RESULTS Sgf73p is an integral component of SAGA in vivo Yeast SAGA is a large multi-protein complex that contains Ada1p, Ada2p, Ada3p, Ada5p/Spt20p, Spt3p, Spt7p, Spt8p, Gcn5p, Tra1p, TAF5p, TAF6p, TAF9p, TAF10p, TAF12p, Ubp8p, Chd1 and Sgf11 (9,27–35). Recently, Sgf73p has been biochemically implicated as a new component of SAGA (10). To conﬁrm whether Sgf73p is a new component of SAGA in vivo, we analyzed recruitment of Sgf73p to the UAS of a well-characterized SAGA-dependent gene, GAL1, using a ChIP assay. We used either a c-myc mouse monoclo- nal antibody and yeast strains expressing a C-terminal myc epitope-tagged Sgf73p or polyclonal antibody to SAGA TAF (e.g. TAF12p). Two sets of promoter-speciﬁc primer-pairs were used that, as shown below in Figure 1A, could distinguish binding to the GAL1 UAS or core promoter. If Sgf73p is an integral component of the SAGA complex, it would be recruited to the GAL1 UAS, but not core promo- ter, since previous ChIP studies (5) have demonstrated recruitment of SAGA to the GAL1 UAS. Figure 1A shows that under condition in which Gal4p activation domain was active (galactose-containing growth medium), the TAF12p subunit of SAGA as well as Sgf73p were present at the GAL1 UAS. Similarly, Gal4p was recruited to the GAL1 UAS (Figure 1A). Signiﬁcantly, these same proteins were not associated with the GAL1 core promoter as well as an irrelevant DNA sequence (GAL4 ORF) (Figure 1A). These observations supported the possibility that Sgf73p is a component of SAGA in vivo. However, because these experiments were performed with the intact GAL1 promoter, it remained possible that other transcription components, such as general transcription factors (GTFs) played a role in recruitment of Sgf73p to the UAS. To address this issue, we asked whether Sgf73p could be recruited by Gal4p to a plasmid bearing only Gal4p-binding sites, but not other promoter elements. Figure 1B shows that Sgf73p was recruited to a plasmid bearing Gal4p-binding sites in galactose-containing growth medium in which Gal4p activation domain was active. Signiﬁcantly, Sgf73p was not associated with the Gal4p- binding sites when Gal4p activation domain was inactive (rafﬁnose-containing growth medium) even though the level of Gal4p recruitment was almost equal to that in galactose- containing growth medium (Figure 1B). Further, our previous studies (5) have demonstrated that, like Sgf73p, other SAGA components, such as Spt3p and Spt20p, were also recruited to the minimal Gal4p-binding sites in the plasmid in galactose- containing growth medium. Signiﬁcantly, the GTFs, such as TBP, TFIIB, Mediator and RNA polymerase II, were not recruited to the minimal Gal4p binding sites in the plasmid (5). Taken together, our data provide evidence that Sgf73p is a new component of the SAGA complex in vivo, consistent with previous biochemical data (9,10). Sgf73p regulates the integrity of SAGA in vivo To understand the role of the newly identiﬁed Sgf73p component of SAGA in transcriptional activation in greater detail, it is necessary to determine its contribution in maintaining the integrity of SAGA. Recently, McMahon Nucleic Acids Research, 2006, Vol. 34, No. 21 6227 Figure 1. Recruitment of Sgf73p to the GAL1 UAS or minimal Gal4p binding site. (A) Recruitment of Sgf73p to the UAS, but not core promoter, of the GAL1 gene. Yeast strain expressing myc-epitope tagged Sgf73p was grown at 30 C in 1% yeast extract containing 2% peptone plus 2% galactose (YPG) up to an OD600 of 1.0 prior to formaldehyde-based in vivo crosslinking. The chromatin (IP) was performed as previously described (23,24). Primer-pair located at the UAS or core promoter of the GAL1 gene (see Materials and Methods section) was used for PCR analysis of the (IP) DNA samples. A PCR fragment corresponding to the GAL4 ORF was used as a control for background binding. (IP) was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology, Inc.) or a polyclonal antibody against TAF12p (from Michael R. Green, University of Massachusetts Medical School). A mouse monoclonal antibody against the DNA binding domain of Gal4p (RK5C1; Santa Cruz Biotechnology, Inc.) was used. The primer-pairs are indicated on the left. The ratio of immunoprecipitate over the input is indicated below each band as %IP. (B) Recruitment of Sgf73p to the minimal Gal4p-binding sites. The plasmid containing minimal Gal4p-binding sites was transformed into the yeast strain expressing myc-epitope tagged Sgf73p. Yeast strain thus generated, was grown at 30 C in 1% yeast extract containing 2% peptone plus 2% galactose or raffinose. The ChIP analysis was performed as described in (A). The primers used for the PCR analysis are adjacent to the Gal4p-binding sites in the plasmid (see Materials and Methods section). The growth media are indicated on the left. et al. (10) have demonstrated biochemically that Sgf73p is required to maintain SAGA integrity. Similarly, if Sgf73p maintains SAGA integrity in vivo, Spt20p and other SAGA components will not be recruited to the GAL1 UAS in the Dsgf73 strain. To test this idea, we analyzed recruitment of SAGA to the GAL1 UAS in the SGF73 deletion mutant. Figure 2A shows that the deletion of SGF73 signiﬁcantly lowered recruitment of the Spt20p and TAF10p components of SAGA to the GAL1 UAS. Similarly, our previous studies (5,6) have also demonstrated that recruitment of SAGA was lost in the SPT20 [that integrates SAGA (3)] deletion mutant strain. On the other hand, recruitment of the SAGA compo- nents such as TAF10p, TAF12p and Spt3p was not altered