6228 Nucleic Acids Research, 2006, Vol. 34, No. 21 in the deletion mutant of GCN5 (5) that is dispensable for the integrity of SAGA (3). Since Spt20p maintains the integrity of SAGA (3,5) and its recruitment was signiﬁcantly reduced in the absence of Sgf73p (Figure 2A), thus Sgf73p, like Spt20p, seems to regulate the structural integrity of the SAGA complex in vivo, consistent with previous biochemical data (10). However, our ChIP data also raised the possibility that Sgf73p might be required for recruitment of Spt20p (and hence SAGA) to the GAL1 UAS by its interaction with the activator, Gal4p. In fact, our previous studies (7) have demonstrated that SAGA is recruited to the GAL1 UAS by interaction of its Tra1 subunit with Gal4p in vivo. Thus, it is more likely that Sgf73p is required to maintain the integrity of SAGA in vivo, consistent with previous biochemical studies (10). Sgf73p facilitates formation of the PIC assembly at the core promoters of the SAGA-dependent genes in vivo To better understand the role of Sgf73p in formation of the PIC assembly and hence transcriptional activation, we analyzed its contribution in recruitment of TBP and RNA polymerase II (two important components of the PIC assem- bly) to the GAL core promoter. Figure 2B shows that recruit- ment of TBP and the largest subunit (Rpb1p) of RNA polymerase II to the GAL1 core promoter was dramatically reduced in the SGF73 deletion mutant strain. Consistently, transcription of GAL1 was signiﬁcantly impaired in Dsgf73 (Figure 2C). Thus, the Sgf73p component of SAGA regulates GAL1 transcription by facilitating formation of PIC at the core promoter. However, formation of the PIC assembly at the GAL1 core promoter was not completely impaired in Dsgf73. On the other hand, our previous studies (5,6) have demonstrated that PIC formation at the GAL1 core promoter was completely impaired in the absence of Spt20p which maintains SAGA integrity. Such a discrepancy in PIC forma- tion in the absence of Sgf73p or Spt20p is due to the fact that the loss of Spt20p leads to complete impairment of SAGA recruitment to the GAL1 UAS (5,6) while SAGA recruitment was not completely lost in Dsgf73 (Figure 2A). Consistently, SAGA has been shown biochemically to be completely disin- tegrated in the absence of Spt20p but not Sgf73p (3,10). Since Sgf73p regulates recruitment of SAGA, the forma- tion of the PIC assembly at the SAGA-dependent promoters in general would be altered in Dsgf73. To test this idea, we analyzed recruitment of TBP and RNA polymerase II to the core promoters of two other SAGA-dependent genes, namely ADH1 and PHO84 (6). Figure 3A shows that recruitment of TBP and RNA polymerase II to the ADH1 and PHO84 core promoters was signiﬁcantly reduced in Dsgf73. Consistently, transcription of these genes was also altered in the SGF73 deletion mutant strain (Figure 3B). As a negative control, we demonstrate that the formation of the PIC assembly (and hence transcription) at a SAGA-independent gene, RPS5, was not altered in Dsgf73 (Figure 3C). Thus, SAGA- associated Sgf73p regulates gene expression by stimulating formation of the PIC assembly in vivo. However, such a func- tion of Sgf73p in PIC formation seems to be mediated by the regulatory role of Sgf73p in recruitment of SAGA or its integrity. In addition, Sgf73p may also contact with the component(s) of the PIC assembly to promote PIC formation. Figure 2. Requirement of Sgf73p for recruitment of SAGA and formation of the PIC assembly at GAL1. (A) Sgf73p is required for recruitment of SAGA to the GAL1 UAS. The SGF73 deletion mutant and its isogenic wild-type equivalent were first grown in raffinose-containing (YPR) and then shifted to galactose-containing (YPG) growth media 90 min before treatment with formaldehyde. (IPs) were performed as described in Figure 1A. A polyclonal antibody against the TAF10p (from Michael R. Green, University of Massachusetts Medical School) was used. Primer-pair located at the GAL1 UAS was used for PCR analysis of the IP DNA samples. The percentage of DNA immunoprecipitated relative to wild-type (%WT) is indicated below each band of the mutant strains. (B) Sgf73p is required for formation of the PIC assembly at the GAL1 core promoter. Yeast strains were grown, crosslinked and immunoprecipitated as in panel A. Primer-pair located at the GAL1 core promoter was used for PCR analysis of the immunoprecipitated DNA samples. IPs were performed using polyclonal antibodies against TBP (obtained from Michael R. Green, University of Massachusetts Medical School) and the mouse monoclonal antibody 8WG16 (Covance) against the carboxy terminal domain of the RNA polymerase II large subunit (Rpb1p). (C) Transcription. Total cellular RNA was prepared from the wild-type or Dsgf73, and mRNA level from the GAL1 gene was quantitated by primer- extension. The percentage transcription relative to wild-type is indicated below. Formation of the PIC assembly is differentially regulated by HAT or histone H3 acetylation in vivo SAGA-associated Gcn5p acetylates primarily histone H3 at K9 (lysine 9) and K14. Histone H3 acetylation or the HAT activity of SAGA has been previously implicated in regula- tion of transcriptional initiation (3,36–41). However, how his- tone H3 acetylation or HAT regulates PIC formation and hence transcriptional initiation is not clearly known in vivo. Thus, we asked whether formation of Sgf73p-mediated PIC assembly at the core promoters of the aforementioned SAGA-dependent genes is dependent on histone H3 acetyla- tion or HAT. To address this question, we analyzed recruit- ment of TBP and RNA polymerase II to the core promoters