SAGA is an essential in vivo target of the yeast acidic activator Gal4p - PubMed (original) (raw)

SAGA is an essential in vivo target of the yeast acidic activator Gal4p

S R Bhaumik et al. Genes Dev. 2001.

Abstract

Despite major advances in characterizing the eukaryotic transcriptional machinery, the function of promoter-specific transcriptional activators (activators) is still not understood. For example, in no case have the direct in vivo targets of a transcriptional activator been unambiguously identified, nor has it been resolved whether activators have a single essential target or multiple redundant targets. Here we address these issues for the prototype acidic activator yeast Gal4p. Gal4p binds to the upstream activating sequence (UAS) of GAL1 and several other GAL genes and stimulates transcription in the presence of galactose. Previous studies have shown that GAL1 transcription is dependent on the yeast SAGA (Spt/Ada/GCN5/acetyltransferase) complex. Using formaldehyde-based in vivo cross-linking, we show that the Gal4p activation domain recruits SAGA to the GAL1 UAS. If SAGA is not recruited to the UAS, the preinitiation complex (PIC) fails to assemble at the GAL1 core promoter, and transcription does not occur. SAGA, but not other transcription components, is also recruited by the Gal4p activation domain to a plasmid containing minimal Gal4p-binding sites. Recruitment of SAGA by Gal4p and stimulation of PIC assembly is dependent on several SAGA subunits but not the SAGA histone acetyl-transferase (HAT) GCN5. Based on these and other results, we conclude that SAGA is an essential target of Gal4p that, following recruitment to the UAS, facilitates PIC assembly and transcription.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Recruitment of SAGA to the GAL1 upstream activating sequence (UAS). Yeast strains were grown at 30°C in 1% yeast extract containing 2% peptone plus 2% glucose or galactose as indicated. Formaldehyde-based in vivo cross-linking/immunoprecipitation was performed as previously described (Li et al. 2000). Primer-pairs located in the UAS or core promoter of GAL1 and RPS5 (see Materials and Methods) were used for PCR analysis of the immunoprecipitated DNA samples. A PCR fragment corresponding to the GAL4 ORF was used as a control for background binding. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz) or a polyclonal antibody against the indicated TAFII. The promoter, primer-pair, and media are indicated on the left. The ratio of immunoprecipite over the input is indicated below each band. Ratios ≤0.01 are designated .01. IP, immunoprecipitate.

Figure 2

Figure 2

Gal4p mediates recruitment of SAGA to the GAL1 upstream activating sequence (UAS). Wild-type and GAL4 deletion strains were first grown in glucose-containing (YPD) and then shifted to galactose-containing (YPG) media 5 h before treatment with formaldehyde. Primer-pairs located in the (A) GAL1 UAS or (B) core promoter were used for PCR analysis of the immunoprecipitated DNA samples. Immunoprecipitations were performed using polyclonal antibodies against a TAFII or TBP, the mouse monoclonal antibody 9E10 (Santa Cruz) against the c-myc epitope-tag, or the mouse monoclonal antibody 8WG16 (Covance) against the CTD domain of the RNA polymerase II large subunit (RPB1).

Figure 3

Figure 3

Requirement of SAGA subunits for GAL1 complex assembly and transcription. Wild-type and SAGA subunit deletion mutants were grown as in Fig. 2 before treatment with formaldehyde. Primer-pairs located in the (A) GAL1 upstream activating sequence (UAS) or (B) core promoter were used for PCR analysis of the immunoprecipitated DNA samples. (C) Transcription. Total cellular RNA was prepared from the wild type or deletion mutants (top), and transcription from the indicated gene (left) was quantitated by primer-extension. The percentage transcription relative to wild type is indicated below. (D) Other Gal4p-dependent genes (as in panel C). (E) Requirement of SAGA following artificial activation of Gal4p (as in panel C).

