Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii
© Vaupotič and Plemenitaš; licensee BioMed Central Ltd. 2007
Received: 05 February 2007
Accepted: 16 August 2007
Published: 16 August 2007
Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG) signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed.
A cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells.
Extremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at high salt concentrations and interacting with HwHog1 MAP kinase, suggesting atypical processes deserving of further study.
When a living organism is subjected to extreme environmental conditions for an extended period of time, an adaptive response may become crucial for its continued existence. The response of eukaryotic cells such as yeast to environmental stress involves complex changes in gene expression which subsequently lead to various metabolic responses to induce adaptation to the new conditions. Fluctuating external osmolarity, like changes in salt concentration, leads to altered transcription of many responsive genes in an effort to counteract the stress with the activity of their protein products. One of the earliest protective biochemical responses is the biosynthesis and accumulation of glycerol as an osmolyte via the activation of corresponding genes. The resulting glycerol accumulates in the cytosol and leads to increased internal osmolarity, thus restoring the osmotic gradient between the cells and their environment . In the salt-sensitive yeast Saccharomyces cerevisiae, the hyperosmotic stress caused by 0.4 M NaCl leads to the transient transcriptional induction of more than 1500 genes, as a consequence of simultaneous action of the general stress response pathway together with the h igh-o smolarity g lycerol (HOG) mitogen-activated protein kinase (MAPK) signaling pathway . A functional HOG pathway is essential for the efficient up-regulation of the vast majority of genes in response to hyperosmotic conditions [3, 4]. The terminal MAPK, Hog1, accumulates in the nucleus within minutes of exposure to high salt concentrations , whereupon it phosphorilates and activates the HOG-specific transcription factors Sko1  and Smp1 , or recruits Hot1  and the general stress-response transcriptional activators Msn1, Msn2 and Msn4 [9, 10] to the promoters of osmoinducible genes. Findings that Hog1 could be an integral part of the upstream activation complex, targeting not only the activators but also components of the general transcription machinery, such as RNA polymerase II [11, 12] together with Hog1-guided recruitment of Rpd3 histone deacetylase to the chromatin , have highlighted the additional level of complexity in the regulation of gene expression during hyperosmotic conditions.
To date, studies on hyperosmotic adaptation and salt tolerance in fungal species have been largely performed with the salt-sensitive model organism S. cerevisiae, for reasons of experimental convenience. However, the cellular machinery of S. cerevisiae is not adapted to the extreme hyperosmolar pressure caused by a salty environment with more than 1–2 M NaCl concentration. Therefore the specially adapted extremely halotolerant yeast-like fungus Hortaea werneckii represents a novel eukaryotic organism for studying cellular responses to extremely elevated environmental salinity. This naturally osmoadaptable species was first isolated from hypersaline crystallizer ponds in salterns , where NaCl concentration fluctuates from 0.5 M up to saturate solution levels (6 M). The increase in surrounding salt concentration is accompanied by increased intracellular glycerol accumulation in H. werneckii . The glycerol accumulation suggested the activation of the HOG-like pathway. The MAPK HwHog1 was later identified, showing its highest activity at a concentration of 4.5 M NaCl . Many cellular and physiological differences were observed between H. werneckii cells growing in the extremely hypersaline environment of 4.5 M NaCl and those growing in a moderate salinity of 3 M NaCl, which has been assigned as the optimal metabolic condition for H. werneckii [15, 17–19]. Therefore the characterization of the differential transcriptional response and identification of HwHog1-target genes in H. werneckii when under extreme hypersaline conditions could provide new insight into eukaryotic saline-response genetics.
In this study we have identified a set of differentially expressed genes in hyperosmotically adapted H. werneckii. Expression profiles of the genes were determined for both hyperosmotically-adapted and hyperosmotically-stressed H. werneckii cells. The identified genes were tested for interaction with HwHog1 and RNA polymerase II. Here we report a new insight into the differential expression of osmoresponsive genes in an extremely halotolerant eukaryotic microorganism and suggest a role for these genes in adaptive metabolism.
