease activity have recently been reported in soybean (3). 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 (4). 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 (5). However, nickel compounds selectively damage heterochromatic regions, and the major cause of nickel-induced carcinogenicity has been explained by an epigenetic mechanism (6). 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 (7). This mechanism seems to be conserved in Saccharomyces cerevisiae, in which Broday et al. (8) 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 (9). 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 (10). 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 (11). 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. In Alyssum bertolonii, Krämer et al. (12) 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. cerevisiae and 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains and Growth Conditions-- The S. cerevisiae strains used in this study were as follows: F113 (Mata, can1, inol-13, ura3-52); UCC1001, a derivative of the YPH250 strain (Mata, ura3-52, ade2-101ochre, trp1-Delta 1, his3-Delta 200, leu2-Delta 1, lys2-801ochre, TELadh4::URA3) with the URA3 gene inserted 1.3 kbp from the telomere on chromosome VII (13); YDS21U, a derivative of the YDS2 strain (Mata, 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 (14); and SRG39 (Delta rpd3), isogenic to YDS21U, but with rpd3::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 (15) 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.

cDNA Cloning and Sequencing-- The F113 yeast strain was transformed by the lithium acetate method (16) with a cDNA library from maize roots (17) constructed in the high copy number expression vector pYPGE15 (18). 106 transformants isolated on SD-Ura medium were pooled and stored at -80 °C in 15% glycerol. 250,000 transformants were then screened for nickel resistance on agar minimum medium containing 0.5 mM NiSO4. After 5 days of growth at 30 °C, the colonies able to grow on this toxic medium were selected and further analyzed. Plasmids were isolated from 98 independent resistant yeast transformants and amplified in Escherichia coli strain JM109 (endA1, recA1, gyrA96, thi, hsdR17 (rk-, mk+), relA1, supE44, Delta (lac-proAB), (F', traD36, proAB, laclqZDelta M15)) (19). Upon retransformation, seven plasmids still conferred nickel resistance to yeast. The inserts were sequenced by the dideoxynucleotide chain termination method. Sequence computer analysis was performed with DNA Strider software and by connecting to the BLAST network service at the NCBI Protein Database. Protein sequence alignments were generated by ClustalW software. The ZmHMG I/Y2 cDNA was subcloned into the centromeric vector pFL38 (20) and into the multicopy number vector pFL61 (21), in which we replaced the URA3 gene with the selective marker TRP1 to express this plasmid in non-URA3 auxotrophic strains.

Northern and Western Analyses of ZmHMG I/Y2-- Yeast RNA was extracted from cell cultures grown in SD medium to mid-log phase using glass beads as described by Sambrook et al. (19). Northern blotting was performed as described by Thoiron and Briat (22). 10 µg of RNA were loaded per lane. Blots were hybridized with the ZmHMG I/Y2 3'-UTR probe obtained by PvuII-XhoI digestion of the cDNA and a yeast PAB1 (poly(A)-binding protein-1) probe kindly provided by Dr. B. Lapeyre (Institut de Génétique Moléculaire, CNRS, Montpellier, France) to control RNA loading. Hybridization was performed overnight at 42 °C in 50% formamide, and washes were done with 0.1% SDS and 0.1× SSC at 42 °C. The filter was then autoradiographed at -80 °C on Royal X-Omat film (Eastman Kodak Co.).

Proteins were extracted from yeast cultures grown to mid-log phase in SD medium. Yeast cells were resuspended in 20% trichloroacetic acid (final concentration), harvested, and then washed with 10% trichloroacetic acid. The pellet was frozen in liquid nitrogen, resuspended in 200 µl of 10% trichloroacetic acid, and disrupted by the addition of glass beads and vortexing for 10 min. The precipitate was collected after centrifugation, resuspended in 1× Laemmli buffer (23), and boiled for 10 min. The samples were pelleted, and the supernatant was collected. Proteins (20 µg) were separated on a 15% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membrane (Hybond C+, Amersham Pharmacia Biotech). The ZmHMG I/Y2 protein was detected using a polyclonal antibody raised against rice PF-1, kindly provided by Professor P. H. Quail (Department of Plant and Microbial Biology, University of California, Berkeley, CA) (24) at a dilution of 1:2500, and the Western blot was revealed using anti-mouse IgG coupled to peroxidase (Sigma) at a dilution of 1:100,000. Immunodetection was performed with the Lumi Light detection system (Roche Molecular Biochemicals).

