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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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-
1,
his3-
200, leu2-
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 (
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,
(lac-proAB), (F',
traD36, proAB,
laclqZ
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.
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RESULTS |
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
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 (
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 ( 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.
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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.
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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 ( 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.
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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.
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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.
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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.
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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
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
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
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
( 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 ( 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.
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DISCUSSION |
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
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
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
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).