Results and Discussion
Since H. werneckii occupies a different ecological niche than S. cerevisiae, it is not surprising that these yeasts perceive hyperosmotic environments differently. In contrast to S. cerevisiae, H. werneckii can actively grow in a wide range of external salt concentrations, which shows that the cells are also well adapted to an extremely hypersaline environment. Differentially expressed genes in H. werneckii cells grown in different salinities therefore represent the transcriptional response of the adapted cells, rather than the stress response. Previous work has shown differences in the activation of HwHog1 between the halotolerant H. werneckii and S. cerevisiae . In the present work, we tried to address two questions: (i) what is the transcriptional response of H. werneckii adapted to a hypersaline environment compared with transcription under optimal growth conditions, and (ii) whether and how the major HOG pathway effector MAPK HwHog1 is associated with the expression of osmoresponsive genes in H. werneckii.
Bioinformatical characterization of the Hw4.5-3 subtracted library revealed over-representation of H. werneckii osmoresponsive genes in functional groups of metabolism and energy
Differentially expressed genes in H. werneckii adaptation to 3 M NaCl or 4.5 M NaCl. BLASTX matches of EST clones derived from the Hw4.5-3 subtraction library and their functional categorizations based on MIPS are collected. Similarities with probability <10-5 were regarded as being significant, and others as not determined (ND). Genes selected for the expression and chromatin immunoprecipitation analyses are printed in bold. aGenBank accession number. bNumbers indicate the functional groups (see Table 3). Numbers in brackets correspond to literature-based arbitrarily-assigned functional categories for ESTs with orthologs in organisms other than S. cerevisiae. cFold induction (fI, positive value) or fold repression (fR, negative value) in 4.5 M NaCl- vs. 3 M NaCl-adapted H. werneckii cells. Numbers indicated are mean values of three independent RT-PCR experiments, and the representative gels are shown in figures 1 and 2.
Characteristic or description
Amino acid permease
Mitochondrial ATPase alpha-subunit
2. 20. 34.
Mitochondrial ATPase beta-subunit
2. 20. 34. 40.
Mitochondrial ATPase gamma-subunit
2. 20. 34.
1. 10. 16. 30. 40. 43.
Septin complex component
10. 16. 34. 40. 42. 43.
Mitochondrial citrate synthase
1. 2. 42.
Cytochrome c oxidase subunit I
Extracellular matrix 33 protein
10. 42. 43.
Eukaryotic translation elongation factor 2 (eEF-2)
Transcription elongation factor
10. 11. 40
p24 component of the COPII-coated vesicles
Mitochondrial acyl-carrier protein
Ferric-chelate reductase-7 transmembrane component
Eukaryotic translation initiation factor 5B (eIF-5B)
NADP(+)-specific glutamate dehydrogenase
1. 2. 16.
1. 2. 16.
FAD-dependent glycerol-3-phosphate dehydrogenase
1. 2. 42.
10. 11. 16.
Heat shock protein 90
1. 2. 10. 14. 32. 34. 43.
10. 11. 16.
Serine-threonine protein kinase and endoribonuclease
1. 11. 14. 30. 32.
Endoplasmic reticulum luminal chaperone
1. 10. 14. 16. 20. 32. 41. 42.
Mitochondrial succinyl-CoA ligase beta-chain
1. 2. 16.
Mitochondrial malate dehydrogenase
1. 2. 42.
ATP adenosine-5'-phosphosulfate 3'-phosphotransferase
Cobalamin-independent methionine synthase
Mitochondrial phosphate transport protein
High mobility group protein A
10. 11. 40. 43.
Unsaturated phospholipid methyltransferase
Protein disulphide isomerase precursor
1. 2. 16.
Plasma membrane proton-exporting ATPase
2. 20. 34.
Pathogenesis-related protein precursor
Serine rich pumilio family RNA binding domain protein
1. 10. 11. 16.
SWI/SNF family DEAD/DEAH box helicase
60S ribosomal protein 10
10. 12. 14.
