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Curr Genet (2017) 63:229–239DOI 10.1007/s00294-016-0628-9
ORIGINAL ARTICLE
HAL2 overexpression induces iron acquisition in bdf1Δ cells and
enhances their salt resistance
Lei Chen1,2 · Mingpeng Wang1,3 · Jin Hou1 · Jiafang Fu1 · Yu
Shen1 · Fanghua Liu2 · Zhaojie Zhang4 · Xiaoming Bao1
Received: 25 March 2016 / Revised: 26 June 2016 / Accepted: 27
June 2016 / Published online: 8 July 2016 © Springer-Verlag Berlin
Heidelberg 2016
acquisition and cellular and mitochondrial remodeling are
induced by HAL2. Overexpression of HAL2 decreases the concentration
of nitric oxide. Mitochondrial iron–sulfur cluster (ISC) assembly
also decreases in bdf1Δ + HAL2. These changes are similar to the
changes of transcriptional profiles induced by iron starvation.
Taken together, our data suggest that mitochondrial functions and
iron homeostasis play an important role in bdf1Δ-induced salt
sensitivity and salt stress response in yeast.
Keywords Bromodomain factor 1 · Iron acquisition · HAL2 · HDA1 ·
Salt stress response
Introduction
Yeast cells have evolved intricate systems to respond to
cel-lular stresses (Ho and Gasch 2015). In Saccharomyces
cer-evisiae, the previous studies have shown that BDF1 plays a role
in multiple stresses, including salt, high temperature, caffeine,
and LiCl (Ferreira et al. 2001). The Bromodomain Factor 1 (Bdf1) is
a transcriptional regulator as a part of the basal transcription
factor TFIID (Lygerou et al. 1994; Matangkasombut et al. 2000). The
BDF1 gene was isolated in a large-scale screen for salt-sensitivity
mutants follow-ing transposon mutugenesis (Ferreira et al. 2001),
and its salt sensitivity can be suppressed by its homologous gene
BDF2 (Liu et al. 2007). Our previous results demonstrate that salt
stress in BDF1 deletion causes abnormal mito-chondrial function
(Liu et al. 2009). We have further dem-onstrated that bdf1Δ is
defective in both autophagy and respiration under salt stress.
These defects can be reversed by overexpression of HAL2, whose
production detoxifies the toxic compound
3′-phosphoadenosine-5′-phosphate (pAp) (Murguia et al. 1995, 1996).
Overexpression of
Abstract The yeast Saccharomyces cerevisiae is capable of
responding to various environmental stresses, such as salt stress.
Such responses require a complex network and adjustment of the gene
expression network. The goal of this study is to further understand
the molecular mechanism of salt stress response in yeast,
especially the molecular mech-anism related to genes BDF1 and HAL2.
The Bromodomain Factor 1 (Bdf1p) is a transcriptional regulator,
which is part of the basal transcription factor TFIID. Cells
lacking Bdf1p are salt sensitive with an abnormal mitochondrial
function. We previously reported that the overexpression of HAL2 or
deletion of HDA1 lowers the salt sensitivity of bdf1Δ. To better
understand the mechanism behind the HAL2-related response to salt
stress, we compared three global transcrip-tional profiles (bdf1Δ
vs WT, bdf1Δ + HAL2 vs bdf1Δ, and bdf1Δhda1Δ vs bdf1Δ) in response
to salt stress using DNA microarrays. Our results reveal that genes
for iron
Communicated by M. Kupiec.
L. Chen and M. Wang have contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00294-016-0628-9) contains supplementary
material, which is available to authorized users.
* Xiaoming Bao [email protected]
1 State Key Laboratory of Microbial Technology, Shandong
University, Jinan 250100, China
2 Yantai Institute of Coastal Zone Research, Chinese Academy of
Sciences, Yantai, Shandong 264003, China
3 School of Municipal and Environmental Engineering, Harbin
Institute of Technology, Harbin 150090, China
4 Department of Zoology and Physiology, University of Wyoming,
Laramie, WY, USA
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230 Curr Genet (2017) 63:229–239
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HAL2 also restores autophagy and mitochondrial function, and
reverses the salt stress resistance of bdf1Δ (Chen et al. 2013).
However, its detailed molecular mechanism is still unknown. The
goal of this study is to better understand the involvement of HAL2
in salt stress response by comparing the global changes of gene
expression between bdf1Δ over-expression and HAL2 overexpression in
bdf1Δ.
Our previous study indicated that deletion of the histone
deacetylase gene HDA1 leads to an increase in salt resist-ance in
bdf1Δ mutants (Chen et al. 2014). Hda1p dimer interacts with
Hda2p–Hda3p to form a histone deacety-lase complex (Lee et al.
2009). This complex reduces his-tone acetylation and leads to
chromosome compaction and repression of gene expression. Deletion
of HDA1 increases histone acetylation (Wu et al. 2001). Hda1p also
deacety-lates subtelomeric domains of chromosomes that contain
normally repressed genes involved in hexose transport, carbon
source utilization, and stress response (Robyr et al. 2002).
In this study, we compare three global transcriptional analysis
profiles: bdf1Δ vs WT, bdf1Δ + HAL2 vs bdf1Δ, and bdf1Δhda1Δ vs
bdf1Δ in response to salt stress using DNA microarrays to better
understand the molecular mech-anism by which HAL2 overexpression in
bdf1Δ reverses the salt sensitivity of bdf1Δ.
Materials and methods
Strains and media
All plasmids and the S. cerevisiae strain used in this study are
listed in Table 1. Yeast cells were routinely grown in YPD media (1
% yeast extract, 2 % peptone, and 2 % glu-cose) or in synthetic
complete (SD) medium (0.17 % yeast nitrogen base, 0.5 % (NH4)2SO4,
and 2 % glucose, supple-mented with the required amino acids).
Gene disruption
The bdf1Δ mutant on the W303-1A strain back-ground was
constructed by transformation with a 2859 bp bdf1::kanMX4 gene
deletion allele. The strain bdf1Δhda1Δ was constructed on the bdf1Δ
strain
background by transformation with a 1700 bp hda1:: URA3 gene
deletion allele (Chen et al. 2014).
Plasmid construction and transformation
The HAL2 gene was amplified from W303-1A genomic DNA using a
pair of PCR primers HAL2-F and HAL2-R (Chen et al. 2013). The
resulting DNA fragments were cloned into a BlnI restriction site of
a plasmid vector pYX242 to yield pYX242–HAL2 (Chen et al. 2013).
The bdf1Δ mutant was transformed with plasmid using the lithium
acetate procedure (Gietz et al. 1995). Gene-spe-cific primers used
in this study are listed in Supplementary Table 1.
