Methods
Yeast strains, plasmids, and general methods
All experiments were conducted in the W303 strain background 1, with the exception of
those involving GBD hybrids (Figure 2), which were carried out in the two-hybrid
reporter strain PJ69-4A 2. Routine manipulations (cell growth, transformation, DNA
preparation) were carried out using standard procedures 3. Unless otherwise indicated,
cells were grown at 30°C, shaking at 150 rpm to a cell density of 2 x 107 cells/mL prior to
being manipulated or harvested. Cell concentration was measured using a CASY cell
counter (Schärfe System). Synthetic medium is SC-URA (0.2% amino acids, 0.5%
ammonium sulphate, 0.17% yeast nitrogen base, 2% Glucose). Rich medium is YPAD
(2% Bactopeptone, 1% yeast extract, 0.01% adenine, 2% Glucose) and YPLG (2%
Bactopeptone, 1% yeast extract, 0.01% adenine, 0.05% Glucose, 2% lactic acid, 3%
Glycerol). The endogenous FHL1 and IFH1 genes were C-terminally tagged by a
13xmyc epitope by transformation with PCR products obtained from the pFA6a-13MycHIS3MX6 and pFA6a-13Myc-TRP1MX6 vectors, respectively 4. Primer sequences used
to generate the tagged genes are available upon request. The resulting Fhl1-myc and
Ifh1-myc strains were indistinguishable in growth rate from the untagged parent strain.
The same PCR-based procedure was used to generate complete ORF deletions of both
FHL1 and IFH1. The strain in which the endogenous IFH1 gene is expressed from the
GAL1 UAS (UASGAL::IFH1) was constructed by transformation with PCR products
obtained from pFA6a-13Myc-HIS3MX6 4 while deleting 100 bp of the endogenous UAS.
This strain showed slow growth on glucose medium and growth comparable to wild-type
on galactose medium. Rapamycin (BIOMOL) was used at a final concentration of 200
ng/ml from a 1 mg/ml stock in 90% ethanol-10% Tween-20. The GBD expression vector
pSS1 was created by inserting oligonucleotides encoding a double HA epitope into
PstI/BglII of pGBDU-C(2) 2. GBD-Fhl1 hybrids were created by inserting EcoRI/SalI-
Schawalder et al.: Ifh1 regulation of ribosomal protein genes
cleaved PCR fragments into the same sites of pSS1. GBD-GAD was created by inserting
an EcoRI/BamHI fragment encoding the Gal4 activation domain (GAD) from plasmid
PM395 into the same sites of pSS1. Expression of all hybrids was controlled by Western
blotting, and binding of all hybrids to the GAL7 UAS was controlled by ChIP.
Expression analysis
Total RNA from 2 x 107 cells was isolated using the RNeasy Mini Kit (QIAGEN). 0.4
g of DNase-treated total RNA were reverse-transcribed 5. Quantitation of cDNAs was
achieved using RealTime PCR on an Applied Biosystems ABI Prism 7700 machine.
Chromatin immunoprecipitation
Chromatin immunoprecipitation experiments were performed as described 6 with the
following exceptions. Cross-linking was performed on 109 cells for 30 min at 30°C.
Dynabeads M280 coupled to sheep anti-mouse IgG or sheep anti-rabbit IgG were used
for the immunoprecipitations. Mouse monoclonal antibodies against the myc epitope
(culture supernatant of 9E10) or HA-epitope (12CA5), or a polyclonal rabbit anti-Rap1
antiserum were used. Precipitates were washed as described 7 with the exception that the
first wash buffer contained 1% Triton instead of 0.01% SDS. Quantitation of
immunoprecipitated DNAs was achieved using RealTime PCR on an Applied Biosystems
ABI Prism 7700 machine. Enrichment of the RPL9A, RPL30, RPL37A, PGK1 and GAL7
promoter regions over an internal control DNA located within the FZF1 gene was
determined after normalization against values obtained with samples precipitated without
antibody (myc or HA) or with preimmune serum (for Rap1). Binding was defined as
(fold enrichment) – 1.
Western blotting
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Schawalder et al.: Ifh1 regulation of ribosomal protein genes
Protein extracts were prepared by resuspending cells in ice-cold ChIP lysis buffer and
mechanical disruption in the presence of zirconium beads (Biospec). Extracts were
clarified by centrifugation in a microcentrifuge (13K rpm) at 4°C for 30 min. Total
protein (50 g per lane) was analyzed by SDS-PAGE and Western blotting according to
standard procedures.
Measurement of transcriptional activation
Transcriptional activation of the pGAL7-LacZ reporter gene in strain PJ69-4A was
measured as described 8. Nine individual transformants from three different, fresh
transformation plates were grown overnight to saturation in SC-URA and diluted into
YPAD and grown for an additional 4 hrs. Average beta-galactosidase values were
normalized to a positive control performed at the same time (GBD/GAD, set to 3500
units).
Genomic intergenic localization (‘ChIP-on-chip’) methodology
Immunoprecipitated DNA (prepared as described above) was processed for microarray
analysis as described previously 9, and references therein. Briefly, DNA fragments were
made blunt-ended, ligated to linker DNAs, and PCR amplified in the presence of either
NHS-ester Cy3 or Cy5 (Amersham Pharmacia biotech) dyes. Control DNAs were
labeled with Cy3, and the DNA prepared from strains expressing either Fhl1-13xmyc or
Ifh1-13xmyc were Cy5 labeled, although dye-swap experiments were done to control for
labeling bias. Labeled DNA was hybridized on yeast intergenic microarrays as described
9
.