60S ribosomal protein 16A
60S ribosomal protein 22A
60S ribosomal protein 2B
60S ribosomal protein 3
60S ribosomal protein 6A
60S ribosomal protein 7B
26S proteasome regulatory particle subunit
11. 40. 30.
40S ribosomal protein 10B
40S ribosomal protein 12
40S ribosomal protein 15
12. 14. 20.
40S ribosomal protein 16A
40S ribosomal protein 17A
40S ribosomal protein 26B
40S ribosomal protein 8A
40S ribosomal protein 8B
Mitochondrial inner membrane protein chaperone
2. 16. 32.
1. 2. 42.
Heat shock protein 70
14. 16. 32.
Catalytic subunit of the oligosaccharyltransferase complex
Cell wall synthesis protein related to glucanases
1. 40. 42.
Eukaryotic elongation factor 1-alpha (EF-1A)
Ekcaryotic translation initiation factor 1-alpha (eIF-4A)
Mitochondrial import receptor translocase
16. 20. 34. 40. 42. 43.
10. 14. 42.
10. 14. 42.
1. 2. 14.
Phosphatidylinositol 3-kinase homolog
1. 14. 20. 30.
Weakly similar to H. sapiens cyclin-dependent kinase 6
Mannose-P-dolichol utilization defect 1 protein
(14. 16. 20.)
Ubiquitin associated protein 2-like protein
(14. 16. 32.)
Histidine-rich glycoprotein precursor
Acetyl xylan esterase
(1. 32. 34.)
(10. 11. 40.)
(1. 32. 34.)
Hyperosmolarity-induced mRNA 18
Weakly similar to yeast stress response transcription factor Crz1
Weakly similar to yeast nitrosoguanidine resistance protein Sng1
Weakly similar to yeast prespliceosomal RNA helicase Prp5
Hyperosmolarity-induced mRNA 22
Weakly similar to yeast Hsp70 protein Ssz1
Weakly similar to yeast t-SNARE protein Sso1
Hyperosmolarity-induced mRNA 25
Weakly similar to yeast transcriptional repressor Rgm1
Hyperosmolarity-induced mRNA 27
Hyperosmolarity-induced mRNA 28
Weakly similar to yeast histone deacetylase complex subunit Pho23
Ten most frequent cDNAs in the Hw4.5-3 subtracted library. GenBank homologies, hit numbers and percentages of the most redundant cDNA clones are presented. aGenBank accession number of the most similar sequence identified by BLASTX alignment. bNumber of clones from Hw4.5-3 subtracted library assigned to the same GenBank accession number.
BLAST hit Acc. No.a
Number of hits b
Plasma membrane proton-exporting ATPase
Heat shock protein 70
Cytochrome c oxidase subunit I
Pathogenesis-related protein precursor
Acetyl xylan esterase
Unsaturated phospholipid methyltransferase
Cobalamin-independent methionine synthase
High mobility group protein A
Sum of top ten redundant clones
Distribution of differentially expressed genes from the Hw4.5-3 library by functional categories. Functional categorization was performed according to the MIPS database [22, 23] and annotated by the FunCat functional annotation scheme for systematic classification [22, 23]. Grouping of genes into indicated groups is shown as percent (%) of total identified EST from Hw4.5-3 library in descending order of abundance.
% N = 95
Cellular transport, transport facilitation and transport routes
Cell-cycle and DNA processing
Protein with binding function or cofactor requirement (structural or catalytic)
Interaction with the cellular environment
Biogenesis of cellular components
Cell rescue, defense and virulence
Cell type differentiation
Cellular communication/signal transduction mechanism
Not determined/Unknown function
The majority of osmoresponsive genes in halotolerant H. werneckii are up-regulated in 4.5 M NaCl
Expression of osmoresponsive genes is different in salt-adapted and salt-stressed H. werneckii cells
Induced and repressed genes in H. werneckii upon acute hypersaline stress. The stress-responsive genes were classified according to the kinetic of the response over the 120 min course of the experiment. Fold induction or repression is indicated as mean value of three independent RT-PCR experiments, and the representative gels are shown in figures 1 and 2.