Nitric oxide (NO) production and DAPI staining
NO production was detected using 3-Amino, 4-aminome-thyl-20,
70-difluorescein diacetate (DAF–FMDA). The cells (108 cells/ml)
were washed twice and suspended in 0.1 M phosphate buffer (pH 7.0)
containing 1 mM EDTA and 0.1 % glucose. Cells were stained with
DAF–FMDA (5 μM) in PBS (pH 7.4) for 20 min at 37 °C. Cells were
then washed three times and resuspended in PBS. Stained cells were
observed using a Nikon ECLIPSE 80i fluorescence microscope (Nikon,
Japan) equipped with either a Plan Apochromat 40× objective (NA =
0.95) or a Plan Apochromat 60× oil objec-tive (NA = 1.40). The
excitation and emission wavelengths were 495 and 515 nm,
respectively. Images were acquired and analyzed using the
NIS-Elements AR 3.1 software. DNA integrity was detected via
2-(4-Amidinophenyl)-6-indolecar-bamidine dihydrochloride (DAPI)
staining. NaCl-treated cells were fixed in 80 % ethanol for 10 min,
washed with phos-phate buffered saline (PBS, pH 7.4), and incubated
with PBS containing 1 µg/ml DAPI for 10 min. The stained cells were
imaged with the same microscope, using different
excitation/emission wavelengths (358/461 nm). Fluorescence
intensity analysis was conducted using the Image J software
(http://rsb.info.nih.gov/ij/).
RNA extraction and quantitative PCR (qPCR)
Cells were cultivated by inoculating a pre-culture in 100 ml
fresh YPD to OD600 = 0.2, and grown to the mid-log phase
Table 1 Yeast strains used in this study
Strain Genotype Reference or source
W303-1A MATa, leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1
can1-100 GAL SUC2 mal0 (Ferreira et al. 2001)
bdf1Δ Derivative of W303-1A: bdf1::kanMX4 (Chen et al. 2014)
bdf1Δ + HAL2 bdf1Δ transformed with pYX242-HAL2 (Chen et al.
2013)bdf1Δhda1Δ Derivative of bdf1Δ: hda1::URA3 (Chen et al.
2014)
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(OD600 = 0.7). The cultures were divided into two 50 ml samples.
Next, 10 ml of sterile water containing 3 M NaCl was added to one
sample (final NaCl concentration of 0.5 M), and the same volume of
sterile water was added to the other sample. After 45 min of
treatment, both cultures were collected by centrifugation and
immediately frozen in liquid nitrogen. The total yeast RNA was
isolated using UNIQ-10 spin column Trizol Total RNA purification
kits (BBI, USA) in accordance with the manufacturer’s
instruc-tions. An aliquot of 5 μg total RNA was treated with
RNA-free DNase I for 30 min at 37 °C. Then, 2 μl of treated RNA was
employed to synthesize the first cDNA strand in a 20 μl reverse
transcription (RT) volume. For PCR ampli-fication, 1 μl of the RT
reaction products were used, utiliz-ing the SYBR Green I monitoring
method. Fold changes in gene expression were calculated using the
2−ΔΔCT method (Chung et al. 2002).
Gene expression profile analysis by microarray
Exponential phase cultures of strains (WT, bdf1Δ, bdf1Δ + HAL2,
and bdf1Δhda1Δ) were diluted in 100 ml of fresh YPD to OD600 = 0.2
and grown to exponential phase (at approximately OD600 = 0.8).
Then, 20 ml 3 M NaCl was added to the sample (final NaCl
concentration of 0.5 M). After 45 min of treatment, the cells were
collected by centrifugation at room temperature and immediately
fro-zen in liquid nitrogen for RNA extraction. The total RNA was
isolated using Trizol reagent (Takara, Tokyo, Japan) and purified
using a NucleoSpin® Extract II Kit (Machery-Nagel Corp., Dueren,
Germany). JINGXIN cDNA amplifi-cation and labeling kits (Capital
Bio Corp., Beijing, China) and a two-channel 6 K yeast genome array
(CapitalBio Corp.) were employed for cy3 or cy5 labeling of the
cDNA and microarray hybridization (Zhang et al. 2002). The array
images were scanned using a LuxScan10KA Micro-array Scanner
(CapitalBio Corp.) and analyzed with Lux Scan 3.0 software
(CapitalBio Corp.). Space- and intensity-dependent normalization
was employed based on a locally-weighted scatter plot smoothing
regression program (Yang et al. 2002). The microarray experiment
was repeated twice (presented data corresponds to the average of
the two experiments). Fold changes in gene transcription ≥2 were
considered significant, whereas changes ≥1.5 were also considered
in the analysis. KegArray (http://www.genome.jp/kegg/expression/)
was used to map the gene expression data to the KEGG BRITE database
for primary functional classification. The gene annotation
information was based on the Saccharomyces Genome Database
(http://www.yeastgenome.org/). GO term Finder was used to group
genes into functional categories, and is found in the MIPS
Functional Catalogue (www.helmholtz-muenchen.de). The microarray
data were submitted to the Gene Expression
Omnibus database (http://www.ncbi.nlm.nih.gov/geo). The
accession number is GSE75828.
Heme staining in gel and western blot analysis
SDS-PAGE was carried out without the addition of DTT. Coomassie
brilliant blue staining was carried out as described previously
(Dumont et al. 1988). 3,3′,5,5′-Tetra-methylbenzidine (TMBZ) was
dissolved in methanol to a final concentration of 6.3 mM. The gel
was covered with a solution of three parts TMBZ and seven parts 0.5
M sodium acetate and incubated in the dark for 20 min. H2O2 was
added to the final concentration of 30 mM for the visible protein
gel bands. Protein sample preparation, SDS-PAGE, and Western blots
were performed as described previously (Camougrand et al. 2003).
The primary antibodies were polyclonal rabbit anti-yeast Rip1p,
anti-yeast Sdh2p, and anti-β-tubulin (ANBO). Semi-quantitative
assessment of the estimated protein expression was performed using
gray values and analyzed using the Image J software
(http://rsb.info.nih.gov/ij/).
Statistical analysis
Data were analyzed using the Origin 8.1 software and the results
expressed in the form: mean ± standard deviation. P < 0.05
(tailed t-student test) is considered to denote sta-tistical
significance.
Results
Deletion of BDF1 affects the transcription of genes related to
amino acid metabolism, electron transport, and rescue and defense
systems.
To better understand the molecular mechanism underly-ing the
salt sensitivity of bdf1Δ, a comparative transcrip-tome analysis
between bdf1Δ mutants and WT was carried out in salty conditions.
The results reveal that 1343 genes had significant changes (≥2
fold). Interestingly, 1295 (96 %) of these genes were
down-regulated, while only 48 were up-regulated in bdf1Δ compared
to WT (Supplemen-tary Table 2).
