6226 Nucleic Acids Research, 2006, Vol. 34, No. 21 in Saccharomyces cerevisiae. Further, we have determined the role of histone H3 acetylation or the HAT activity of SAGA in regulation of the PIC formation at the SAGA- dependent promoters in vivo. Our results reveal that Sgf73p is required for SAGA recruitment and stimulates formation of the PIC assembly at the SAGA-dependent promoters. Consistently, transcription of the SAGA-regulated genes is signiﬁcantly impaired in Dsgf73. Interestingly, PIC formation at the SAGA-regulated genes is differentially regulated by histone H3 acetylation or the HAT activity of SAGA in vivo. MATERIALS AND METHODS Plasmids Plasmid SGP4 was generated by cloning a DNA fragment containing three Gal4p-binding sites into the low copy number plasmid pRS416 (5). The plasmid pFA6a-13Myc- KanMX6 (20) was used for genomic myc epitope-tagging of the proteins of interest. The Plasmid pRS416 (21) was used in the PCR-based gene disruption. Yeast strains and media Yeast strain harboring mutation in GCN5 (FY1370), and its isogenic wild-type equivalent FY1369 were obtained from Fred Winston (Harvard Medical School, Boston, MA) (2). Multiple myc-epitope tags were added at the original chromo- somal locus of SGF73 in W303a to generate ASY3 (Sgf73p- myc, Kan). The plasmid SGP4, carrying three Gal4p-binding sites, was transformed into ASY3 to generate ASY6. The endogenous SGF73 gene of W303a was disrupted using the PCR-based gene knock-out method (22) to generate ASY9 (Dsgf73::URA3). Multiple myc-epitope tags were added at the original chromosomal locus of SPT20 in W303a and ASY9 to generate ASY10 (Spt20p-myc, Kan) and ASY13 (Spt20p-myc, Kan; Dsgf73), respectively. For the studies at the GAL1 promoter in Dsgf73 and Dgcn5 mutant strains, and their isogenic wild-type equivalents, cells were ﬁrst grown in YPR (yeast extract containing peptone plus 2% rafﬁnose) to an optical density at 600 nm (OD600) of 0.9 and then transferred to YPG (yeast extract–peptone plus 2% galactose) for 90 min at 30 C prior to formaldehyde crosslinking. Minimal media containing either 2% galactose or rafﬁnose were used for the strain (ASY6) with reporter plasmid, SGP4. The yeast strains were grown in YPD (yeast extract containing peptone plus 2% dextrose) to an OD600 of 1.0 at 30 C for the studies at the ADH1, PHO84 and RPS5 promoters. ChIP assay The ChIP assay was performed as described previously (23,24). Brieﬂy, yeast cells were treated with 1% formalde- hyde, collected and resuspended in lysis buffer. Following sonication (that generates the average size of 400 bp of sheared DNA), cell lysates (400 ml lysate from 50 ml of yeast culture) were precleared by centrifugation, and then 100 ml lysate was used for each immunoprecipitation (IP). Immunoprecipitated (IP) protein–DNA complexes were treated with proteinase K, the crosslinks were reversed, and the DNA was puriﬁed. IP DNA was dissolved in 20 ml TE 8.0 [10 mM Tris–HCl (pH 8.0) and 1 mM EDTA], and 1 ml of IP DNA was analyzed by PCR. The PCR (a total of 23 cycles) contained [a-32P]dATP (2.5 mCi for each 25 ml reaction) and the PCR products were detected by autoradio- graphy after separation on a 6% polyacrylamide gel. As a control, ‘input’ DNA was isolated from 5 ml lysate without going through the IP step and suspended in 100 ml TE 8.0. To compare the PCR signal arising from the IP DNA with the input DNA, 1 ml of input DNA was used in PCR analysis. For analysis of histone H3 levels at the promoters, we modiﬁed the above ChIP protocol as follows. Eight hundred microlitre lysate was prepared from 100 ml of yeast culture. Four hundred microlitre lysate was used for each IP (using 5 ml of anti-histone H3 antibody from Abcam and 100 ml of protein A/G plus agarose beads from Santa Cruz Biotech- nology, Inc.), and IP DNA sample was dissolved in 10 ml TE 8.0 of which 1 ml was used in PCR analysis. In parallel, the PCR for ‘input’ DNA was performed using 1 ml DNA that was prepared by dissolving puriﬁed DNA from 5 ml lysate in 1000 ml TE 8.0. Serial dilutions of the input and IP DNAs were used to asess the linear range of PCR ampliﬁcation as described pre- viously (6). The PCR data presented in this paper are within the linear range of PCR analysis. All the ChIP experiments were repeated at least three times and consistent results were obtained. Primer-pairs used for PCR analysis were as follows: GAL1(UAS): 50-CGCTTAACTGCTCATTGCTATATTG-30, 50-TTGTTCGGAGCAGTGCGGCGC-30; GAL1(Core): 50-ATAGGATGATAATGCGATTAGTTTTTTAGCCTT-30, 50-GAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCA- 30; ADH1(Core): 50-GGTATACGGCCTTCCTTCCAGT- TAC-30, 50-GAACGAGAACAATGACGAGGAAACAA- AAG-30; PHO84(Core): 50-GATCCACTTACTATTGTG- GCTCGT-30, 50-GTTTGTTGTGTGCCCTGGTGATCT-30; RPS5(Core): 50-GGCCAACTTCTACGCTCACGTTAG-30, 50-CGGTGTCAGACATCTTTGGAATGGTC-30; GAL4 [open reading frame (ORF)]: 50-CTTGTTCAATGCAGTC- CTAGTACCC-30, 50-CACAAGTCTGGATTTTAAAAGT- GGCC-30. Primers ﬂanking Gal4p-binding sites in the plasmid SGP4 are 50-GGTGGCGGCCGCTCTAGAACTAGT-30 and 50-TTGACCGTAATGGGATAGGTCACG-30. Autoradiograms were scanned and quantitated by the National Institutes of Health image 1.62 program. The ratio of IP DNA over the input was presented as %IP. For mutant strains, DNA IP relative to wild-type (WT) was presented as %WT. Primer extension analysis Primer extension analysis was performed as described previously (5–7,23,25,26). Brieﬂy, total RNA was prepared from 10 ml of yeast culture. Fifteen micrograms of total RNA was used in the primer extension analysis using 32P-labeled primer and AMV reverse transcriptase (Promega). The primers used for analysis of GAL1, ADH1, PHO84 and RPS5 mRNAs were as follows: GAL1: 50-CCTTGACGTTACC- TTGACGTTAAAGTATAGAGG-30; ADH1: 50-TATCCTT- GTGTTCCAATTTACCGTGG-30; PHO84: 50-GAAGACT- TCTTTCAGCAACATG-30; RPS5: 50-GACTGGGGTGAAT- TCTTCAACAACTTC-30.