These data demonstrate that in contrast to the response of the adapted cells, only a portion of the identified differentially expressed genes in H. werneckii transcriptionally responded to acute hypersaline stress as well. Interestingly, HwFRE7, HwKAR2, HwNHP6A, HwSTT3, SOL18 and SOL24 were up-regulated in cells adapted to extreme hypersaline conditions, but they were actually repressed after acute hypersaline stress. Additionally, HwCDC3, HwSUN4 and SOL11 were down-regulated in cells adapted to extreme hypersaline conditions, but induced during stress. This observation indicates an additional level of complexity in regulation of gene expression in halotolerant yeast when compared with the stress response of salt-sensitive S. cerevisiae.
It is noteworthy that the expression profile of H. werneckii adapted to 4.5 M NaCl is very different from the hypersaline stressed cells of S. cerevisiae. In comparison with the microarray studies performed with S. cerevisiae [2, 25, 26], using SSH-MOS we have identified only 18 gene orthologs that were clearly up-regulated in adapted or stressed H. werneckii and also in salt-stressed wild-type S. cerevisiae: HwBMH1, HwCIT1, HwGND2, HwGUT2, HwHSP82, HwKGD2, HwMDH1, HwOPI3, HwRPL16A, HwRPS10B, HwRPS15, HwRPS8A, HwSHY1, HwSSA4, HwTDH1, HwTIF1, HwTKL1, and HwUGP1. However, some discrepancies were observed between the compared microarray data in S. cerevisiae studies mentioned above. Among upregulated genes in salt-stressed S. cerevisiae, only HwGUT2, HwOPI3, HwTKL1 and HwUGP1 were also salt-stress responsive in H. werneckii. The mRNA levels of HwECM33, HwEFT2, HwFAS1, HwFUN12, HwGDH1, HwMET14, HwMET17, HwMET6, HwMIR1, HwNUC1, HwPMA2, HwPRY1 HwRPL6A, HwRPS16A, HwSAM2, HwSTT3, HwTEF1, HwTIF1 and HwTOM40 were induced in H. werneckii adapted to 4.5 M NaCl, whereas these levels diminished or remained unaffected in salt-stressed S. cerevisiae. Two genes, DBP2 and SUN4 were substantially downregulated in both, the hypersalinity-adapted H. werneckii and salt stressed S. cerevisiae. It was also found that GND2, GPD1 and SSA4 belong to a common environmental response (CER) genes in S. cerevisiae, which were affected not only by high salinity but various stresses such as heat, high or low pH, oxidative stress and sorbitol  and therefore might also present the CER response in H. werneckii. Interestingly, the induction of SSA4 was extremely stress-responsive in S. cerevisiae (47-fold induction), whereas it was only slightly affected by high salt concentration in H. werneckii (1.8-fold in 4.5 M NaCl).
One theory is that the main difference between salt-sensitive and halotolerant organisms in the expression of osmoresponsive genes relates to the inducibility and/or maintenance of the transcription level of protective genes. The perception threshold for the extracellular "hyper"-osmolarity in extremely halotolerant yeasts must be set at concentrations higher than salt-sensitive unicellular eukaryotes could even survive. In H. werneckii adapted to these extreme conditions, the protective response remains "on", meaning that there is a long-term up-regulation program of specific genes, and this up-regulation does not decrease over time.