We have previously shown that the overexpression of HAL2
reversed defects in bdf1Δ (Chen et al. 2013). Thus, a comparative
transcriptome analysis between bdf1Δ + HAL2 and bdf1Δ in salt
conditions was car-ried out. The results reveal that 110 genes had
significant changes (Fig. 1a; Supplementary Table 2). The qPCR
anal-ysis of randomly selected genes OYE2, ACO1, and MEF1 further
confirmed the microarray data (Table 2).
Genes with significant changes in the transcriptomic analysis
were classified using the MIPS database. The most
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significant change between bdf1Δ and WT was the transla-tion
machinery, including ribosome biogenesis, ribosomal proteins, and
translation (Supplementary Table 3). Other significant changes in
expression are mainly related to amino acid metabolism (e.g.,
lysine and leucine), electron transport, rescue and defense (e.g.,
stress response, osmotic and salt stress response), and subcellular
structures (e.g., cytoplasm, endoplasmic reticulum, and
mitochondrion) (Supplementary Table 3). The changes in the
translation machinery might be a secondary response to other
changes,
such as salt stress response, or amino acid metabolism. In
comparison between bdf1Δ + HAL2 and bdf1Δ mutants, the most
significant change in gene expression in response to salt stress
was the iron-responsive genes (up to 7.74 fold) (Supplementary
Tables 2 and 4).
A previous study has shown that the deletion of HDA1 in the
bdf1Δ mutant increases its salt resistance (Chen et al. 2014). To
further confirm the possible involve-ment of the iron-responsive
genes in salt resistance, the global transcriptional profiles were
compared between
Fig. 1 Venn diagrams showing a number of differentially
expressed genes (up-/down-regulated) in response to salt treatment
(0.5 M NaCl, 45 min) in microarrays for bdf1Δ + HAL2 vs bdf1Δ
and
bdf1Δhda1Δ vs bdf1Δ. Functional categories overrepresented in
the subset of genes present in the overlapping part of the Venn
diagram (b)
Table 2 qPCR analysis of the expression changes of OYE2, ACO1,
and MEF1 in bdf1Δ vs WT, bdf1Δhda1Δ vs bdf1Δ, and bdf1Δ + HAL2 vs
bdf1Δ after salt treatment (0.5 M NaCl, 45 min)
Data were presented as mean ± SD from three experiments
Gene Fold changes
bdf1Δ vs WT bdf1Δ + HAL2 vs bdf1Δ bdf1Δhda1Δ vs bdf1Δ
Microarray qPCR Microarray qPCR Microarray qPCR
OYE2 0.231 ± 0.046 0.187 ± 0.010 1.567 ± 0.059 2.098 ± 0.120
2.012 ± 0.023 2.030 ± 0.010ACO1 0.658 ± 0.024 0.642 ± 0.058 0.651 ±
0.004 0.650 ± 0.020 0.475 ± 0.015 0.340 ± 0.050MEF1 0.321 ± 0.085
0.305 ± 0.013 0.775 ± 0.043 0.832 ± 0.022 1.521 ± 0.063 1.720 ±
0.150
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bdf1Δhda1Δ and bdf1Δ under the same salt-stressed con-ditions
(0.5 M NaCl, 45 min). One striking feature of our comparison is
that the iron metabolism genes were the most highly up-regulated in
bdf1Δhda1Δ compared to bdf1Δ, similar to the profile of bdf1Δ +
HAL2 compared to bdf1Δ under the same conditions. Our results show
that 89 genes displayed significant changes (≥2 fold) (Fig. 1a;
Supplementary Tables 2 and 5). Among the genes of ≥2-fold changes,
11 genes encoding iron transportation were found up-regulated (≥2
fold) in both bdf1Δ + HAL2 vs bdf1Δ and bdf1Δhda1Δ vs bdf1Δ (Fig.
1a; Supple-mentary Table 2).
Genes with a fold change of 1.5 in both the profiles bdf1Δ +
HAL2 vs bdf1Δ and bdf1Δhda1Δ vs bdf1Δ were also analyzed. It was
found that some of these genes are involved in pathways that may be
important in restoring the bdf1Δ salt sensitivity. Of the 536
changed genes (1.5–7 folds) in bdf1Δ + HAL2 vs bdf1Δ and the 342
changed genes in bdf1Δhda1Δ vs bdf1Δ, 35 genes were common to both
profiles (Supplementary Table 2). These 35 genes are involved in
five pathways with similar upward trends, including iron
acquisition, heme degradation, iron–sulfur cluster biosynthesis,
and sterol biosynthesis. Two path-ways presented different
tendencies, including sulfur
assimilation and ammonium transport (Figs. 1b, 2). The processes
are all linked, directly or indirectly, to cellular iron and/or
heme metabolism.
The MIPS categories in these three profiles (bdf1Δ vs WT, bdf1Δ
+ HAL2 vs bdf1Δ, and bdf1Δhda1Δ vs bdf1Δ) are all involved in:
amino acid metabolism, energy, NAD/NADP binding; transported
compounds (substrates); cell rescue, defense and virulence; and
interaction with the environment (Supplementary Tables 3–5). This
suggests that HAL2 and HDA1 may play a compensation role and help
bdf1Δ to response to salt stress by launching these pathways.
Overexpression of HAL2 in bdf1Δ increases gene expression in the
iron‑acquisition pathway
Iron-related gene expression in the transcriptional profile of
bdf1Δ vs WT is different from that of bdf1Δ + HAL2 vs bdf1Δ or
bdf1Δhda1Δ vs bdf1Δ. Many iron-related genes, such as, FIT2 (0.16
fold), FIT3 (0.49 fold), FET3 (0.20 fold), SIT4 (0.48 fold), SMF3
(0.17 fold), FTH1 (0.46 fold), MRS3 (0.42 fold), YLR126C (0.33
fold), and CUP5 (0.26 fold), were down-regulated in bdf1Δ (Fig. 2;
Supple-mentary Table 6).
Fig. 2 Heat map showing the significantly (based on signal log2
ratio) differentially expressed genes (up-/down-regulated) in
response to salt treatment (0.5 M NaCl, 45 min) in microarrays. (1)
bdf1Δ + HAL2 vs bdf1Δ, (2) bdf1Δhda1Δ vs bdf1Δ, and (3) bdf1Δ vs
WT
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Our results suggest that either HAL2 overexpression or HDA1
deletion in bdf1Δ causes a similarly strong induc-tion of genes (up
to 16.92-fold change) related to iron acquisition. These include
those related to siderophore–iron chelate transferal (FIT1-3,
ARN1-2), the ferrous-specific oxidation and transmission system
(FET3/FTR1), ferric iron salt reduction (FRE1/2), and
ferrous-specific transport system (SMF3) (Fig. 2; Supplementary
Table 7).
