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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 bxm@sdu.edu.cn

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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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)

http://rsb.info.nih.gov/ij/http://rsb.info.nih.gov/ij/

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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

http://www.genome.jp/kegg/expression/http://www.genome.jp/kegg/expression/http://www.yeastgenome.org/http://www.yeastgenome.org/http://www.helmholtz-muenchen.dehttp://www.ncbi.nlm.nih.gov/geohttp://rsb.info.nih.gov/ij/http://rsb.info.nih.gov/ij/

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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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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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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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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).

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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