HwHog1 associates with 36 novel osmoresponsive genes in chromatin of long-term adapted cells
The transcriptional induction or repression of approximately 500 genes in S. cerevisiae that are strongly responsive to salt stress was highly or fully dependent on the hyperosmolarity-responsive MAPK Hog1, indicating that the Hog1-mediated signaling pathway plays a key role in global gene regulation under saline stress conditions [2, 28]. It has been shown that following exposure to salt stress, Hog1 is retained in the nucleus and becomes associated with the chromatin of target genes . We approached the study of endogenous HwHog1 interaction with the chromatin regions of identified up-regulated genes in adapted H. werneckii cells by a chromatin immunoprecipitation (ChIP) PCR assay. Lacking information on promoter regions for the identified differentially-expressed genes in H. werneckii, a ChIP-coding region PCR amplification was performed. It has recently been shown that the activated Hog1 in S. cerevisiae is associated with elongating RNA polymerase II and is therefore recruited to the entire coding region of osmoinducible genes . HwGPD1A was used as a positive control for the HwHog1-ChIP. As negative controls for the association of HwHog1 with DNA, the HwCOB1 gene encoded in mitochondrial DNA and the 26S rRNA gene (Hw26SRR) transcribed by RNA polymerase I were used.
The results of the HwHog1-DNA interactions determined by ChIP are shown in Figure 2, third column. As evidenced by PCR, the protein HwHog1 cross-linked with the coding region of the positive control HwGPD1A and 36 of the differentially expressed genes (50%), but not with the negative control genes HwCOB1 and Hw26SRR. As seen from the PCR product level, for 34 up-regulated genes the interaction with HwHog1 was stronger in cells adapted to 4.5 M NaCl. In contrast, for 2 down-regulated genes (HwDBP2 and SOL11), the HwHog1-ChIP signal was stronger in cells adapted to 3 M NaCl.
Genome-wide expression profiling studies using wild-type and hog1 mutant S. cerevisiae cells were performed to comparatively identify genes whose up-regulation of expression was dependent on Hog1. Of several hundred genes whose RNA levels were Hog1-dependent, a relatively small subset of approximately 40 high-osmolarity induced genes had a strong requirement for Hog1 for their induction [26, 28]. Among them, only the UGP1 ortholog was induced in 4.5 M NaCl adapted and salt-stressed H. werneckii cells. Other yeast orthologs of HwHog1-ChIP positive genes in H. werneckii were reported for the first time in the present study in connection with MAPK Hog1. However, the HwHog1-ChIP did not confirm the HwHog1 interaction with the HwUGP1 gene in salt-adapted H. werneckii. The relative distribution of HwHog1-dependent genes was approximately equivalent among functional categories, except in the case of transcription, cellular transport, signal transduction mechanism and cell fate categories (MIPS categories 11, 20, 30 and 41, respectively), where the HwHog1-ChIP positive genes represented more than 70% of tested genes. Only 2 of 10 tested SOL genes (SOL23 and SOL28) with unknown functions (ND in table 1) were HwHog1-ChIP positive. Caution must be applied to the interpretation of genes which were HwHog1-ChIP negative, since many of them were salt-stress responsive. These genes could still be regulated by HwHog1. It is possible that during long-term adaptation the continuous interaction of HwHog1 with their genomic region is not obligatory, and thus HwHog1 was not cross-linked with the chromatin. Alternatively, HwHog1 could activate responsible factors more distal to the chromatin, thereby avoiding the cross-linking range.
HwHog1 and RNA polymerase II co-localization in coding regions of osmoresponsive genes is reflected by elevated levels of corresponding transcript in adapted cells
It has been previously shown that in the HOG response, the nuclear retention and chromatin association of Hog1 in S. cerevisiae depends on co-localization with general transcription machinery components [11, 12]. We further asked whether HwHog1 cross-linking occurs with co-localization of RNA polymerase II in the case of HwHog1-dependent genes. A sequential HwHog1-ChIP analysis (SeqChIP) using primers specific for the genes identified as HwHog1-positive was therefore performed after the primary RNAPolII-ChIP. If the interaction of HwHog1 and RNAPolII existed within the same genomic region of HwHog1-positive genes, the PCR signal should be obtained in eluates of SeqChIP. As shown in Figure 2, fourth column, the co-localization of HwHog1 and RNA polymerase II existed in 17 cases out of 36 HwHog1-ChiP positive differentially expressed genes. Similarly, if the co-occupancy of HwHog1 and RNAPolII favored gene expression, then the relative ratio of amplified PCR products from SeqChIP eluates should reflect the relative ratio of RT-PCR results from the gene expression in adapted cells. As shown in Figure 2, the ratio of amplified PCR products in SeqChIP-positive genes does indeed reflect the ratio of mRNA levels observed by RT-PCR in both conditions of adaptation. Co-occupation of HwHog1 and RNA polymerase II in target genes resulted in an increased PCR signal in SeqChIP, with an accompanying increased level of corresponding transcript in RT-PCR analyses.