Iron transfer requires the carrier siderophore to be in the
cell. Proteins encoded by FIT1, FIT2, and FIT3 may cap-ture the
siderophores with ferric salts and then transfer the ferric
chelates to the reductive system in the membrane (Protchenko et al.
2001). Consistent with this notion, it is found that FIT2 and FIT3
were up-regulated (>3 fold) in both the sets of transcriptome
data. This suggests that HAL2 overexpression or HDA1 deletion in
bdf1Δ causes acquisition of ferric salts.
The ferric chelates can be recognized and reduced to the ferrous
state in the membrane. The reductive system is composed of the FRE
family of genes, which includes FRE1 to FRE8 (Dancis et al. 1990).
FRE1 and FRE2 encode flavocytochromes that catalyze the reductive
release of iron from siderophores. FRE1 is also induced by copper
depletion via Mac1p. FRE3 encodes a plasma membrane reductase that
catalyzes the reductive uptake of iron bound to hydroxamate
siderophores (Philpott and Protchenko 2008). Fre6p, which localizes
to the vacuole, transports reduced iron and copper from the vacuole
into the cytosol (Singh et al. 2007). In our comparative
transcriptome data, FRE1 (3.09 fold), FRE2 (2.74 fold), FRE3 (1.67
fold), and FRE4 (2.27 fold) were induced in bdf1Δ + HAL2 vs bdf1Δ.
FRE1 and FRE2 in bdf1Δhda1Δ vs bdf1Δ were induced by 3.67 fold and
4.10 fold, respectively (Fig. 2; Supplementary Table 7).
In addition, four other iron-related genes, including FET3,
FTR1, SMF3, and MRS4, were up-regulated in both bdf1Δ + HAL2 and
bdf1Δhda1Δ (Fig. 2; Supple-mentary Tables 4, 5, and 7). FET3
encodes a multicopper ferroxidase, and FTR1 encodes a permease.
These results suggest that the overexpression of HAL2 or deletion
of HDA1 in bdf1Δ facilitates the reduction of ferric salts car-ried
by siderophore ferrous salts and this reduction may help to
increase the salt resistance. Smf3p is regulated by iron and
functions with Fre6p to transport ferric iron to the cytoplasm
(Singh et al. 2007). MRS4 encodes an iron transporter and mediates
Fe2+ transport across the inner mitochondrial membrane and is
active under low-iron con-ditions (Li et al. 2014).
SIT1 and ENB1 are also up-regulated, both in bdf1Δ + HAL2 and
bdf1Δhda1Δ (Fig. 2; Supplementary Tables 4, 5, and 7). Sit1p is a
Ferrioxamine B transporter that specifically recognizes
siderophore–iron chelates and is induced during iron deprivation
and diauxic shift. ENB1
encodes an endosomal ferric enterobactin transporter and is
expressed under conditions of iron deprivation (Deng et al. 2009).
Both Sit1p and Enb1p require specific substrates. They undergo a
degradation process when no peculiar sub-strates are available
(Froissard et al. 2007; Kim et al. 2006).
In bdf1Δ, overexpression of HAL2 causes reduced levels of heme
and iron–sulfur proteins
In yeast, the two major iron-consuming processes are heme
biosynthesis and iron–sulfur protein assembly. Our results show
that HMX1, which encodes yeast heme oxygenase and releases iron
from heme during iron deficiency (Kim et al. 2006; Protchenko and
Philpott 2003), is up-regulated in both bdf1Δ + HAL2 (1.39 fold)
and bdf1Δhda1Δ (3.96 fold) (Fig. 2; Supplementary Table 7).
HEM15 encodes a mitochondrial inner-membrane protein
ferrochelatase that catalyzes the insertion of fer-rous iron into
protoporphyrin IX (Ihrig et al. 2010). It is down-regulated in both
bdf1Δ + HAL2 (0.73 fold) and bdf1Δhda1Δ (0.60 fold) (Fig. 2;
Supplementary Table 7), although the fold changes are relatively
small. Heme lev-els were determined to further confirm whether or
not the down-regulation is significant. Our results show that the
overexpression of HAL2 in bdf1Δ causes a significant decrease in
the heme level compared to bdf1Δ under salty conditions. The heme
levels in bdf1Δ + HAL2, with or without salt stress, are lower than
that in bdf1Δ (Fig. 3a, d).
The expression levels of mitochondrial iron–sulfur pro-teins
Sdh2p and Rip1p in WT, bdf1Δ, and bdf1Δ + HAL2 were measured using
the Western blot method. The expres-sion levels of Rip1p were
similar in bdf1Δ, WT, and bdf1Δ + HAL2. However, the Sdh2p level is
lower in bdf1Δ + HAL2 (Fig. 3c, d), compared to bdf1Δ. It seems as
if these two major iron-consuming processes (heme bio-synthesis and
iron–sulfur protein assembly) are ‘blocked’ in bdf1Δ + HAL2 and
that the cells start to transcribe iron-acquisition genes to
acquire more iron ions for the biosynthesis of heme and iron–sulfur
proteins. This result is consistent with the microarray data, which
showed that iron-acquisition genes are up-regulated in bdf1Δ + HAL2
vs bdf1Δ.
Compared to WT, a higher level of heme and Sdh2p proteins was
observed in bdf1Δ (Fig. 3). This might be a response of the bdf1Δ
cells, at the transcriptional level, to stop the acquisition of
additional iron. The profile bdf1Δ vs WT shows that
iron-acquisition and iron–sulfur pro-tein synthesis-related genes
are inhibited in bdf1Δ vs WT, including FIT2 (0.16 fold), FIT3
(0.49 fold), FET3 (0.20 fold), SIT4 (0.48 fold), MRS3 (0.42 fold),
SMF3 (0.17 fold), FTH1 (0.46 fold), NFU1 (0.24 fold), ISU2 (0.14
fold), YPL251 W (0.03 fold), CFD1 (0.03 fold), ERV1 (0.02 fold),
and YAH1 (0.02 fold) (Fig. 2; Supplementary
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Table 6). This suggests that the deletion of BDF1 leads to
excessive heme and iron–sulfur protein production, and this might
induce an imbalance in the iron homeostasis com-pared to the wild
type. Thus, we speculate that the overex-pression of HAL2 in bdf1Δ
may recover the iron homeosta-sis and then lead to reversal of the
sensitivity of bdf1Δ.