Green Fluorescent Protein (GFP) Localization-- A GFP-tagged ZmHMG I/Y2 protein was constructed by inserting the 0.75-kbp GFP DNA fragment in frame with the N-terminal region of the ZmHMG I/Y2 cDNA at the XbaI site in the pYPGE15 plasmid. The GFP DNA fragment was amplified by polymerase chain reaction using primers 5'-GCTCTAGAATGTCTAAAGGTGAAGAATTATTC-3' and 5'-GCTCTAGACCCGGGTTATTTGTACAATTCAT-3' from the YCplac111-PAB-GFP plasmid provided by Dr. B. Lapeyre. As a control, the 0.75-kbp GFP DNA fragment was subcloned downstream of the phosphoglycerate kinase promoter into the pYPGE15 plasmid at the XbaI site. Yeast cells transformed with the GFP-expressing plasmids were grown in SD medium to mid-log phase and then collected by centrifugation. Yeast pellets were washed with 1× PBS (2.7 mM KCl, 0.15 M NaCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4) and then fixed with 70% EtOH for 20 min at room temperature. Yeast cells were washed again with 1× PBS and then incubated with 4,6-diamidino-2-phenylindole (1 mg/ml) at a dilution of 1:1000 for 12 min. Yeast cells were pelleted and resuspended in 250 µl of 1× PBS. Images were captured with a Leica Model DMR microscope equipped with a ×100 oil objective and coupled to a Hamamatsu CCD camera. GFP was imaged using a 440-470-nm excitation filter and a 520-550-nm emission filter; 4,6-diamidino-2-phenylindole was imaged using a UV channel 340-480 nm excitation filter and a 425-nm emission filter; and the cells were imaged by Nomarski optics. Images were mounted using Adobe Photoshop.

Nickel Content in Yeast-- Cells were grown for 24 h in SD medium containing various NiSO4 concentrations. Yeast cells were harvested by centrifugation and washed with 50 mM EDTA and then H2O. Cell pellets were digested in 500 µl of 6 M nitric acid at 100 °C for 2 h. After digestion, the samples were diluted 10 times with water and flamed in a SpectrAA220 atomic absorption spectrometer. Yeast cells were counted with an electronic particle counter (ZM, Coultronics). The particle size distribution was displayed by Coulter Channelyzer 256 analysis.

Flow Cytometry-- Yeast cells were grown to mid-log phase and fixed in 70% EtOH for 1 h. Cell pellets were washed with 1 ml of 50 mM Tris, pH 7.8, and digested successively with RNase A (0.25 mg/ml) for 1 h at 37 °C and proteinase K (40 µg/ml) for 1 h at 55 °C. Yeast cells were pelleted, washed with 1× PBS, and sonicated. They were then pelleted and resuspended in 500 µl of 1× PBS containing propidium iodide (55 µg/ml). Fluorescence was analyzed using a Becton Dickinson FACScan.