Taken together, these observations indicate a stimulating role for HwHog1 and RNA polymerase II co-localization on the efficiency of transcription of indicated genes, even in long-term high-salt adapted cells. Regarding the coding region positioning of primer pairs used in this study, we can say that in H. werneckii HwHog1 also associates with the elongating RNA polymerase II, as has recently been shown in S. cerevisiae . In our study, H. werneckii cells used for chromatin immunoprecipitation analyses were completely adapted to extremely high environmental salt concentration by long-term growth in media containing either 3 M or 4.5 M NaCl. These results thus reflect HwHog1/RNAPolII-chromatin interactions, relevant for the extremely high saline conditions which until now could not be studied in salt-sensitive organisms. Moreover, to date this study is the first large-scale exploration of Hog1 interaction with target genes by chromatin immunoprecipitation in an organism with unavailable genomes. All similar studies have been performed using tagged and over-expressed proteins. Our study relays information based entirely on the cross-linking of endogenous HwHog1 and RNA polymerase II with their downstream targets on the chromatin, showing actual physiological interactions never studied before in eukaryotic cells adapted to such an extremely hyperosmotic condition.
An integrative model of osmoresponsive gene action through functional modules in H. werneckii
In our study, we have identified a set of 95 osmoresponsive genes in the extremely halotolerant black yeast H. werneckii adapted to a moderately saline environment of 3 M NaCl or an extremely saline environment of 4.5 M NaCl. Among them, more than half were related with general metabolism and energy. Thirteen unclassified SOL genes represent a specific transcriptional response unique to H. werneckii. A novel offset of 36 genes was shown as Hog1-dependent in long-term adaptation to extreme environments, previously not assigned as such in the salt-sensitive model organism S. cerevisiae. The combined data indicate important differences in the cellular processes of osmoadaptation between halotolerant and salt-sensitive yeasts. The novel set of osmoresponsive genes probably represents only a portion of actual differentially expressed genes in H. werneckii; however, we believe that valuable information was obtained concerning genes related to the hypersaline adaptation of extremely halotolerant eukaryotes.
Cell growth conditions
The H. werneckii strain MZKI B736 was obtained from culture collections of the Slovenian National Institute of Chemistry. Cells were grown at 28°C and 180 rpm in the defined YNB medium (0.17% (w/v) yeast nitrogen base, 0.08% (w/v) complete supplement mixture, 0.5% (w/v) ammonium sulphate, 2% (w/v) glucose in deionized water, pH 7.0), supplemented with the indicated NaCl concentration. Cells were harvested in the mid-exponential phase (OD600 nm 0.6–0.8) and frozen in liquid nitrogen. For hypersaline stress, H. werneckii cells were grown in YNB with 1 M NaCl to OD600 nm 0.8 and then transferred to the medium containing 4.5 M NaCl. Aliquots of the culture were removed before the stress was induced, and then at 10, 30, 60, 90 and 120 min after the stress. Cells were separated from the growth medium by fast filtration through a 0.45 μm-pore filters and then frozen in liquid nitrogen.