ChIP-on-chip data analysis and interpretation
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Schawalder et al.: Ifh1 regulation of ribosomal protein genes
Hybridized arrays were scanned using a GenePix 4000A scanner (Axon Instruments,
Inc.) and fluorescence ratio measurements were determined with the GenePix Pro 4.0
software (Axon Instruments, Inc.) 9. Spots were considered for analysis if more than 55%
of the pixels fluoresced with intensities above the median fluorescence of the background
plus one standard deviation, and if they contained less than 5 saturated pixels 9. Data
analysis was performed using Gene Spring 6.0 software (Silicon Genetics) and Excel
(Microsoft) essentially as described 9. For each experiment, 3 to 5 independent replicates
were obtained depending on the quality of the immunoprecipitates and hybridizations in
each case. Each chip was normalized to the 50th percentile rank of the measurement
taken from that chip, and the median log enrichment ratios were then determined for each
locus. For each experiment, the median log enrichment ratio of each locus was
distributed in centiles defining the median percentile rank of each locus. We also filtered
the normalized data for a minimal log enrichment ratio of 1.8, and for p-values <0.001 (as
calculated by GeneSpring 6.0’s t-test p-value and Benjamini and Hochberg false
discovery rate) to obtain the final list of enriched loci in each experiment (see
Supplementary Information, Table 1). The complete raw data set is available at
http://www.ncbi.nlm.nih.gov/geo (GEO accession: GSE1944).
Oligonucleotide microarray analysis
Isogenic wild-type (YSS37, IFH1-13myc:TRP1, MATa) and UAS_GAL1-IFH1
strains (YSS89, UASGAL::IFH1-13myc:TRP1, MATa) cells were grown to exponential
phase (2x107 cells/mL) at 30°C in YPLG medium and t = 0 samples (2mL) were
harvested. Galactose was then added to 2% and samples (2mL) harvested 20, 30, 40 and
60 min later. All cells were harvested by rapid centrifugation at 4°C then flash frozen in
liquid nitrogen. RNA was extracted from either 4x107 cells using the RNeasy Protect
mini kit (Quiagen, Basel, Switzerland). For both cell lines three independent experiments
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Schawalder et al.: Ifh1 regulation of ribosomal protein genes
were performed. 5 g of RNA was converted into double-stranded cDNA using a cDNA
synthesis kit (Superscript Choice; Invitrogen) with a special oligo(dT)24 primer
containing a T7 RNA promoter site added 3' to the poly-T tract. Biotinylated cRNAs were
generated from cDNAs using the GeneChip IVT labelling kit (Affymetrix) and
subsequently purified with the RNeasy kit (Qiagen). Eighteen µg of cRNA probe was
fragmented and hybridised to the Affymetrix Yeast Genome S98 array (Affymetrix, High
Wycombe, United Kingdom) using standard hybridisation conditions. The Yeast
Genome S98 array contains 9335 probe sets interrogating approximately 6100 ORFs. 16
pairs of unique 25-mer composed of perfect matches and their corresponding mismatch
oligonucleotide probes represent each ORF. Arrays were washed and stained using an
Affymetrix GeneChip Fluidics Station 450 and standard protocols. Arrays were
visualized on a GCS3000 scanner (Affymetrix) and image files were processed using
GCOS (Affymetrix), with scaling target to 100 on all probe sets and normalisation value
of 1. Only ORFs with a detection call “P” (according to GCOS detection calls) present at
least in two out of three replicates were subsequently used for comparison analysis. The
software calculates “signal” values and provides “detection” calls, “present” (P),
“marginal” (M) or “absent” (A), for each probe set. Detection calls are determined from
statistical calculations of the difference in hybridisation signals between perfect match
oligonucleotides and their corresponding control mismatch sequence (probe set). Only
ORFs with a detection call “P” in both control and exp were subsequently used for
comparison analysis. To identify differentially expressed transcripts, pairwise
comparison analyses were carried out with Affymetrix GCOS. Each of the experimental
samples (NE) was compared with each of the reference samples (NR), resulting in (NE X
NR) pair wise comparisons. This approach, which is based on the Mann-Whitney
pairwise comparison test, allows the ranking of results by concordance, as well as the
calculation of significance (P value -  = 0.05) of each identified change in gene
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Schawalder et al.: Ifh1 regulation of ribosomal protein genes
expression 10,11. Genes for which the concordance in the pairwise comparisons exceeded
a threshold were considered to be statistically significant. This conservative analytical
approach was used to limit the number of false-positives. A 77% cut off in consistency
of change (at least 7 out 9 comparisons were either increase or decrease) was then applied
to identify potential interesting genes. Regulated genes were organized and visualized
into functional clusters using the GeneSpring Software version 6.1 (Silicon Genetics,
Redwood City, USA) and R 1.9 software. The complete raw data set is available at
http://www.ncbi.nlm.nih.gov/geo (GEO accession: GSE1934). Genes specifically
induced (2 compared to t = 0) at t = 30, 40 and 60 in UAS_GAL1-IFH1 were grouped
and are shown in Fig 3A and Supplementary Information, Fig. S3.
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