Overexpression of HAL2 in bdf1Δ causes a decrease in nitric
oxide production
Nitric oxide (NO) is an important cellular signaling mol-ecule
involved in many physiological and pathological processes. In S.
cerevisiae, intracellular NO is involved in cell death (Almeida et
al. 2007). NO affects transcriptional
regulation of genes involved in iron acquisition and iron–sulfur
cluster assembly and repair (Jones-Carson et al. 2008). To
investigate if NO is involved in the salt sensitiv-ity of bdf1Δ, NO
content was measured in W303, bdf1Δ, and bdf1Δ + HAL2. As shown in
Fig. 4, about 50 % of the bdf1Δ cells were detected with NO.
Overexpression of HAL2 (bdf1Δ + HAL2) reduced the NO level to the
wild-type (W303) level under salt stress (P < 0.05) (Fig. 4b),
suggesting that the bdf1Δ underwents cell death, which is also
supported by our previous study (Chen et al. 2013).
Chromatin fragmentation was also investigated to further confirm
the cell death status. The bdf1Δ strain displayed dispersive
nuclear or chromatin fragments, whereas the wild type and bdf1Δ +
HAL2 showed
Fig. 3 Heme staining and Western blot results for the detection
of Rip1p and Sdh2p protein expression levels. a SDS-PAGE is carried
out without the addition of DTT. TMBZ was solubilized in methanol
to a final concentration of 6.3 mM. The gel was covered with a
solu-tion of three parts TMBZ and seven parts 0.5 M sodium acetate
and incubated in the dark for 20 min; H2O2 was added to the final
concen-tration of 30 mM for the visible protein gel bands. ‘+’
denotes cells with 0.5 M NaCl (45 min) treatment, and ‘–’ denotes
cells with water (45 min) treatment. b Loading control with
Coomassie blue staining. c For Western blot detection of Rip1p and
Sdh2p protein expression level, the cells were grown in YPD lipid
medium until log phase and then incubated for 45 min with or
without 0.5 M NaCl. Cells were
collected and broken in PBS. After centrifugation at 13,000 rpm
for 15 min, supernatant was used for the whole-cell protein
concentra-tion assay to enable all samples having the same
concentration of the whole-cell protein. Rabbit anti-β-tubulin
(ANBO) was used as the control; rabbit anti-yeast Rip1p and
anti-yeast Sdh2p were for immu-nodetection purposes. d Expression
of heme, Rip1p, Sdh2p, and tubu-lin were estimated using gray
values. The gray values were quanti-fied using the Image J software
and averaged over three experiments. All experiments were repeated
three times. The error bars denote the standard deviation (SD). *P
< 0.05, **P < 0.01 vs strains without HAL2 overexpression for
the same treatment
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236 Curr Genet (2017) 63:229–239
1 3
relatively intact nuclei (Fig. 4a). As shown in Fig. 4, a higher
proportion of cells with altered nuclei was detected in bdf1Δ under
salt stress. When HAL2 was overex-pressed in bdf1Δ, the proportion
of cells with altered nuclei decreased significantly (P < 0.01)
(Fig. 4b). This again demonstrates that bdf1Δ undergoes cell death,
which is likely to be apoptotic (Chen et al. 2013). In addi-tion,
overexpression of HAL2 in bdf1Δ reverses, at least in part, the
occurrence of cell death.
Overexpression of HAL2 in bdf1Δ up‑regulates sterol
biosynthesis
Sterol biosynthesis is associated with mitochondrial
mor-phogenesis (Schneiter 2007) and is important to mito-chondrial
function. In our comparative transcriptome data, genes related to
sterol biosynthesis were up-regulated in bdf1Δ + HAL2 vs bdf1Δ,
e.g., ERG1, ERG10, ERG11,
ERG12, ERG13, ERG2, ERG25, ERG26, ERG27, ERG3, ERG5, ERG6, and
ERG8 (Fig. 2; Supplementary Tables 4 and 7).
Similarly, genes related to sterol biosynthesis in bdf1Δhda1Δ vs
bdf1Δ were up-regulated, e.g., ERG1, ERG10, ERG11, ERG12, ERG13,
ERG2, ERG25, ERG26, ERG27, ERG3, ERG5, ERG6, and ERG8 (Fig. 2;
Supple-mentary Tables 5 and 7). This might be one of the reasons
that the function of the mitochondria in bdf1Δ + HAL2 and
bdf1Δhda1Δ is improved (Chen et al. 2013, 2014).
In our comparative transcriptome data for bdf1Δ vs WT, genes
involved in sterol biosynthesis were down-regulated, e.g., ERG10,
ERG13, ERG2, ERG20, ERG26, ERG27, ERG3, ERG5, ERG9, and ERG4 (Fig.
2; Supplementary Tables 3 and 6). This suggests that the deletion
of BDF1 may also inhibit the expression of genes involved in sterol
biosynthesis and that HAL2 overexpression induces their
transcription.
Fig. 4 DAF-FMDA and DAPI staining results. Yeast cells (W303,
bdf1Δ, and bdf1Δ + HAL2) with or without 0.5 M NaCl 45 min
treatment in YPD. a NO production was detected via DAF-FMDA. Cells
were washed twice and suspended in 0.1 M phosphate buffer (pH 7.0)
containing 1 mM EDTA and 0.1 % glucose. Cells were loaded with
DAF-FMDA (5 μM) in PBS (pH 7.4) for 20 min at 37 °C. Thereafter,
cells were washed three times and resuspended in PBS. ‘+’ denotes
cells with 0.5 M NaCl (45 min) treatment and ‘–’ denotes cells with
water only (45 min) treatment. Integrity of the DNA was detected
via DAPI staining. Cells were collected and resus-
pended in PBS and then 80 % ethanol for 10 min. After washing,
DAPI was added to a final concentration of 1 μg/ml and kept in the
dark for 15 min. ‘+’ denotes cells with 0.5 M NaCl (45 min)
treat-ment and ‘–’ denotes cells with water only (45 min)
treatment. b Flu-orescence intensity and proportion of cells with
altered nuclei were quantified using the Image J software and
analyzed using ~50 cells. All experiments were repeated three
times. The error bars denote the standard deviation (SD). *P <
0.05, **P < 0.01 vs strains without HAL2 overexpression for the
same treatment
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237Curr Genet (2017) 63:229–239
1 3
Overexpression of HAL2 in bdf1Δ causes down‑regulation of sulfur
assimilating genes
Hal2p participates in the sulfate assimilation pathway in which
sulfur, an important element in iron–sulfur clusters (ISCs), is
produced. Our microarray data show that the overexpression of HAL2
in bdf1Δ causes down-regulation of the genes involved in the
sulfate assimilation pathway, including MET2 (0.30 fold), MET3
(0.40 fold), MET10 (0.46 fold), MET14 (0.53 fold), MET16 (0.62
fold), MET17 (0.30 fold), MET28 (0.48 fold), and STR3 (0.28 fold)
(Fig. 2; Supplementary Tables 4 and 7). Hal2p is a prod-uct of the
sulfate assimilation pathways. Its overexpression may cause
feedback inhibition, which could down-regulate genes in this
pathway. The feedback inhibition may also result in less sulfur and
iron–sulfur proteins.