Suppression subtractive hybridization, mirror orientation selection and differential screening of subtracted library
Total RNA from cells of H. werneckii was isolated using TRI Reagent (Sigma-Aldrich) according to the manufacturer's instructions from mid-exponential phase cells grown in YNB media with 3 M or 4.5 M NaCl. The poly(A)RNA was isolated using the Oligotex mRNA Mini Kit (Qiagen). 1 μg of poly(A)RNA from each sample was used for reverse transcription to perform the cDNA suppression subtractive hybridization using the Clontech PCR-select cDNA Subtraction Kit (BD Bioscience) and Advantage cDNA PCR Polymerase Mix (BD Bioscience) according to the manufacturer's protocols. cDNA from a sample of cells growing in YNB medium with 4.5 M NaCl was used as a tester, while a sample from the 3 M NaCl medium was used as a driver in forward subtraction (and vice versa for reverse subtraction). Mirror orientation selection was implemented as described by Rebrikov et. al.  with some modifications. Briefly, after subtractive hybridization, the primary PCR of 27 cycles was performed in 5 parallel tubes. The samples were then combined, diluted 500-fold and subsequently amplified by 12 additional PCR cycles using the same primer and conditions as described for the primary PCR. A secondary PCR was then performed as described in the manufacturer's protocol. 150 ng of the resultant subtracted cDNA samples were digested with 10 U of Cfr9 I (Fermentas) for 1 h at 37°C in total volume of 20 μL. 1 μL of Cfr9 I-digested cDNA was mixed with 1 μL of 4 × hybridization buffer (2 M NaCl, 200 mM HEPES pH 8.3, 0.8 mM EDTA), 2 μL of water, denatured at 98°C for 1.5 min, and then hybridized at 68°C for 4 h. Samples were then diluted with 200 μL of the dilution buffer (50 mM NaCl, 20 mM HEPES pH 8.3, 0.2 mM EDTA) and heated at 70°C for 7 min. 1 μL of diluted cDNA was taken for 20 μL tertiary PCR with 0.6 μM adapter-specific primer NP2Rs (5'-GGTCGCGGCCGAGGT-3') by the following temperature program: 2 min at 72°C for initial extension of 3'-ends, followed by 23 cycles with 7 s at 95°C, 20 s at 62°C, and 2 min at 72°C. 1.5 μL of PCR products was cloned into pGEM-T Easy Vector (Promega) and transformed in JM109 competent cells (Promega). After blue/white selection on LB-Ampicillin/IPTG/X-Gal plates, white colonies were picked and arrayed on LB-Ampicillin plates, including 2 blue colonies as negative hybridization controls for the differential screening. Two identical colony lifts were made from each plate onto nitrocellulose membranes (Sigma), soaked with denaturation solution (0.5 M NaOH, 1.5 M NaCl), neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.4) and finally with washing solution (2 × SSC, 0.5 M Tris-HCl, pH 7.4), and then fixed by baking for 1–2 hours at 80°C. Forward and reverse subtracted hybridization probes were prepared from SSH-MOS secondary PCR products digested with Rsa I, Cfr 9I and Eae I to remove and degrade adaptors. After clean-up, 100 ng of each probe was labeled with [γ-32P]dCTP. Colony lifts were pre-hybridized with hybridization solution (5 × SSC, 0.5% SDS, 5 × Denhard's reagent, 0.1% SDS, 150 μg/mL salmon sperm DNA) for 1 h at 72°C, and then hybridized overnight at 72°C with labeled probes. Membranes were then washed once with low-stringency solution (2 × SSC, 0.5% SDS), twice with high-stringency solution (0.2 × SSC, 0.5% SDS), each for 20 min at 68°C, and then exposed to BioMax MR film (Kodak) overnight. For the forward-subtracted cDNA library (Hw4.5-3), clones that hybridized only to the forward-subtracted probe but not to the reverse-subtracted probe and clones that hybridized to both subtracted probes with a difference in signal intensity >5-fold were assigned as truly differential and chosen for sequencing. Plasmid DNA from positive clones was isolated using the Wizard Plus Minipreps Purification System (Promega), sequenced and analyzed by BLAST algorithms.
