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Supplementary Information
Methods
Yeast strains. The following yeast strains were used: TM101 (MATa ura3 leu2 his3) and its derivatives
YEN108 (rpd3::KAN-MX), YEN115 (sin3::HIS3), YEN100 (hda1::KAN-MX) and YEN123
(hog1::LEU2). L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) and its derivatives YEN96
(rpd3::KAN-MX), YEN116 (sin3::HIS3), YEN98 (hda1::KAN-MX). Strains YEN130 (PKY501
ura3::KAN-MX), YEN143 (PKY501 rpd3::KAN-MX), YEN135 (LDY722 ura3::KAN-MX) and YEN137
(LDY107 ura3::KAN-MX) were generated from PKY501, LDY722 and LDY107 strains described in
Durrin et al. 1991. TM141 and YEN114 strains were generated as described before (de Nadal et al.
2003). All strains for ChIP were derivatives of K699 (leu2-3, 112 ura3-1 his3-11 trp1-1 can100). Strains
PAY320 (W303 MATalpha SIN3-HA-HIS3), PAY318 (W303 Mata SIN3-HA6-HIS3 hog1 ::KanMX),
PAY225 (W393 Mata RPB1-myc18-TRP1), PAY324 (W393 Mata RPB1-myc18-TRP1 rpd3 ::HIS3) and
PAY185 (W303 Mata hog1 ::KanMX) were obtained in this work.
Plasmids and Oligonucleotides. Full-length wild type RPD3 (PEN149) and a catalytically inactive allele
of RPD3, which contains a double amino acid substitution in residues His150 and His151 to Ala
(PEN153) were cloned into pRS415 centromeric plasmid and expressed under the native promoter.
Construction of RPD3 site directed mutant was carried out by PCR-directed mutagenesis. PEN80
(pBTM116-Hog1) is described previously (Alepuz et al.2003). pSH18-8 plasmid (2/URA3; 8 lexA ops)
is a gift from S.D. Hanes. Full-length RPD3 (PEN138), HDA1 (PEN141) and HOG1 (PEN111) were
cloned into the pGEX-4T, pGEX-3T and pSETC vectors respectively by PCR. To express GST proteins
in yeast, HDA1 (PEN139) and RPD3 (PEN140) genes were cloned into pRS426TEG2. Oligonucleotides
that covered the promoter region used for ChIP analysis are as follow: CTT1 (-450 /-150), STL1 (-372 / 112 and –181 / +117), GRE2 (-300 / +30), ALD3 (-570 / -250 and –180 / +173), YLR164w (-300 / +20),
YKR033c (-300 / + 20) and RPB11 (-410 / -120).
Expression and purification of epitope-tagged proteins
His-tagged Hog1 was expressed in E. coli BL21(DE3) cells, purified using TALON metal affinity resin
(Clontech) and eluted using imidazole buffer, according to the manufacturer's instructions. GST fusion
proteins encoding Hda1 and Rpd3 were expressed in E. coli DH5, and purified using glutathione-Sepharose
beads (Pharmacia) in Buffer B as described (Posas et al., 1996).
In vitro and In vivo binding assays.
To characterize the interaction of Hog1 and Rpd3 proteins in vivo, GST pull down experiments were carried out
from cells subjected or not to stress (0.4 M NaCl, 5 min). 1 mg of yeast extract in buffer A (50 mM Tris HClpH 7.5, 15 mM EDTA, 15 mM EGTA, 0.1% Triton-X100, 150 mM NaCl, 2 mM dithiothreitol plus
antiproteases and phosphatase inhibitors) was incubated with 50 µl of glutathione-Sepharose beads overnight at
4ºC. Beads were washed extensively with buffer A, resuspended in loading buffer and resolved by SDS-PAGE.
For in vitro binding assay a GST fusion proteins encoding Rpd3, Hda1 or His-tagged Hog1 were expressed in
E. coli and purified using glutathione-Sepharose beads or TALON matrix as described before. Approximately 1
g of GST His-tag proteins were incubated for three hours at 4º C and then washed extensively. Bound proteins
were analyzed by western blotting.