Discussion
Iron is an essential element for all eukaryotes. It can readily
gain or lose electrons by alternating between two valence states by
oxidation/reduction reactions (Kaplan et al. 2006). It participates
in various reactions in the form of elemental iron, oxoiron or
oxoiron-zinc, heme, and iron–sulfur clus-ters (Kaplan and Kaplan
2009). As a result, iron plays a key role in respiration and heme
regulation.
There are two mechanisms by which iron can traverse the cell
membrane. One system completes iron uptake to cytoplasm after
binding to a siderophore. In yeast, this system requires the cell
wall mannoproteins Fit1p, Fit2p, and Fit3p to capture siderophores
(Protchenko et al. 2001). These proteins can retain siderophores
that have a high affinity for Fe3+ in the cell wall (Kaplan and
Kaplan 2009). In addition to the cytoplasm and vacuole, iron is
also pre-sent in the endosome and mitochondria, where iron is
inserted into heme and iron–sulfur clusters (ISCs). These proteins
constitute cellular iron pools.
Bdf1p is a transcriptional regulator and part of the basal
transcription factor TFIID (Lygerou et al. 1994; Matang-kasombut et
al. 2000). Bdf1p binds to histone H4 with a preference for
acetylated forms (Matangkasombut and Buratowski 2003). CK2
phosphorylation of Bdf1p may regulate RNA polymerase II
transcription and/or chroma-tin structure (Sawa et al. 2004). No
details are given in the previous studies about the function of
iron in the bdf1Δ mutant.
In the current study, we performed comparative tran-scriptomics
to obtain a better understanding of the molec-ular mechanism by
which HAL2 overexpression in bdf1Δ reverses the salt sensitivity of
bdf1Δ. We found that several pathways are involved in the reversal
of salt sensitivity by HAL2 overexpression or HDA1 deletion. These
pathways
include iron acquisition, heme degradation, sterol
biosyn-thesis, sulfur assimilation, and nitrogen metabolism.
The genes that changed significantly are mostly linked to
cellular iron and/or heme metabolism either directly or indirectly.
The iron response at transcriptional level is similar to
iron-deprived conditions reported previously, in which the most
notable up-regulated genes are those related to iron acquisition
(Hausmann et al. 2008). By com-paring the heme levels in WT, bdf1Δ,
and bdf1Δ + HAL2, it was revealed that the heme level was decreased
in bdf1Δ + HAL2 compared to bdf1Δ (Fig. 3). It is not clear how the
decreased heme level facilitates the recovery of salt sensitivity
of bdf1Δ.
In Saccharomyces cerevisiae, sulfate and ATP are used to produce
adenosine 5′-phosphosulfate (ApS) and then 3′-phosphoadenosine-
5′-phosphosulfate (pApS) for sul-fate assimilation. Sulfite and pAp
are produced by the pApS reductase reaction. Sulfide and methonine
are formed through these reactions (Todeschini et al. 2006). HAL2
encodes a nucleotidase, which catalyzes 3′-phosphoadeno-sine
5′-phosphate (pAp) to AMP and 3′-phosphoadenosine 5′-phosphosulfate
(pApS) to APS in the sulfate assimila-tion process (Todeschini et
al. 2006). It appears that HAL2 overexpression might cause feedback
inhibition, which could down-regulate genes in this pathway. Its
feedback inhibition might cause reduction of the iron–sulfur
pro-tein Sdh2p. The previous studies have shown that the
dys-function of Fe–S protein assembly leads to cellular and
mitochondrial global remodeling, and defects in ISC bio-genesis
affect genetic changes in the genome (Flury et al. 1976; Rasmussen
et al. 2003). This will induce cellular and mitochondrial
transcriptional remodeling (Hausmann et al. 2008). Thus, a relative
decline in the Sdh2p protein level could also cause a global
remodeling of the transcriptome of bdf1Δ + HAL2 vs bdf1Δ. ISC
machinery defects induce regulation among cellular Fe/S protein
maturation, respira-tion, heme biosynthesis, and the regulation of
cellular iron homeostasis.
The high levels of heme and Sdh2p proteins in bdf1Δ (Fig. 3)
might inhibit cells from acquiring additional iron. Thus, we found
that genes related to iron acquisition and iron–sulfur protein
synthesis were inhibited in bdf1Δ vs WT. We can speculate that the
deletion of BDF1 causes an imbalance in iron homeostasis and that
the overexpression of HAL2 in bdf1Δ might recover the iron
homeostasis and then lead to a reversal of the sensitivity of
bdf1Δ.
Biosynthesis of sterol is associated with mitochondrial
morphogenesis (Schneiter 2007). Genes in the ERG fam-ily are found
to have a wide range of increased activity in bdf1Δ + HAL2 vs
bdf1Δ, suggesting that the overexpres-sion of HAL2 makes a large
change in the morphology of the mitochondria. This is consistent
with the results of our previous study (Chen et al. 2013). Abnormal
mitochondrial
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238 Curr Genet (2017) 63:229–239
1 3
function affects ISC biogenesis (Flury et al. 1976; Rasmus-sen
et al. 2003). We found that the mitochondria in bdf1Δ are
dysfunctional and overexpression of HAL2 restored the mitochondrial
functions, e.g., membrane potential, and decreased levels of
reactive oxygen species (ROS) (Chen et al. 2013). We have further
shown in this study that the overexpression of HAL2 decreases the
heme and ISC levels close to those found in wild type under salty
conditions.
NO is formed in mitochondria (Zorov et al. 2007) and is
cytotoxic, reversibly inhibiting respiration (Brown 2001). NO can
react with ferric iron (Fe3+) without any kinetic restriction
(Bindoli et al. 2008; Grant et al. 1996). It can also stimulate
transcriptional up-regulation of genes involved in iron acquisition
and Fe/S cluster assembly and repair (Jones-Carson et al. 2008).
