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. 2012 Feb 7;109(6):1997-2002.
doi: 10.1073/pnas.1116340109. Epub 2012 Jan 23.

Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4

Ling Lin  1 , Lynn Chamberlain, Lihua J Zhu, Michael R Green
Affiliations

Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4

Ling Lin et al. Proc Natl Acad Sci U S A. .

Abstract

Promoter-specific transcriptional activators (activators) stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the "target." The identification of direct in vivo targets of activators has been a major challenge. Previous studies have provided evidence that the Tra1 subunit of the yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is the target of the yeast activator Gal4. However, several other general transcription factors, in particular the mediator complex, have also been implicated as Gal4 targets. Here we perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo [Gal4 interaction defective (GID) mutants]. In contrast to WT Tra1, Tra1 GID mutants are not recruited by Gal4 to the promoter and cannot support Gal4-directed transcription, demonstrating the essentiality of the Gal4-Tra1 interaction. In yeast strains expressing a Tra1 GID mutant, binding of Gal4 to the promoter is unexpectedly also diminished, indicating that Gal4 and Tra1 bind cooperatively. Consistent with cooperative binding, we demonstrate that the Gal4-Tra1 interaction occurs predominantly on the promoter and not off DNA. Finally, we show that although Tra1 is targeted by other activators, these interactions are unaffected by GID mutations, revealing an unanticipated specificity of the Gal4-Tra1 interaction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Isolation of tra1 mutants that cannot support growth on galactose. (A) Growth of tra1 mutants 1–13 on YPD and YPG media supplemented with antimycin. Growth of WT TRA1 and gal4-Δ strains are shown as controls. Cells were spotted as 10-fold serial dilutions. (B) qRT-PCR analysis monitoring expression of GAL1 and GAL3 in strains expressing WT TRA1 or tra1 mutant grown in raffinose or galactose. Expression of each gene was normalized to that observed in the WT TRA1 strain grown in raffinose, which was set to 1. The fold induction in galactose in the WT TRA1 strain is indicated. Error bars indicate SD.
Fig. 2.
Fig. 2.
Development of a BiFC assay for detecting interactions between activators and Tra1 in vivo. (A) Schematic diagram depicting the BiFC assay. Tra1 is tagged at the C terminus with the N-terminal Venus fragment (VN), and the activator (Act) is tagged at the C terminus with the C-terminal Venus fragment (VC). (B) BiFC assay monitoring interaction between Tra1 and Gal4 in vivo, as evidenced by intense YFP signal (arrowheads) in YPG. The Tra1–Gal4 interaction occurs in the nucleus, as evidenced by colocalization (arrowheads) with the DNA stain DAPI. Tra1 was tagged at either the C terminus (Upper) or N terminus (Lower). (C) BiFC assay monitoring interaction between Tra1 and Gcn4 in vivo, as evidenced by intense YFP signal in response to amino acid starvation (−His + 3-AT media).
Fig. 3.
Fig. 3.
Identification of Tra1 mutants that are unable to interact with Gal4. (A) BiFC assay monitoring the interaction between Gal4 and the mutant Tra1 proteins. (B) Coimmunoprecipitation assay. Spt20-HA was immunoprecipitated with an anti-HA antibody, and the immunoprecipitate analyzed for the presence of Tra1. The levels of Spt20 and Tra1 in the input extract are shown.
Fig. 4.
Fig. 4.
Gal4 and Tra1 bind cooperatively to the GAL1 promoter. (A) ChIP assay monitoring recruitment of Tra1, Spt20, and Gal4 to the GAL1 and RPS0B promoters in strains expressing WT TRA1, tra1-mut1, or tra1-mut8 and grown in media containing galactose or raffinose. Error bars indicate SD. (B) ChIP assay monitoring recruitment of Gal4 to the GAL1 promoter in a WT SPT20 or spt20-Δ strain. (C) Schematic diagram of the BiFC-based strategy to detect whether the Gal4–Tra1 interaction occurs predominantly on or off DNA. (D) BiFC assay monitoring the interaction between Tra1-VN and LexA(DBD)-Gal4(AD)-VC in the three yeast strains grown in galactose or raffinose. (E) BiFC assay monitoring the interaction between Gal80-VN and LexA(DBD)-Gal4(AD)-VC in the three yeast strains grown in galactose or raffinose.
Fig. 5.
Fig. 5.
Gcn4 functionally interacts with the Tra1 GID mutants. (A) Growth of tra1-mut1 and tra1-mut8 on His-lacking media containing or lacking 3-AT. Growth of WT TRA1 and gcn4-Δ strains are shown as controls. (B) BiFC assay monitoring the interaction between Tra1-VN and Gcn4-VC in His-lacking media containing or lacking 3-AT.
Fig. 6.
Fig. 6.
The Gal4-interaction site on Tra1 is highly selective. (A) qRT-PCR analysis monitoring expression of 11 Tra1- and SAGA-dependent genes in tra1-ts, spt20-Δ, tra1-mut1, and tra1-mut8 strains. Gene expression is presented relative to that observed in a WT strain, which was set to 1 (indicated by the red line). Error bars indicate SD. (B) BiFC analysis monitoring the ability of WT Tra1, Tra1-mut1, or Tra1-mut8 to interact with various activators. Only merged images are shown (see also Fig. S6B). (C) Scatter plot analyses comparing gene expression in a WT TRA1 strain and tra1-mut1 strain (Upper) or tra1-mut8 strain (Lower). The red line represents no change in gene expression; the blue dotted line represents twofold down-regulation. Gray circles represent all of the genes on the array; black circles represent genes with a P value <0.05; red circles represent genes with a P value <0.05 and down-regulated more than twofold.
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