Telomeric Silencing of the URA3 Marker-- To avoid the addition of histidine to the culture medium, leading to modified nickel resistance of transformed yeast cells, the UCC1001, YDS21U, and SRG39 yeast strains were transformed with a HIS3 fragment. The HIS3 gene fragment was amplified by polymerase chain reaction using primers 5'-AGATTGTACTGAGAGTGCAC-3' and 5'-CTGTGCGGTATTTCACACCG-3' from the pRS413 plasmid (Stratagene) to make these strains autotrophic for histidine. The UCC1001 and YDS21U strains were grown overnight in SD-Ura-Trp medium for low silencing of the URA3 gene. The cells were then inoculated at 0.01 A600 nm in SD+Ura-Trp medium containing various nickel concentrations and grown for 40 h at 30 °C. 10 µl of 5-fold serial dilutions were spotted onto SD plates with or without 5-FOA (1 g/liter). Growth was recorded after 3 days of incubation at 30 °C. To calculate the number of 5-FOA-resistant colonies, ~200 cells were spread onto SD medium with or without 5-FOA (1 g/liter) and counted after 3 days of growth at 30 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of a ZmHMG I/Y2 cDNA Conferring Nickel Resistance to S. cerevisiae-- To isolate plant genes conferring nickel resistance to yeast, we transformed the F113 yeast strain with a maize root cDNA library. The resistant transformants were selected on a modified minimum medium containing 0.5 mM NiSO4, an inhibitory concentration for yeast growth (pYPGE15 and Delta HMG I/Y in Fig. 1A. Of the cDNAs isolated from this screen, seven still conferred nickel resistance to yeast after retransformation. Partial sequencing of the seven clones revealed three identical cDNAs sharing identities with cDNAs encoding HMG proteins (see below). One of them was named ZmHMG I/Y2 and was further characterized. Transformation of the wild-type yeast strain F113 with the ZmHMG I/Y2 cDNA cloned into a high copy number plasmid (pYPGE15) as well as into a low copy number plasmid (pFL38) (Fig. 1A) conferred growth on 0.5 mM NiSO4-containing medium. In contrast, when cloned in a high copy number plasmid (pYPGE15) devoid of the phosphoglycerate kinase promoter, the maize cDNA did not confer nickel resistance to the transformed yeast cells, indicating that transcription of the cDNA was required for resistance (Delta HMG I/Y in Fig. 1A). Nickel resistance of yeast cells transformed with the ZmHMG I/Y2 cDNA was shown to be dose-dependent and was observed with up to 0.9 mM NiSO4 in the medium (Fig. 1B). Specificity of the nickel-resistant phenotype was addressed by growing yeast cells transformed with the ZmHMG I/Y2 cDNA on medium supplemented with growth-inhibiting concentrations of various metals. A slight resistance to copper and no resistance to cobalt, cadmium, and zinc were observed (Fig. 1C).


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Fig. 1.   Expression of the ZmHMG I/Y2 cDNA in yeast confers specific resistance to nickel. A, the F113 yeast strain was transformed with two empty plasmids (multicopy number pYPGE15 and centromeric pFL38), with the same plasmids containing the ZmHMG I/Y2 cDNA (pG15-HMG I/Y and pFL38-HMG I/Y), and with the constitutive phosphoglycerate kinase promoterless pYPGE15-ZmHMG I/Y2 construct (Delta HMG I/Y). Transformed strains were grown overnight in SD medium, and 5-fold serial dilutions were spotted onto agar minimum medium plates containing 0.5 mM NiSO4. Growth was recorded after 5 days of incubation at 30 °C. B and C, the F113 strain was transformed with the pYPGE15 empty plasmid or with pYPGE15 containing the ZmHMG I/Y2 cDNA (HMG I/Y) and grown in SD medium for 24 h. 5-Fold serial dilutions of transformed yeast cells were spotted onto agar minimum medium plates containing increasing NiSO4 concentrations (B) or onto agar minimum medium plates containing various metals at concentrations that inhibit yeast growth (C). The plates were incubated for 5 days at 30 °C.

Structural Features of ZmHMG I/Y2-- Complete sequencing of the ZmHMG I/Y2 cDNA revealed an open reading frame of 193 amino acids encoding a protein with a predicted molecular mass of 19.8 kDa. This coding sequence is flanked by a 3'-UTR of 413 base pairs and by a 5'-UTR of 90 base pairs (Fig. 2A). The deduced amino acid sequence shares common features with plant proteins of the HMG I/Y family. The N-terminal region is similar to the globular domain of histone H1 (Fig. 2A), and four AT hook motifs were observed within the sequence; these motifs allow the protein to interact with plant promoters containing AT-rich sequences (25). The ZmHMG I/Y2 protein shares 95% identity with a previously reported maize HMG I/Y sequence (Fig. 2B) (26). However, the two proteins are likely to be encoded by two different genes since the 5'- and 3'-UTRs of the ZmHMG I/Y2 cDNA are not fully conserved (40 and 90% identities, respectively) in the corresponding regions of the previously reported maize HMG I/Y cDNA. A high degree of sequence identity of the ZmHMG I/Y2 protein was also observed with other plant HMG I/Y proteins (Fig. 2B): 67% identity to PF-1 from rice (24), 66% to PF-1 from oat (27), and 43% identity to Arabidopsis thaliana HMG I/Y (28).