Thus, the up-regulation of iron-acquisition genes by overexpression
of HAL2 in bdf1Δ could help obtain more iron that can be used for
elimination of the excess NO in bdf1Δ. On the other hand,
iron–sulfur clusters in proteins are targets for reactive nitrogen
species (Jones-Carson et al. 2008). Higher con-centrations of NO
can displace iron from iron–sulfur cent-ers causing loss of iron,
which inhibits complexes in the respiratory chain (Brown 2001). We
speculate that iron–sulfur centers have no activity in light of the
higher propor-tion of cells with NO in bdf1Δ. Overexpression of
HAL2 in bdf1Δ may cause an influx of vast amounts of iron into the
cell which can react with the excess of NO in bdf1Δ. Owing to the
decreased proportion of cells with NO in bdf1Δ + HAL2, most cells
return to normal and then the synthesis of the iron–sulfur protein
Sdh2p is similar to that in the wild type. Removing NO may also
reversibly restore respiration (Brown 2001). That is why
overexpression of HAL2 restores the normal function of the
mitochondria (Chen et al. 2013), which is important for osmotic
toler-ance and adaption (Pastor et al. 2009). In addition, NO can
inhibit autophagosome formation (Sarkar et al. 2011). In our
previous data, bdf1Δ is apparently autophagy-defec-tive (Chen et
al. 2013), and this defect may be caused by higher levels of NO. In
addition, we also found that BDF1 deletion raises the proportion of
cells with NO and with altered nuclei under salty conditions. HAL2
overexpres-sion in bdf1Δ decreases these two proportions. This
sug-gests that NO accumulation in bdf1Δ has a lethal effect and
HAL2 overexpression in bdf1Δ functions to reverse this effect.
In summary, our comparative transcriptome analyses reveal that
overexpression of HAL2 in bdf1Δ induces genes for iron-acquisition
and mitochondria remodeling, which decreases the mitochondrial
iron–sulfur cluster assembling. Our results suggest that HAL2
overexpression in bdf1Δ restores mitochondrial functions,
especially those linked to iron homeostasis. This restoration, in
return, facilitates reversal of the salt sensitivity of bdf1Δ. Our
study showed
new light on the molecular mechanism of salt stress response in
yeast.
Acknowledgments This research was supported by the Young
Scien-tists Fund (No. 41401285) of the National Natural Science
Founda-tion of China and the Open Foundation of State Key
Laboratory of Estuarine and Coastal Research (SKLEC-KF201412). This
work was also supported by the National Natural Science Foundation
of China (Nos. 30170021, 30671143, 30570031, 41371257 and
41573071).
References
Almeida B, Buttner S, Ohlmeier S, Silva A, Mesquita A,
Sampaio-Marques B, Osorio NS, Kollau A, Mayer B, Leao C, Laranjinha
J, Rodrigues F, Madeo F, Ludovico P (2007) NO-mediated apop-tosis
in yeast. J Cell Sci 120:3279–3288
Bindoli A, Fukuto JM, Forman HJ (2008) Thiol chemistry in
per-oxidase catalysis and redox signaling. Antioxid Redox Signal
10:1549–1564
Brown GC (2001) Regulation of mitochondrial respiration by
nitric oxide inhibition of cytochrome c oxidase. Biochim Biophys
Acta 1504:46–57
Camougrand N, Grelaud-Coq A, Marza E, Priault M, Bessoule J-J,
Manon S (2003) The product of the UTH1 gene, required for
Bax-induced cell death in yeast, is involved in the response to
rapamycin. Mol Microbiol 47:495–506
Chen L, Liu L, Wang M, Fu J, Zhang Z, Hou J, Bao X (2013) Hal2p
functions in Bdf1p-involved salt stress response in Saccharomy-ces
cerevisiae. PLoS One 8:e62110
Chen L, Wang M, Hou J, Liu L, Fu J, Shen Y, Zhang Z, Bao X
(2014) Regulation of Saccharomyces cerevisiae MEF1 by Hda1p affects
salt resistance of bdf1Δ mutant. FEMS Yeast Res 14:575–585
Chung J, Bachelder RE, Lipscomb EA, Shaw LM, Mercurio AM (2002)
Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF
translation: a survival mechanism for carcinoma cells. J Cell Biol
158:165–174
Dancis A, Klausner RD, Hinnebusch AG, Barriocanal JG (1990)
Genetic evidence that ferric reductase is required for iron uptake
in Saccharomyces cerevisiae. Mol Cell Biol 10:2294–2301
Deng Y, Guo Y, Watson H, Au WC, Shakoury-Elizeh M, Basrai MA,
Bonifacino JS, Philpott CC (2009) Gga2 mediates sequential
ubiquitin-independent and ubiquitin-dependent steps in the
traf-ficking of ARN1 from the trans-Golgi network to the vacuole. J
Biol Chem 284:23830–23841
Dumont ME, Ernst JF, Sherman F (1988) Coupling of heme
attach-ment to import of cytochrome-C into yeast
mitochondria—stud-ies with heme lyase-deficient mitochondria and
altered apocy-tochromes-C. J Biol Chem 263:15928–15937
Ferreira MCD, Bao XM, Laize V, Hohmann S (2001) Transposon
mutagenesis reveals novel loci affecting tolerance to salt stress
and growth at low temperature. Curr Genet 40:27–39
Flury F, von Borstel RC, Williamson DH (1976) Mutator activity
of petite strains of Saccharomyces cerevisiae. Genetics
83:645–653
Froissard M, Belgareh-Touze N, Dias M, Buisson N, Camadro JM,
Haguenauer-Tsapis R, Lesuisse E (2007) Trafficking of sidero-phore
transporters in Saccharomyces cerevisiae and intracellular fate of
ferrioxamine B conjugates. Traffic 8:1601–1616
Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on
the transformation of intact yeast cells by the LiAc/SS-DNA/PEG
procedure. Yeast 11:355–360
Grant CM, MacIver FH, Dawes IW (1996) Glutathione is an
essential metabolite required for resistance to oxidative stress in
the yeast Saccharomyces cerevisiae. Curr Genet 29:511–515
-
239Curr Genet (2017) 63:229–239
1 3
Hausmann A, Samans B, Lill R, Muhlenhoff U (2008) Cellular and
mitochondrial remodeling upon defects in iron-sulfur protein
biogenesis. J Biol Chem 283:8318–8330
Ho YH, Gasch AP (2015) Exploiting the yeast stress activated
signal-ing network to inform on stress biology and disease
signaling. Curr Genet 61:503–511
Ihrig J, Hausmann A, Hain A, Richter N, Hamza I, Lill R,
Muhlenhoff U (2010) Iron regulation through the back door:
iron-dependent metabolite levels contribute to transcriptional
adaptation to iron deprivation in Saccharomyces cerevisiae.
Eukaryot Cell 9:460–471
Jones-Carson J, Laughlin J, Hamad MA, Stewart AL, Voskuil MI,
Vazquez-Torres A (2008) Inactivation of [Fe–S] metalloproteins
mediates nitric oxide-dependent killing of Burkholderia mallei.
