In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

Abstract

Our understanding of eukaryotic transcriptional activation mechanisms has been hampered by an inability to identify the direct in vivo targets of activator proteins, primarily because of lack of appropriate experimental methods. To circumvent this problem, we have developed a fluorescence resonance energy transfer (FRET) assay to monitor interactions with transcriptional activation domains in living cells. We use this method to show that the Tra1 subunit of the SAGA (Spt/Ada/Gcn5/acetyltransferase) complex is the direct in vivo target of the yeast activator Gal4. Chromatin-immunoprecipitation experiments demonstrate that the Gal4-Tra1 interaction is required for recruitment of SAGA to the upstream activating sequence (UAS), and SAGA, in turn, recruits the Mediator complex to the UAS. The UAS-bound Mediator is required for recruitment of the general transcription factors to the core promoter. Thus, our results identify the in vivo target of an activator and show how the activator-target interaction leads to transcriptional stimulation. The FRET assay we describe is a general method that can be used to identify the in vivo targets of other activators. Transcription initiation by RNA polymerase II involves the assembly of general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). A variety of studies indicate that promoter-specific activator proteins (activators) work, at least in part, by increasing PIC formation (Orphanides et al. 1996;Roeder 1996;Ptashne and Gann 1997;. Activator-mediated stimulation of PIC assembly is believed to result from a direct interaction between the activation domain (AD) and one or more components of the transcription machinery, termed the "target." The unambiguous identification of the direct in vivo targets of activators has been a major challenge in the field.Transcriptional induction of genes involved in galactose utilization (GAL genes) has been a model experimental system for studying transcriptional activation mechanisms. The well-characterized acidic activator Gal4 is responsible for the transcriptional stimulation of GAL genes, such as GAL1, which contain Gal4-binding sites in their promoters (Johnston 1987;Johnston and Carlson 1992;Dudley et al. 1999). A variety of transcriptional components have been proposed to be the target of Gal4 including TBP (Melcher and Johnston 1995;Wu et al. 1996), TFIIB (Wu et al. 1996), Srb4 (Koh et al. 1998Park et al. 2000), Gal11 (Jeong et al. 2001), the Swi/Snf complex (Neely et al. 2002), and the SAGA (Spt/Ada/ Gcn5/acetyltransferase) complex (Bhaumik and Green 2001;Brown et al. 2001;Larschan and Winston 2001). These proposals are based on either in vitro protein interaction studies or inferences from various indirect in vivo experiments, and to date there is no definitive evidence that Gal4 directly interacts with any of these putative targets in vivo. The major obstacle has been the lack of an experimental strategy to measure direct interactions with transcriptional ADs in vivo.The development of spectra...

Orchestration of chromatin-based processes: mind the TRRAP

“…Given that TRRAP is the only shared element between HAT complexes within the same family and also between different HAT families (GNAT and MYST), TRRAP is likely to play a unique and nonredundant role in these complexes. This idea is supported by elegant studies from Workman's and Green's laboratories demonstrating that TRRAP/Tra1-containing complexes with either histone H3 (SAGA, GCN5, PCAF) or histone H4 (NuA4, Tip60) specificity contact transcription activators exclusively through TRRAP/Tra1 (Brown et al, 2001;Bhaumik et al, 2004). The TIP49 (Pontin) and TIP48 (Reptin) ATPase/helicase proteins, components of TRRAP-containing Tip60 complex, were found to directly associate with transcription factors such as c-Myc and b-catenin and regulate embryonic development, oncogenic transformation and metastatic potential Etard et al, 2005;Kim et al, 2005).…”

“…How are HATs recruited to their target promoters? Biochemical studies revealed that TRRAP/Tra1, the only common component of many HAT complexes, mediates the targeting of these complexes to promoters (Brown et al, 2001;Bhaumik et al, 2004) (Figure 4). Direct interaction between several activators and Tra1 was demonstrated in yeast and this interaction is essential for efficient transcriptional activation (Brown et al, 2001).…”

“…TRRAP was originally identified in the Michael Cole's laboratory as an interacting partner of c-Myc , and subsequent studies showed that several other transcription factors including E2F and nuclear receptors interact with TRRAP Lang et al, 2001;Yanagisawa et al, 2002;Fan et al, 2004). Interestingly, TRRAP/Tra1 appears to be the only component through which HAT complexes (either histone H3 acetylating complexes or those with preference for histone H4) contact transcription activators (Brown et al, 2001;Bhaumik et al, 2004). The evidence that simultaneous enrichment of TRRAP, transcription factors and HATs at chromatin is associated with promoter-specific chromatin hyperacetylation Bouchard et al, 2001;Frank et al, 2001;Herceg et al, 2003;Li et al, 2004;Taubert et al, 2004) led to the suggestion that TRRAP functions to recruit HAT complexes to transcription factors bound to their target promoters in chromatin.…”

Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus

“…Gal4 activation does require Tra1 and other subunits of SAGA. Rather than influencing histone acetylation at the GAL promoters, the Gal4 interaction with SAGA through Tra1 seems to function by stimulating the binding of Mediator complex (Bhaumik et al, 2004) and TBP (Larschan and Winston, 2001) to the GAL promoters. Consequently, E1A CR1 interactions with TRAPP might influence the expression of a more specific set of genes than might be expected from an affect on a HAT.…”

On the Mechanism of Constitutive Pdr1 Activator-mediated PDR5 Transcription in Saccharomyces cerevisiae

“…Fluorescence resonance energy transfer experiments demonstrated that the Gal4 activation domain targets the Tra1 subunit of SAGA, Gal4-Tra1 interaction is required for recruitment of SAGA to the UAS, and SAGA, in turn, recruits the Mediator complex to the UAS (22). To address the role of Mediator in regulating PDR5 transcription, we analyzed the effect of inactivating Srb4 on PDR5 transcription.…”