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Fig. 2.   Characteristics of the ZmHMG I/Y2 protein. A, nucleotide and deduced amino acid sequences of the ZmHMG I/Y2 cDNA. The protein is shown starting from the putative initiator methionine. The underlined sequences indicate sequence homology to the histone H1 globular domain, and the boxes delimit the AT hook motif. B, amino acid sequence alignment of HMG I/Y proteins from maize (ZmHMG I/Y (GenBankTM/EBI accession number AJ131371) and ZmHMG I/Y2 (from this work; accession number AF291748)), oat (AsPF-1; accession number L24391), rice (OsPF-1; accession number L24390), A. thaliana (AtHMG I/Y; accession number X99116), and human (HsHMG Y (accession number X14958) and HsHMG I (accession number X14957)). Identical amino acid residues are shown in black boxes, and similar ones are shown in gray boxes.

The ZmHMG I/Y2 Protein Is Synthesized in Yeast and Targeted to the Nucleus-- Northern and Western blot analyses of total RNA or protein extracts revealed the presence of the ZmHMG I/Y2 transcript and protein specifically in cells transformed with the ZmHMG I/Y2 cDNA, but not in cells transformed with the pYPGE15 empty vector or with the ZmHMG I/Y2 cDNA cloned in the phosphoglycerate kinase promoterless vector (Fig. 3, A and B). Equal amounts of RNA loaded in each lane were validated by hybridizing the same blot with a PAB1 probe. The sizes of the transcript (1.1 kbp) and of the polypeptide (19.8 kDa) were consistent with the sizes of the isolated cDNA and of the open reading frame it contained (Fig. 2A).


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Fig. 3.   The ZmHMG I/Y2 protein is localized in the nucleus. A, total RNA was extracted from yeast expressing the pYPGE15 empty plasmid or pYPGE15 containing the ZmHMG I/Y2 cDNA (HMG I/Y) or with the phosphoglycerate kinase promoter deleted (Delta HMG I/Y). The blots were hybridized with a 32P-radiolabeled ZmHMG I/Y2 3'-UTR probe and a yeast PAB1 probe to serve as RNA loading control. B, proteins were extracted from yeast cells expressing the pYPGE15 empty plasmid or pYPGE15 containing the ZmHMG I/Y2 cDNA or with the phosphoglycerate kinase promoter deleted. The ZmHMG I/Y2 protein was immunodetected with a polyclonal anti-PF-1 antibody. C, yeast cells expressing ZmHMG I/Y-GFP or GFP alone under the control of the phosphoglycerate kinase promoter from the pYPGE15 plasmid (pYPGE15-GFP) were analyzed by direct fluorescence microscopy. The GFP, 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, and Nomarski images are shown. Bar = 3 µm.

HMG I/Y proteins are targeted to the nucleus, where they bind to DNA sequences and may act as transcriptional trans-acting factors (29). Before addressing the mechanisms by which ZmHMG I/Y2 is able to confer nickel resistance to yeast cells, it was therefore important to determine its subcellular localization. The N-terminal part of the ZmHMG I/Y2 protein was fused in frame to the GFP, and expression of the fused protein was analyzed in living yeast cells. Expression of the ZmHMG I/Y2-GFP construct in yeast conferred nickel resistance to the yeast cells (data not shown). The GFP activity of the fusion protein colocalized with 4,6-diamidino-2-phenylindole staining, revealing its nuclear localization (Fig. 3C). As a negative control, the pYPGE15 vector containing the GFP sequence under the control of the phosphoglycerate kinase promoter without any ZmHMG I/Y2 cDNA was expressed in yeast. Expression of this construct in yeast cells revealed a random spreading of the fluorescence throughout the cell (Fig. 3C).

ZmHMG I/Y2 Does Not Affect Nickel Accumulation in Transformed Yeast Cells-- To understand how ZmHMG I/Y2 could confer nickel resistance to yeast cells, we hypothesized that this protein could modify the nickel concentration of cells either by limiting or decreasing nickel accumulation or, alternatively by facilitating sequestration of nickel ions. Chromatography on a Ni2+-nitrilotriacetic acid column of a yeast protein crude extract expressing the ZmHMG I/Y2 protein resulted in the quantitative recovery of the protein in the flow-through, ruling out a direct interaction between the ZmHMG I/Y2 protein and nickel (data not shown). Furthermore, expression of the ZmHMG I/Y2 cDNA in yeast cells grown at three various nickel concentrations did not lead to a change in intracellular nickel concentrations compared with control cells transformed with a empty plasmid (Fig. 4). These results indicate that ZmHMG I/Y2-dependent nickel resistance of yeast cells is not related to alterations in nickel accumulation.


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Fig. 4.   Nickel accumulation of yeast cells expressing the ZmHMG I/Y2 cDNA. Yeast cells expressing the pYPGE15 empty plasmid or pYPGE15 containing the ZmHMG I/Y2 cDNA (HMG I/Y) were grown in SD medium containing various NiSO4 concentrations. After 24 h of growth, the intracellular nickel content of cells was determined by an atomic absorption spectrometer. Values are the mean of three independent experiments, and bars represent S.D. values.

Several mechanisms have been suggested for resistance to nickel toxicity and imply an increase in free histidine, which is an effective chelator of nickel, and nickel storage in the vacuole (30, 31). Our data presented in Fig. 4 do not rule out the possibility that the ZmHMG I/Y2 protein could enhance histidine biosynthesis and/or nickel targeting to the vacuole without affecting the total nickel content of the cell. To clarify this point, we measured the effect of ZmHMG I/Y2 on free histidine concentration and on the expression of various genes involved in histidine biosynthesis or transport and in nickel transport into the vacuole. HPLC measurements revealed a slightly lower free histidine concentration (30% less) in ZmHMG I/Y2-transformed yeast cells compared with control cells (data not shown). Northern blot analysis showed that transcript abundance of HIS3 (a gene encoding a biosynthetic component for histidine biosynthesis), VMA1 (a gene encoding a subunit of a vacuolar H+-ATPase putatively coupled to a tonoplast nickel transporter), and HIP1 (a gene encoding a high affinity histidine permease) (30, 31) was unchanged in yeast cells expressing ZmHMG I/Y2 compared with control cells transformed with the empty plasmid (data not shown).

ZmHMG I/Y2 Counterbalances the Nickel Effect on the Yeast Cell Cycle-- In the case of iron toxicity, Philpott et al. (32) have shown that yeast cells grown under toxic conditions have their cell cycle blocked in G1. To test if nickel could alter the cell cycle, which could be rescued upon expression of the ZmHMG I/Y2 cDNA, yeast cells were grown in various nickel concentrations and analyzed by fluorescence-activated cell sorting (Fig. 5). Although no major changes in the progression through the cell cycle were observed in response to nickel treatment, a slight decrease in the ability of yeast cells to move into M phase was detected at 0.2 or 0.4 mM NiSO4 in the culture medium. Expression of the ZmHMG I/Y2 cDNA in yeast cells restored progression into M phase, hence rescuing the effect of nickel. However, this faint effect of nickel on the progression through the cell cycle is unlikely to explain the totality of the nickel-resistant phenotype.


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Fig. 5.   ZmHMG I/Y2 improves cell cycle progression in the presence of nickel. The F113 yeast strain transformed either with the pYPGE15 empty vector (left panels) or with pYPGE15 containing the ZmHMG I/Y2 cDNA (HMG I/Y; right panels) were grown for 24 h in SD medium in the presence or absence of nickel at the indicated concentrations. The DNA content of the yeast cells was measured by flow cytometry. The experiments were repeated three times with similar results.

ZmHMG I/Y2-mediated Nickel Resistance Correlates with Increased Chromatin Condensation-- Nickel toxicity has been reported to be linked to chromatin condensation in yeast, leading to increased gene silencing at the telomere (8). When expressed in yeast, the ZmHMG I/Y2 cDNA localized in the nucleus (Fig. 3C), where this class of protein is known to bind AT-rich sequences with low sequence specificity (29). We therefore hypothesized that the ZmHMG I/Y2 protein could confer nickel resistance to yeast cells by interfering with nickel-induced chromatin condensation.

We first addressed this point by using yeast strains in which the URA3 gene was inserted adjacent to the telomere on chromosome VII (strain UCC1001 in Fig. 6) or on chromosome V (strain YDS21U in Fig. 6). Expression of the URA3 gene inhibits cell growth on a medium containing the suicide substrate 5-FOA. This enables the measurement of URA3 transcriptional repression due to telomeric silencing dependent on chromatin structure (13).


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Fig. 6.   ZmHMG I/Y2 increases yeast telomeric URA3 silencing. A, yeast strains were transformed with the pFL61 empty plasmid or with pFL61 containing the ZmHMG I/Y2 cDNA (HMG I/Y). The UCC1001 strain contains the URA3 marker inserted 1.3 kbp from the telomere on chromosome VII. The YDS21U strain contains the URA3 marker inserted 2.1 kbp from the telomere on chromosome V. Yeast strains were grown for 24 h in SD medium without uracil and were then diluted into SD medium containing various NiSO4 concentrations for 40 h. 5-Fold serial dilutions were spotted onto SD plates with or without 5-FOA (1 g/liter). Growth was recorded after 3 days of incubation at 30 °C. B, 200 cells were spread onto SD plates with or without 5-FOA (1 g/liter). The number of colonies was counted after 3 days of growth at 30 °C. Values are the mean of three independent experiments, and bars represent S.D. values.

We then tested whether silencing at telomeres could be modified by ZmHMG I/Y2 in either the absence or presence of various nickel concentrations (Fig. 6). Expression of the ZmHMG I/Y2 cDNA led to 1.8- and 3-fold increases in 5-FOA resistance in the absence of nickel when URA3 was inserted on chromosome VII or on chromosome V, respectively. These results indicate that the ZmHMG I/Y2 protein increases chromatin condensation in yeast, leading to telomeric silencing. Treatment with either 50 or 75 µM NiSO4 of UCC1001 yeast cells expressing the empty plasmid, with URA3 inserted on chromosome VII, led to a 1.6-fold increase in 5-FOA resistance, but nickel had no effect on URA3 silencing in yeast cells expressing the ZmHMG I/Y2 cDNA. When URA3 was inserted on chromosome V, no increase in 5-FOA resistance to nickel treatment was observed in YDS21U cells expressing the empty vector. However, treatment with 50 and 75 µM NiSO4 of YDS21U yeast cells expressing the ZmHMG I/Y2 cDNA led to a drop in 5-FOA resistance from 3-fold to 2- and 1.5-fold, respectively. It is important to note that cells from the UCC1001 and YDS21U strains transformed with the ZmHMG I/Y2 cDNA were resistant to 0.5 mM NiSO4 (data not shown). It can be concluded from these experiments that gene silencing at the telomere can be increased by ZmHMG I/Y2 and that nickel, at least in some cases, modifies this silencing. Therefore, it can be postulated that nickel resistance mediated by the ZmHMG I/Y2 protein could be linked to chromatin structure modification, interfering with the effect of nickel on DNA condensation.

The method used above limits the observation of silencing to a specific region of the genome, i.e. the telomere. To further document the relationship between chromatin condensation and nickel resistance in yeast cells expressing the ZmHMG I/Y2 cDNA, we used a yeast mutant strain carrying a deletion of the histone deacetylase RPD3 gene that was reported to decondense chromatin in an "open" structure (14). The S. cerevisiae Delta rpd3 mutant was more sensitive to nickel compared with the YDS21U strain, and its growth was almost completely inhibited by 0.4 mM NiSO4 (Fig. 7A). Expression of the ZmHMG I/Y2 cDNA in the yeast Delta rpd3 mutant restored nickel tolerance, in contrast to transformation with the pFL61 empty vector (Fig. 7B). The same level of nickel resistance was observed between the wild-type strain and the yeast Delta rpd3 mutant. These data support the hypothesis that expression of the ZmHMG I/Y2 cDNA in yeast increases DNA condensation and confers nickel resistance to yeast cells.