PLoS One 3:e1976
Kaplan CD, Kaplan J (2009) Iron acquisition and transcriptional
regu-lation. Chem Rev 109:4536–4552
Kaplan J, McVey Ward D, Crisp RJ, Philpott CC (2006)
Iron-depend-ent metabolic remodeling in S. cerevisiae. Biochim
Biophys Acta 1763:646–651
Kim D, Yukl ET, Moenne-Loccoz P, Montellano PROd (2006) Fungal
heme oxygenases: functional expression and characterization of Hmx1
from Saccharomyces cerevisiae and CaHmx1 from Can-dida albicans.
Biochemistry 45:14772–14780
Lee J-H, Maskos K, Huber R (2009) Structural and functional
studies of the yeast class II Hda1 histone deacetylase complex. J
Mol Biol 391:744–757
Li L, Miao R, Jia X, Ward DM, Kaplan J (2014) Expression of the
yeast cation diffusion facilitators Mmt1 and Mmt2 affects
mito-chondrial and cellular iron homeostasis: evidence for
mitochon-drial iron export. J Biol Chem 289:17132–17141
Liu X, Zhang X, Wang C, Liu L, Lei M, Bao X (2007) Genetic and
comparative transcriptome analysis of bromodomain factor 1 in the
salt stress response of Saccharomyces cerevisiae. Curr Microbiol
54:325–330
Liu X, Yang H, Zhang X, Liu L, Lei M, Zhang Z, Bao X (2009)
Bdf1p deletion affects mitochondrial function and causes apoptotic
cell death under salt stress. FEMS Yeast Res 9:240–246
Lygerou Z, Conesa C, Lesage P, Swanson RN, Ruet A, Carlson M,
Sentenac A, Seraphin B (1994) The yeast Bdf1 gene encodes a
transcription factor involved in the expression of a broad class of
genes including snRNAs. Nucleic Acids Res 22:5332–5340
Matangkasombut O, Buratowski S (2003) Different sensitivities of
bromodomain factors 1 and 2 to histone H4 acetylation. Mol Cell
11:353–363
Matangkasombut O, Buratowski RM, Swilling NW, Buratowski S
(2000) Bromodomain factor 1 corresponds to a missing piece of yeast
TFIID. Gene Dev 14:951–962
Murguia JR, Belles JM, Serrano R (1995) A salt-sensitive
3′(2′),5′-bisphosphate nucleotidase involved in sulfate activation.
Science 267:232–234
Murguia JR, Belles JM, Serrano R (1996) The yeast HAL2
nucle-otidase is an in vivo target of salt toxicity. J Biol Chem
271:29029–29033
Pastor MM, Proft M, Pascual-Ahuir A (2009) Mitochondrial
function is an inducible determinant of osmotic stress adaptation
in yeast. J Biol Chem 284:30307–30317
Philpott CC, Protchenko O (2008) Response to iron deprivation in
Saccharomyces cerevisiae. Eukaryot Cell 7:20–27
Protchenko O, Philpott CC (2003) Regulation of intracellular
heme levels by HMX1, a homologue of heme oxygenase, in
Saccharo-myces cerevisiae. J Biol Chem 278:36582–36587
Protchenko O, Ferea T, Rashford J, Tiedeman J, Brown PO,
Botstein D, Philpott CC (2001) Three cell wall mannoproteins
facilitate the uptake of iron in Saccharomyces cerevisiae. J Biol
Chem 276:49244–49250
Rasmussen AK, Chatterjee A, Rasmussen LJ, Singh KK (2003)
Mito-chondria-mediated nuclear mutator phenotype in Saccharomyces
cerevisiae. Nucleic Acids Res 31:3909–3917
Robyr D, Suka Y, Xenarios I, Kurdistani SK, Wang A, Suka N,
Grunstein M (2002) Microarray deacetylation maps determine
genome-wide functions for yeast histone deacetylases. Cell
109:437–446
Sarkar S, Korolchuk VI, Renna M, Imarisio S, Fleming A, Williams
A, Garcia-Arencibia M, Rose C, Luo S, Underwood BR, Kro-emer G,
O’Kane CJ, Rubinsztein DC (2011) Complex inhibitory effects of
nitric oxide on autophagy. Mol Cell 43:19–32
Sawa C, Nedea E, Krogan N, Wada T, Handa H, Greenblatt J,
Bura-towski S (2004) Bromodomain factor 1 (Bdf1) is phosphorylated
by protein kinase CK2. Mol Cell Biol 24:4734–4742
Schneiter R (2007) Intracellular sterol transport in eukaryotes,
a con-nection to mitochondrial function? Biochimie 89:255–259
Singh A, Kaur N, Kosman DJ (2007) The metalloreductase Fre6p in
Fe-efflux from the yeast vacuole. J Biol Chem 282:28619–28626
Todeschini AL, Condon C, Benard L (2006) Sodium-induced GCN4
expression controls the accumulation of the 5′ to 3′ RNA
degra-dation inhibitor, 3′-phosphoadenosine 5′-phosphate. J Biol
Chem 281:3276–3282
Wu J, Carmen AA, Kobayashi R, Suka N, Grunstein M (2001) HDA2
and HDA3 are related proteins that interact with and are essential
for the activity of the yeast histone deacetylase HDA1. Proc Natl
Acad Sci USA 98:4391–4396
Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP
(2002) Normalization for cDNA microarray data: a robust composite
method addressing single and multiple slide systematic variation.
Nucleic Acids Res 30:e15
Zhang L, Zhang Y, Zhou Y, An S, Zhou Y, Cheng J (2002) Response
of gene expression in Saccharomyces cerevisiae to amphotericin B
and nystatin measured by microarrays. J Antimicrob Chem-other
49:905–915
Zorov DB, Isaev NK, Plotnikov EY, Zorova LD, Stelmashook EV,
Vasileva AK, Arkhangelskaya AA, Khrjapenkova TG (2007) The
mitochondrion as janus bifrons. Biochemistry (Mosc)
72:1115–1126
HAL2 overexpression induces iron acquisition in bdf1Δ cells
and enhances their salt resistanceAbstract
IntroductionMaterials and methodsStrains and mediaGene
disruptionPlasmid construction and transformationNitric oxide
(NO) production and DAPI stainingRNA extraction
and quantitative PCR (qPCR)Gene expression profile analysis
by microarrayHeme staining in gel and western blot
analysisStatistical analysis
ResultsOverexpression of HAL2 in bdf1Δ increases gene
expression in the iron-acquisition pathwayIn bdf1Δ,
overexpression of HAL2 causes reduced levels of heme
and iron–sulfur proteinsOverexpression of HAL2
in bdf1Δ causes a decrease in nitric oxide
productionOverexpression of HAL2 in bdf1Δ up-regulates
sterol biosynthesisOverexpression of HAL2 in bdf1Δ causes
down-regulation of sulfur assimilating genes
DiscussionAcknowledgments References