Figure 4

Figure 4

Recruitment of SAGA to minimal Gal4p-binding sites. (A) Gal4p recruits SAGA but not other transcription factors to minimal Gal4p-binding sites. Yeast strains were grown, and in vivo cross-linking analysis was performed as described in Fig. 1. Immunoprecipitation was performed using polyclonal antibodies against the indicated TAFII or TBP, or a mouse monoclonal antibody against the c-myc epitope-tag, HA epitope-tag, or TFIIB. The primers used for the PCR analysis are adjacent to the Gal4p-binding sites in the plasmid. (B) Dependence of SAGA recruitment on the Gal4p activation domain. As in panel A except strains were used that expressed the Gal4p derivative indicated on the left. (C) In vitro interaction between the Gal4p activation domain and SAGA. Whole-cell extracts were prepared from a wild-type strain or an SPT20 deletion mutant and incubated with immobilized GST or GST-34 in buffer A for 30 min at 4°C. The eluate was analyzed by immunoblotting using the c-myc mouse monoclonal antibody against Spt3p-myc. The position of Spt3p-myc is indicated.

Figure 5

Figure 5

Kinetic analysis of transcription complex assembly on the GAL1 promoter. (A) Association of Gal4p, SAGA, and GTFs with the GAL1 promoter in different carbon sources. The carbon source is indicated on the left and association of transcription factors (top) is given with the upstream activating sequence (UAS) or core region of the GAL1 promoter analyzed by formaldehyde-mediated cross-linking/immunoprecipitation. (B) Kinetics of Gal4p binding on switch from glucose to raffinose. Cells were grown in glucose and shifted to raffinose for the times (includes 15 min cross-linking) indicated on the left. Binding of Gal4p to the GAL1 UAS was analyzed by formaldehyde-mediated cross-linking/immunoprecipitation. (C) Kinetics of complex assembly on switch from raffinose to galactose. Cells were grown in raffinose and shifted to galactose for the times indicated on the left. Association of the indicated transcription factor (top) with the UAS or core region of the GAL1 promoter was analyzed by formaldehyde-mediated cross-linking/ immunoprecipitation. (D) Kinetics of complex assembly on switch from glucose to galactose. As in panel C except that cells were grown in glucose and shifted to galactose. (E) Plot of TBP and RNA polymerase II association with the GAL1 core promoter upon switch from glucose to galactose. The data from an independent experiment (inset) was quantitated and plotted. Each point was normalized to the maximum cross-linking signal (assigned value of 100%).

Figure 6

Figure 6

A stepwise pathway of transcription complex assembly on the GAL1 promoter. Deletion strains and their isogenic wild-type counterparts were first grown in glucose, switched to galactose for 5 h, and treated with formaldehyde. Temperature-sensitive mutant stains and their isogenic wild-type versions were grown in galactose to an OD600 of 0.85 at 23°C, switched to 37°C for 1 h, and treated with formaldehyde. Association of Gal4p (A) and SAGA (TAFII68; B) with the GAL1 upstream activating sequence (UAS) or TBP and RNA polymerase II (C) with the GAL1 core promoter was analyzed by formaldehyde-mediated cross-linking/immunoprecipitation.

Figure 7

Figure 7

Summary of transcription complex assembly on the GAL1 promoter. The entry of SAGA and TBP were not kinetically resolvable, but the results of mutational experiments (Fig. 6) indicate that SAGA can associate with GAL1 in the absence of TBP. Although not analyzed here, it has been shown previously that other GTFs are associated with the transcriptionally active GAL1 promoter (Li et al. 1999, 2000). The model shows the other general transcription factors (GTFs) entering subsequent to TBP, but it is possible that some GTFs may be recruited to the promoter simultaneously with TBP (see Li et al. 1999, 2000).

Similar articles

Cited by

References

    1. Ansari AZ, Reece RJ, Ptashne M. A transcriptional activating region with two contrasting modes of protein interaction. Proc Natl Acad Sci. 1998;95:13543–13548. - PMC - PubMed
    1. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD. Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998;14:115–132. - PubMed
    1. Brown CE, Lechner T, Howe L, Workman JL. The many HATs of the transcriptional coactivators. Trends Biochem Sci. 2000;25:15–19. - PubMed
    1. Carey M, Lin Y-S, Green MR, Ptashne M. A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives. Nature. 1990;345:361–364. - PubMed
    1. Davidson I, Xiao JH, Rosales R, Staub A, Chambon P. The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence. Cell. 1988;54:931–942. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources