6230 Nucleic Acids Research, 2006, Vol. 34, No. 21 caused by the elevated levels of histone H3 at the core promoters of these genes, possibly because of reduction of histone H3 eviction in the absence of the PIC formation or active transcription (37). To test this possibility, we analyzed the levels of histone H3 at the GAL1, ADH1 and PHO84 core promoters in the SGF73 deletion mutant strain and its isogenic wild-type equivalent. Figure 5B shows that the levels of histone H3 at the core promoters of GAL1, ADH1 and PHO84, but not RPS5, were elevated in Dsgf73. Interest- ingly, the increase in the level of histone H3 acetylation is remarkably similar to that of histone H3 at each of the SAGA-dependent promoters in Dsgf73. Thus, Sgf73p does not seem to elevate the relative level of histone H3 acetyla- tion or the HAT activity of Gcn5p in vivo. Nonetheless, our data reveal that the loss of Sgf73p lowers histone H3 eviction from the SAGA-dependent promoters in vivo. DISCUSSION Although Sgf73p has been shown to be a new component of SAGA on the basis of the biochemical studies (9,10), it is not known whether Sgf73p is a SAGA component in vivo. If so, it would be recruited along with other SAGA components to the GAL1 UAS but not to its core promoter, since SAGA is spe- ciﬁcally recruited to the GAL1 UAS by the activator Gal4p (5,7). Here, we show that, like SAGA, Sgf73p is recruited to the GAL1 UAS, but not core promoter, or even to the minimal Gal4p-binding sites in a plasmid, implicating Sgf73p as a SAGA component in vivo. Furthermore, we show that the deletion of SGF73 signiﬁcantly impairs recruit- ment of the SAGA complex, consistent with biochemical requirement of Sgf73p for SAGA integrity (10). Thus, our results support the fact in vivo that Sgf73p is a SAGA component and is required for the structural integrity of SAGA, consistent with recent biochemical data (9,10). Since Sgf73p is essential for recruitment of SAGA, the PIC would thus not be formed at the SAGA-dependent promoters in Dsgf73. Indeed, our study demonstrates that recruitment of the PIC components such as TBP and RNA polymerase II is signiﬁcantly impaired at the core promoters of the three representative SAGA-dependent genes, GAL1, ADH1 and PHO84, in the absence of Sgf73p. Consistently, transcription of these genes is altered in Dsgf73. However, whether Sgf73p plays a dual role in assembling PIC by interacting with the component(s) of PIC as well as maintaining structural integrity of SAGA to regulate gene expression remains to be elucidated. Previous studies (3,38–41) have demonstrated the func- tional role of histone H3 acetylation at the promoters in reg- ulation of transcriptional initiation. Here, we demonstrate that all three SAGA-dependent promoters are acetylated at his- tone H3. Acetylation of histone H3 at these promoters is sig- niﬁcantly lost in Dgcn5. However, formation of the PIC assembly at the GAL1 and ADH1 promoters is not altered in Dgcn5. Thus, our study reveals that histone H3 acetylation status does not regulate PIC formation at GAL1 and ADH1. In contrast, PIC formation is signiﬁcantly reduced at the PHO84 promoter in Dgcn5, demonstrating the role of histone H3 acetylation in regulation of the PIC formation, consistent with previous studies (3,38–41). Thus, our study reveals Figure 5. Elevation of the levels of histone H3 as well as histone H3 acetylation at the core promoters of GAL1, ADH1 and PHO84 in Dsgf73. (A) Analysis of the levels of histone H3 acetylation at the promoters of GAL1, ADH1, PHO84 and RPS5 in the SGF73 deletion mutant strain. The wild-type and SGF73 deletion mutant strains were grown, crosslinked and immuno- precipitated as in Figure 4A. The percentages of DNA immunoprecipitated in Dsgf73 relative to wild-type (%WT) at the core promoters of GAL1, ADH1, PHO84 and RPS5 are plotted in the form of a histogram. (B) Analysis of the levels of histone H3 at the promoters of GAL1, ADH1, PHO84 and RPS5 in the SGF73 deletion mutant strain. The wild-type and SGF73 deletion mutant strains were grown and crosslinked as in panel A. IP was performed using an anti-histone H3 antibody (Ab1791; Abcam) and a modified ChIP protocol as described in Materials and Methods section. that histone H3 acetylation differentially regulates formation of the PIC assembly. However, the molecular basis for such differential roles of histone H3 acetylation in regulation of the PIC formation remains unknown. According to the ‘histone-code’ hypothesis (42), it is likely that histone H3