Reverse transcription PCR
The total RNA was isolated as described above and treated with DNase I (Fermentas). 1 μg of RNA was used for 20 μl of reverse transcription reaction using Superscript III Reverse Transcriptase (Invitrogen, USA) and random hexamer-primers (Promega) according to the manufacturer's protocols. PCR with Gotaq DNA polymerase (Promega) was performed using 0.5 μl of cDNA in a 20 μl PCR reaction with 15 nmol of specific primers listed in Additional file 1. Thermal cycling was programmed for 23 cycles, each consisting of 30 sec at 94°C, 30 sec at 60°C and 30 sec at 72°C. The cycle number of 23 was empirically determined in the optimal linear range of amplification by measuring the concentration of PCR products after the 20th, 22nd and 24th cycles. PCR products were resolved in agarose gels, documented by MiniBis (DNR BioImaging Systems), and the band intensities quantified with the TotalLab gel analysis program (Nonlinear Dynamics).
Immunoprecipitation of cross-linked chromatin (ChIP) was performed as described by Hecht et al. and Proft et al., and sequential ChIP (SeqChIP) as described by Geisberg et al.  with some modifications. Briefly, cells of H. werneckii growing in YNB media with 3 M or 4.5 M NaCl were cross-linked at OD600 0.8 using formaldehyde in a final concentration of 1% for 15 min at room temperature. Cross-linking was stopped with glycin at a final concentration of 0.125 M for 5 min at room temperature. Cells were harvested, washed twice with ice-cold PBS, pelleted, frozen in liquid nitrogen and broken with a dismembrator. 2.5 g of powdered cells were re-suspended in 10 mL of ChIP-L buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) containing a cocktail of fungal protease inhibitors (Sigma) and mixed by inversion for 30 min on ice. The samples were sonicated for five 15-s pulses, resulting in DNA fragments with an average length of 500 bp, and then centrifuged for 15 min at 10.000 × g to remove insoluble debris. For each ChIP experiment, 100 μL of supernatant was frozen as an input and 1 mL of supernatant was diluted in 9 mL of ChIP-D buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) with inhibitors. Chromatin solutions were pre-cleared with the salmon sperm DNA-precoated protein G-sepharose (Amersham) and then incubated with 5 μg of rabbit anti-Hog1 antibody (Santacruz) or mouse monoclonal anti-RNAPol II antibody 4H8 (Abcam) overnight at 8°C. 250 μL of precoated protein G-sepharose was added to each sample. Immunocomplexes were precipitated for 2 h at 8°C with shaking, pelleted 1 min at 1000 × g, and then pellets were washed with 2 mL of the following washing buffers: twice with ChIP-W1 (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1), once with ChIP-W2 (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1), once with ChIP-W3 (250 mM LiCl, 1% Na-deoxycholate, 1 mM EDTA, pH 8.0) and twice with ChIP-W4 (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Immunocomplexes were eluted twice with 200 μL of ChIP-E buffer (1% SDS, 100 mM NaHCO3) for 10 min at 65°C and eluates were collected. For SeqChIP, the RNAPolII-ChIP eluates (400 μL) were diluted with 3.6 mL of ChIP-dilution buffer and incubated for 2 h at room temperate with 1 μg of rabbit anti-Hog1 antibodies, and then processed as described above. ChIP eluates and inputs were reverse cross-linked in 0.2 M NaCl for 5 h at 65°C, incubated with 20 μg of RNase for 30 min at 37°C, followed by treatment with 10 μg of proteinase K. DNA was purified using the Wizard PCR Clean-up Purification System (Promega) and eluted with 150 μL of water for Hog-ChIP samples or with 50 μL for SeqChIP samples. PCR with Gotaq DNA polymerase was performed using 1 μl of eluted DNA from immunoprecipitated samples or 1 μl of 100-fold diluted input in 20 μl PCR reaction with Gotaq DNA polymerase and 15 nmol of specific primers (Additional file 1). Thermal cycling was programmed for 30 cycles, each consisting of 30 sec at 94°C, 30 sec at 55°C and 60 sec at 72°C.
We would like to thank Prof. S. A. Lukyanov from Evrogen for useful suggestions in MOS technique and Prof. M. B. Peterlin from UCSF for his support in ChIP methodology training. This work was supported in part by research grant P1-0170 and in part by a Young Researcher Fellowship from the Slovenian Research Agency.
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