ABSTRACT: A genome-wide location analysis by ChIP on chip of the gene occupancy by Sub1 was undertaken to define the gene targets of Sub1 in vivo. Chromatin immunoprecipitation (ChIP) assays were performed on epitope-tagged Sub1-3HA cross-linked chromatin from exponentially growing cells. Immunopurified DNA and DNA from whole-cell extracts were fluorescently labelled and competitively hybridized to DNA microarrays harbouring ORFs and intergenic regions. The ratio of fluorescence intensities at each site in the microarray provided a measure of the extent of Sub1 binding to a specific genomic locus. Data from three independent experiments were compiled. Many loci were found to be significantly enriched in active growth conditions where Sub1 is not essential for cell viability. Approximately one-fourth of the enriched loci were located within ORF and the others corresponded to intergenic regions and to genes encoding non-translated RNAs. The ACT1, PMA1, PYK1, ADH1 and snoRNA genes previously identified as DNA targets of Sub1 were indeed enriched in our data. Sub1 was also preferentially bound to a subset of Pol II-transcribed genes encoding constituents of the cell wall, the nucleosome and the ribosome. Remarkably, these Pol II genes represented only two-third of the enriched loci. The other ones corresponded to Pol III-transcribed genes present on the arrays or to the intergenic regions and ORFs adjacent to these genes, suggesting the association of Sub1 to all Pol III-transcribed genes in conditions of active growth. The arrays used had a poor coverage of the rDNA gene locus. Nevertheless, we found a significant enrichment of the different loci corresponding to that region suggesting that Sub1 associates at many locations throughout the rDNA gene. Altogether, the results suggested that Sub1 could be a regulator of all three transcription systems. Keywords: ChIP-Chip Chromatin immunoprecipitation Chromatin immunoprecipitations were performed essentially as described by Ren et al. (2000) with some modifications described below. Briefly, overnight yeast cultures were used to inoculate fresh YPD medium at 0.1 OD600. The cells were grown in 100 mL of YPD to 1 OD600 and fixed with formaldehyde at a final concentration of 1% for 15 min at room temperature. Fixation was stopped by the addition of glycine (final concentration 340 mM) and incubation for 5 min. The fixed cells were harvested by centrifugation and washed twice with cold TBS. The cell pellets were frozen and stored at –80°C for further use. The cell pellets were thawed, resuspended in 700 µL of lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM PMSF, O Complete protease inhibitor (Roche)) and transferred to 1.5 mL Eppendorf tubes. 0.5 mL glass beads (425-600 µm, Sigma) were then added to each tube, and the yeast cells were lysed at 4°C for 2 hours using an Eppendorf Thermomixer Comfort at 1400 rpm. Each tube was pierced at the bottom and the cell lysate was collected by centrifugation in a fresh tube. The lysate was then sonicated 4 times for 10 s with 20 s cooling on ice between each sonication. The power of the Vibra Cell™ sonicator (Sonics & Materials Inc., Danbury, Connecticut, USA) was set to 4 with 60% duty cycle. Sonication resulted in chromatin fragments of 0.25–1 kb in size with a mean size of 600 base pairs as determined by gel electrophoresis. After 5 min of centrifugation in 1.5 mL Eppendorf tubes at 15 000 rpm, the supernatant (named whole cell extract, WCE) was transferred to another tube on ice. The preparation of magnetic beads, immunoprecipitation (with anti-HA 12CA5 or anti-myc 9E10), elution from beads and reversal of crosslinking, DNA precipitation, DNA blunting and ligation of blunted DNA to linkers were done exactly as described in Ren et al. (2000). Construction of yeast microarrays The S. cerevisiae ORF and intergenic regions were amplified from S288C genomic DNA with oligonucleotides from ResGen. The primers allow the amplification of the sequence located on either side of elements such as open reading frames, tRNAs, small nuclear RNAs, Ty elements, solo δ, etc. A complete description of the primers can be obtained on the web site (ftp://ftp.resgen.com/pub/genepairs/yeast_intergenic). The PCR products were purified by ethanol precipitation and their size and concentration were measured by agarose gel electrophoresis. 95 % of the regions were correctly amplified (as evidenced by gel electrophoresis) and purified at a mean plate concentration of 70 ng/µL. The purified DNAs were spotted onto amino-silane coated glass slides (GAPS II, Corning) using an automated arrayer (MicroGrid II, BioRobotics) with a spotting success percentage higher than 96 %. The final success rate is 93-94 %. LM-PCR, probe labeling and hybridization Ligation-mediated PCR was done as described by Ren et al. (2000) except that amino-allyl conjugated dUTP (150 µM final) was used instead of Cy3-dUTP or Cy5-dUTP and only 30 cycles of PCR were performed. After PCR amplification, 5 μl of the reaction mixtures was run on 1.5% agarose gel to measure the amount of amplified DNA. The PCR product size ranged from 200 to 1000 bp with an average size of 300 bp. The PCR products were purified using a Microcon YM-30 filter (Amicon/Millipore). Amino-allyl modified DNA was recovered with 20 µL of H2O and the DNA was lyophilized. The DNA pellet was resuspended in 9 µL of 100 mM sodium bicarbonate, pH 9.0. This sample DNA was used to dissolve a dry pellet of monofunctional NHS-ester Cy3 or Cy5 (1/16e of the quantity delivered from the mono-reactive dye pack PA23001 or PA25001 respectively from Amersham) and the mixture was incubated for 1 hour at room temperature in the dark. The coupling reaction of the Cy dyes to the amino-allyl dUTP was stopped by quenching with the addition of 4.5 µL of 4M hydroxylamine (Sigma) and the solution was further incubated for 15 min in the dark. Cy3-immunoprecipitated DNA and Cy5-WCE control DNA were mixed and unincorporated/quenched Cy dyes were removed using a Microcon YM-30 filter (Amicon/Millipore). DNA was recovered with 60 µL of TE and ethanol precipitated. Prehybridization and hybridization conditions were those described on P. Brown's web site (http://brownlab.stanford.edu/protocols.html) with few minor modifications. The labeled precipitated DNA samples were recovered in hybridization buffer (50% formamide, 5X Denhardt’s, 0.5% SDS, 7X SSPE, 10 µg herring sperm DNA) and hybridized to the DNA microarray in a Corning slide chamber at 42°C overnight. Arrays were washed at room temperature once with 0.1X SSC + 0.1% SDS, twice with 0.1X SSC, then dried by centrifugation. Data analysis Hybridized arrays were scanned using a GenePix 4000A scanner (Axon Instruments, Inc.) and fluorescence ratio measurements were determined with the GenePix Pro 6.0 software (Axon Instruments, Inc.). Array analyses were undertaken using the Limma package (Smyth, 2005) from the R/Bioconductor software (R-Development-Core-Team, 2007). Data from three independent experiments were compiled. ChIP-chip spot intensities have been normalized using the weighted median as implemented in the normalizeWithinArrays function of the Limma package. Normalized measures served to compute the log2-ratio for each probe. To exhibit enriched probes, we have used a moderated t-test. The moderated t test applied here was based on an empirical Bayes analysis and was equivalent to shrinkage (or expansion) of the estimated sample variances towards a pooled estimate, resulting in a more stable inference, but we have considered a one-sided alternative hypothesis, since we expect higher intensity levels in the IP-enriched hybridizations than in the negative control hybridizations.
