Published online 7 November 2006 Nucleic Acids Research, 2006, Vol. 34, No. 21 6225–6232 doi:10.1093/nar/gkl844 SAGA-associated Sgf73p facilitates formation of the preinitiation complex assembly at the promoters either in a HAT-dependent or independent manner in vivo Abhijit Shukla, Pratibha Bajwa and Sukesh R. Bhaumik* Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL-62901, USA Received July 7, 2006; Revised and Accepted October 9, 2006 ABSTRACT Although Sgf73p, a yeast homologue of human Sca7p, has recently been implicated as a new com- ponent of Spt-Ada-Gcn5-acetyltransferase (SAGA), its association with SAGA and functional role in regulation of transcription remain unknown in vivo. Here, using a chromatin immunoprecipitation (ChIP) assay, we show in vivo that, like SAGA, Sgf73p is recruited to the upstream activating sequence (UAS) of a SAGA-dependent gene, GAL1, in an activator- dependent manner. Further, Sgf73p is required for recruitment of SAGA to the GAL1 UAS, and facili- tates formation of the preinitiation complex (PIC) assembly at the GAL1 promoter. When PIC is not formed in Dsgf73, histone H3 is not evicted from the GAL1 promoter. Interestingly, PIC formation at GAL1 is not regulated by histone H3 acetylation or histone acetyltransferase (HAT) activity of SAGA. Similarly, Sgf73p facilitates PIC formation at another SAGA-dependent gene, ADH1, independent of his- tone H3 acetylation or HAT. In contrast, Sgf73p stimulates PIC formation at PHO84 (a SAGA- dependent gene), in a HAT-dependent-manner. Collectively, our data reveal that Sgf73p is required for SAGA recruitment, and stimulates PIC formation either in a HAT-dependent or -independent manner, providing significant information on how Sgf73p and possibly human Sca7p function physiologically. INTRODUCTION Spt-Ada-Gcn5-acetyltransferase (SAGA), a large multi- protein complex with Gcn5p-HAT (histone acetyltransferase) activity, is required for the normal transcription of 10% of yeast genes (1). The role of SAGA in transcriptional activation has been studied extensively at GAL1 (2–8). At the GAL1 promoter, the activator Gal4p ﬁrst recruits SAGA to the upstream activating sequence (UAS), and then the UAS-bound SAGA facilitates recruitment of TATA-box binding protein (TBP) to the core promoter, thereby stimulat- ing formation of preinitiation complex (PIC) and hence transcription (5,7,8). Recently, Sgf73p has been biochemically identiﬁed as a new component of SAGA (9,10). Sgf73p is a yeast homolo- gue of the human Spinocerebellar ataxia type 7 (SCA7) gene product, ataxin-7 or Sca7p. It is one of the eight known autosomal dominant neurodegenerative disorders which are caused by CAG (that encodes glutamine, Q) nucleotide repeat expansions from <35 (non-pathogenic) to an extreme of 300 (pathogenic) copies (11–15). However, it is not known how CAG nucleotide repeat expansion within ataxin-7 causes neurodegenerative diseases. Recent biochemical studies (16,17) have demonstrated that, like Sgf73p, wild-type ataxin-7 is a component of mammalian SPT3-TAFII31- GCN5L acetylase (STAGA) transcription coactivator com- plex (18,19), a human homologue of yeast SAGA complex, and it regulates HAT activity of STAGA and its interaction with activators. The poly(Q)-expanded ataxin-7 gets incorpo- rated into STAGA as well as SAGA (10,16), but inhibits Gcn5p-HAT activities of both STAGA and SAGA (10,16), and thus alters gene expression, providing signiﬁcant information on the molecular basis of poly(Q) disorders. Although previous biochemical studies (10,16,17) have deﬁned the functional roles of Sgf73p and ataxin-7 in main- taining the overall structural integrities of SAGA and STAGA, respectively, and their HAT activities, the molecular role of Sgf73p or ataxin-7 in transcriptional regulation remains mostly unknown in vivo. Here, using a formal- dehyde-based in vivo crosslinking and chromatin immunopre- cipitation (ChIP) assay, we have analyzed the role of Sgf73p in recruitment of SAGA and formation of the PIC assembly (and hence transcription) at the SAGA-dependent promoter *To whom correspondence should be addressed. Tel: +1 618 453 6479; Fax:+1 618 453 6440; Email: sbhaumik@siumed.edu 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.