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Fig. 7.   ZmHMG I/Y2 rescues nickel hypersensitivity of a yeast histone deacetylase RPD3 mutant strain. A, the YDS21U (wild-type (wt)) and SRG39 (Delta rpd3) strains were grown in SD medium for 24 h. 5-Fold serial dilutions were then spotted onto agar minimum medium plates containing various NiSO4 concentrations. Growth was recorded after 3 days of incubation at 30 °C. B, the YDS21U (wild-type) and SRG39 (Delta rpd3) strains were transformed with the pFL61 empty plasmid or with pFL61 containing the ZmHMG I/Y2 cDNA (HMG I/Y) and were grown in SD medium for 24 h. 5-Fold serial dilutions were then spotted onto agar minimum medium plates containing 0.5 mM NiSO4. The plates were incubated at 30 °C for 5 days.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this work, we isolated a maize cDNA encoding a ZmHMG I/Y2 protein that confers nickel resistance when expressed in yeast. This protein was identified by expressing a maize cDNA library in S. cerevisiae and screening for growth of transformed cells on a medium containing 0.5 mM NiSO4. Expression of the ZmHMG I/Y2 cDNA in yeast enabled cells to grow at up to 0.9 mM NiSO4, and the conferred resistance was specific to nickel. However, no changes in the nickel content of transformed yeast cells were observed. This prompted us to hypothesize that the ZmHMG I/Y2 protein could counteract nickel toxicity. One possibility for such a resistance could be that the binding of the ZmHMG I/Y2 protein to chromatin would have specific effects on gene expression, in particular on those genes encoding proteins involved in histidine biosynthesis and/or transport and in nickel transport into the vacuole (30, 31). However, no significant variations in histidine content and in HIS3, VMA1, and HIP1 transcript abundance were detected between ZmHMG I/Y2-expressing cells and control cells (see "Results"; data not shown). It is therefore unlikely that ZmHMG I/Y2 expression in yeast up-regulates transcription of genes involved in nickel detoxification through histidine chelation and/or nickel compartmentalization within the vacuole. However, at this point, it cannot be ruled out that expression of other genes could be activated through ZmHMG I/Y2 binding to chromatin, the product of which is responsible for nickel resistance.

Nickel compounds produce oxidative damage in cells preferentially in the heterochromatic regions of chromosomes (33). This is achieved through the formation of nickel complexes with cellular components such as specific subsequences of histone H3 (34) or H4 (35). In animal cells, nickel is a widespread carcinogen altering gene expression through DNA compaction and methylation, leading, for example, to the silencing of tumor suppressor genes (7). On the other hand, HMG I/Y proteins are considered to function as architectural elements, modifying chromatin structure (29). These proteins contain four AT hook motifs that mediate their binding to the minor groove of AT-rich DNA with low sequence specificity. The maize HMG I/Y protein is located in the nucleus of transformed yeast cells (Fig. 3), leading us to postulate that nickel resistance could be due to chromatin structure modification mediated by this plant protein.

In S. cerevisiae, nickel compounds have been reported to induce URA3 gene silencing at the telomere and to result in a stable epigenetic switch (8). The level of silencing was dependent upon the distance of the reporter gene from the telomere. In agreement with this statement, we also observed silencing at telomeres by nickel ions only when the URA3 gene was inserted 1.3 kbp from the telomere on chromosome VII, but not when the URA3 gene was positioned 2.1 kbp on chromosome V (Fig. 6). Expression of the ZmHMG I/Y2 cDNA in yeast also increased silencing at the same level as nickel ions, but was not dependent upon the distance of the URA3 gene insertion from the telomere (Fig. 6). The ZmHMG I/Y2 protein promotes URA3 gene silencing at the telomere, as does nickel, but without being detrimental to growth. This may suggest a role for this protein in preventing the detrimental effect of nickel binding to chromatin. When the URA3 gene was inserted 2.1 kbp from the telomere on chromosome V, its silencing in response to expression of the ZmHMG I/Y2 cDNA was decreased in the presence of nickel ions, evidencing an antagonist interaction of these two compounds in the process of telomeric silencing.

