of the GAL1, PHO84 and ADH1 genes in the GCN5 deletion mutant and its isogenic wild-type equivalent. Figure 4A shows that the GAL1, PHO84 and ADH1 core promoters were acetylated at histone H3. Histone H3 acetylation at these core promoters were signiﬁcantly reduced in Dgcn5. Interestingly, such a reduction in the level of histone H3 acet- ylation did not alter recruitment of RNA polymerase II and TBP to the GAL1 and ADH1 core promoters (Figure 4B). Thus, at these two promoters, Sgf73p facilitates formation of the PIC assembly in a HAT-independent-manner. In con- trast, recruitment of RNA polymerase II and TBP to the PHO84 core promoter was dependent on histone H3 acetyla- tion or Gcn5p-HAT (Figure 4A and B), demonstrating the Figure 3. Sgf73p is required for PIC formation (and hence transcriptional activation) at the ADH1 and PHO84 promoters. (A) The analysis of the PIC assembly at the ADH1 and PHO84 core promoters in Dsgf73. The yeast strains were grown in YPD at 30 C up to an OD600 of 1.0 prior to crosslinking. IPs were performed as described in Figure 2B. Primer-pairs located at the ADH1 and PHO84 core promoters were used for PCR analysis of the immunoprecipitated DNA samples. (B) Transcription. Total cellular RNA was prepared from the wild-type or Dsgf73, and mRNA levels from the ADH1 and PHO84 genes were quantitated by primer-extension. (C) Sgf73p is not required for PIC formation (and hence transcription) at the RPS5 promoter. Left panel: Analysis of recruitment of RNA polymerase II at the RPS5 core promoter in the wild-type and SGF73 deletion mutant strains. Both the wild-type and deletion mutant strains were grown, crosslinked and immunoprecipitated as in panel A. Primer-pair located at the RPS5 core promoter was used for PCR analysis of the IP DNA samples. Right panel: Transcription. Total cellular RNA was prepared from the wild- type or Dsgf73, and mRNA level from the RPS5 gene was quantitated by primer-extension. Nucleic Acids Research, 2006, Vol. 34, No. 21 6229 requirement of histone H3 acetylation or HAT for formation of the PIC assembly. Thus, our data demonstrate that histone H3 acetylation or the HAT activity of SAGA is differentially required for formation of the PIC assembly in vivo, revealing a complex regulation of the PIC formation. Role of Sgf73p in regulation of histone H3 acetylation or histone H3 eviction at the SAGA-dependent promoters We next asked whether Gcn5p-HAT activity of SAGA is regulated by Sgf73p in vivo. Previous biochemical studies (36) have demonstrated that Gcn5p-HAT activity on nucleo- somal histone H3 is dependent on the Ada2p and Ada3p com- ponents of SAGA. Similarly, deletion of SGF73 has been shown to biochemically impair the HAT activity of SAGA (10). However, it is not known whether Sgf73p regulates his- tone H3 acetylation at the promoters of the SAGA-dependent genes in vivo. To address this question, we analyzed the levels of histone H3 acetylation at the GAL1 core promoter in the SGF73 wild-type and deletion mutant strains. Since Sgf73p is required for recruitment of Gcn5p as well as SAGA, histone H3 acetylation at the GAL1 core promoter is expected to be signiﬁcantly impaired in Dsgf73. Interest- ingly, our ChIP data demonstrate the elevated level of histone H3 acetylation at the GAL1 core promoter in Dsgf73 when compared with wild-type equivalent (Figure 5A). Likewise, the elevated levels of histone H3 acetylation were also observed at the ADH1 and PHO84 core promoters in Dsgf73 (Figure 5A). In contrast, histone H3 acetylation level at the core promoter of a SAGA-independent RPS5 gene was not altered in the absence of Sgf73p (Figure 5A). Since H3-K9/14 acetylation antibody also recognizes unace- tylated histone H3 (Upstate biotechnology, Inc.; Figure 4A), the increase in the levels of histone H3 acetylation at the core promoters of GAL1, ADH1 and PHO84 in Dsgf73 may be Figure 4. Histone H3 acetylation differentially regulates formation of the PIC assembly at the core promoters of the SAGA-dependent genes. (A) The levels of histone H3 acetylation at the GAL1, ADH1 and PHO84 promoters are significantly reduced in the GCN5 deletion mutant. The wild-type and GCN5 deletion mutant strains were grown and crosslinked as described in Materials and Methods section. IP was performed using a rabbit polyclonal antibody against Lys-9/14 di-acetylated H3 (06–599, Upstate Biotechnology, Inc.). (B) Gcn5p is required for formation of the PIC assembly at the PHO84 core promoter, but not GAL1 and ADH1 core promoters. Yeast strains were grown and crosslinked as in (A). IPs were performed as in Figure 2B.