-galactosidase assays
Exponentially growing cells (optical density at 660 nm of 0.5-0.8) were then subjected or not to osmotic stress
(0.4 M NaCl for 30 min), permeabilized by ethanol/toluene treatment, and -galactosidase measured as in 18.
Results are presented as mean values obtained from at least three independent transformants measured in
duplicate.
Microarray analysis
Starting from eight single culture extracts (respectively derived from wild type and rpd3 yeast grown in the
presence and absence of NaCl for 5 or 20 min), four types of two-color microarray hybridizations were
performed, confronting treated versus untreated conditions. Each comparison was performed in triplicate for
a total of twelve hybridization experiments: treated versus untreated wild type (wtA_5min, wtB_5min,
wtC_5min, wtA_20min, wtB_20min, and wtC_20min) and treated versus untreated rpd3 (rpd3A-5min,
rpd3B_5min, rpd3C_5min, rpd3A_20min, rpd3B_20min, and rpd3D_20min). Twenty micrograms of total
RNA were labelled using the indirect dye labelling method. The Atlas Glass Array labelling kit (Clontech,
Beckton Dickinson) was used following the manufacturers instructions, except that the aminoallyl-dUTP
incorporation reaction was performed for 2 hours using Superscript II reverse transcriptase (Invitrogen) in
the presence of Rnasin Rnase inhibitor (Promega), and that crosslinking to NHS-Cyanine dye esters of Cy3
and Cy5 (Amersham) was carried for 3 hours. Glass microarrays holding 12800 features representing the
whole genome of S. cerevisiae spotted in duplicate onto CMT-GAPS slides (Corning) by the microarray
facility at University Health Network (Toronto, Canada) were preblocked in 1% BSA, 0.5% SDS 45 min. at
42ºC and hybridized to labelled samples in the presence of blocking agents (20 ug tRNA, 4 ug salmon sperm
DNA and 20 ug poly-dA -1:1 60mer:40mer) in a buffer containing 50% formamide, 6X SSC, 0.5% SDS,
5XDenhardts at 42ºC (set 1: wtA_20min, wtB_20min , rpd3A_20min, and rpd3B_20min), or alternatively
(set 2: chips wtA_5min, wtB_5min, wtC_5min, rpd3A-5min, rpd3B_5min, rpd3C_5min,wtC_20min and
rpd3D_20min) in DigEasyHyb (Roche) at 37ºC, for 16 hours. After hybridization they were washed
extensively in 0.1X SSC, 0.1% SDS at room temperature with a final rinse in 0.05X SSC at room
temperature; alternatively (for set 2) they were washed in 1X SSC at room temperature, followed by 1X
SSC, 0.1% SDS at 50ºC with a final rinse at room temperature in 0.1X SSC. Fluorescent images were
obtained using a GMS417 Array (Affymetrix) or a Scanarray 4000 (PerkinElmer) scanner (set 2 except
wtC_20min and rpd3D_20min), and images were quantified using GenePix4.0 (Axon) software. Gene
intensities were background subtracted (taking mean of channel intensities and median of background).
Spots with low intensities were excluded from normalization (<150 in either or both channels).After
intensity filtration only 2503 of 6155 ORFs were present in all 12 microarrays compared. An intensity
dependent normalization was applied using lowess with a smoothing factor f=20% for all experiments
(Cleveland et al., 1988) and replicate spot average values of normalized log2ratios were calculated for each
hybridization. To be able to include measures for spots with low intensities in only one of the two channels,
log2ratios from spots excluded from normalization were adjusted using a correction factor calculated by
dividing the local regression coefficient of the median of normalization features before and after
normalization. The median value of the three hybridization experiments was used as the final measure of
difference in gene expression between osmostressed and untreated samples for each genotype and time of
treatment.
Data were visualized and filtered using Array File Maker 4.0 to generate the color graph (Breitkreutz et
al., 2001). Final list selection was performed through visual inspection of filtered rank ordered data. We
considered genes that showed a two-fold gene induction in the treated wild type, and either were not
regulated, down-regulated or were only mildly up-regulated in NaCl-exposed rpd3 mutant (with a smaller
fold-difference than in the wild type).