Furthermore, assessment of the effect of nickel and ZmHMG I/Y2 was conducted in a yeast strain carrying a deletion of the histone deacetylase RPD3 gene (Fig. 7). This mutant strain is characterized by increased acetylation at lysines 5 and 12 of histone H4 that correlates with increased transcriptional activity (14), but it also exhibits an increased silencing at telomeric loci. In this genetic background, the increased nickel sensitivity observed might be associated with increased spreading of heterochromatin. However, nickel treatment of the Delta rpd3 strain did not increase silencing of the URA3 gene at the telomere (data not shown). Another explanation of the high nickel sensitivity of the Delta rpd3 strain could involve a more accessible chromatin structure to nickel ions due to increased histone acetylation. Expression of the ZmHMG I/Y2 cDNA in the yeast Delta rpd3 mutant restored a wild-type level of nickel sensitivity (Fig. 7). This result is in agreement with a role of ZmHMG I/Y2 in chromatin condensation restricting nickel accessibility to DNA.

HMG I/Y protein, as well as a synthetic reiterated AT hook peptide (MATH20), binds to scaffold-associated regions (36). As a result, compacting proteins such as histone H1 are displaced, resulting in chromatin accessibility (37, 38). Moreover, plant HMG I/Y proteins contain an N-terminal region homologous to the histone H1 globular domain (25). It is therefore tempting to speculate that this domain might be involved in the interaction of plant HMG I/Y proteins with chromatin (39). In yeast, the HHO1 gene could encode a protein functionally similar to histone H1, but it is not yet clear whether the protein functions as a true histone H1 (40). In addition, although no HMG I/Y proteins have been identified in yeast, some yeast nuclear proteins known to interact with DNA contain one or two AT hook motifs (41). These proteins include Swi2p/Snf2p, involved in chromatin remodeling (42); Swi5p, an activator protein (43); and Ash1p, a member of the Trithorax group proposed to regulate chromatin decondensation (44). Therefore, the ZmHMG I/Y2 protein can probably compete for the same DNA-binding sites as these proteins, displacing them and altering chromatin structure. In S. cerevisiae, mutations in the histone H3 and H4 genes (45) and mutations in the SIN1 gene, which encodes an HMG1-like protein (46), suppress the transcriptional defects due to inactivation of the SWI·SNF complex. This is likely achieved by disruption of histone-DNA interactions, leading to increased accessibility of nucleosomal DNA to transcription factors (47).

In conclusion, we have characterized a ZmHMG I/Y2 protein that confers nickel resistance to yeast cells, likely by interfering with the chromatin structure. These results remain to be assessed in plants, in which the mechanisms involved in chromatin remodeling are probably conserved since different types of histone deacetylase (48) have recently been identified as well as DDM1, a SWI/SNF-like protein (49).

    ACKNOWLEDGEMENTS

We thank Professors M. Grunstein and D. E. Gottschling for providing indispensable yeast strains, Dr. B. Lapeyre for the PAB1 and GFP plasmids, and Professor P. H. Quail for the anti-PF-1 antibody. We are grateful to Dr. P. Pasero for helpful discussions and advice on the flow cytometric analysis and GFP imaging and to Dr. B. Touraine for the HPLC measurements.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF291748.

Dagger To whom correspondence should be addressed. Tel.: 33-4-67-14-47-99; Fax: 33-4-67-14-36-37; E-mail: lebrun@arpb.univ-montp2.fr.

Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M007462200

    ABBREVIATIONS

The abbreviations used are: HMG, high mobility group; kbp, kilobase pair(s); UTR, untranslated region; GFP, green fluorescent protein; PBS, phosphate-buffered saline; 5-FOA, 5-fluoroorotic acid; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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