Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae

Abstract

Background: The human adenovirus type 5 early region 1A (E1A) gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two regions that can independently induce transcription when fused to a heterologous DNA binding domain. When expressed in Saccharomyces cerevisiae, each of these regions of E1A also acts as a strong transcriptional activator. This allows yeast to be used as a model system to study mechanisms by which E1A stimulates transcription.

Results: Using 81 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, CSR, INO80, ISW1, NuA3, NuA4, Mediator, PAF, RSC, SAGA, SAS, SLIK, SWI/SNF and SWR1 transcriptional regulatory complexes on E1A dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on transcriptional activation.

Conclusion: Our analysis indicates that the two activation domains of E1A function via distinct mechanisms, identify new factors regulating E1A dependent transcription and suggest that yeast can serve as a valid model system for at least some aspects of E1A function.

Figure 1

Map of the major adenovirus type 5 E1A proteins and transcriptional activation by LexA-E1A fusions. A) The two major products of E1A are 289 and 243 residues (R) in length and differ only by the presence of an additional 46 amino acids unique to the larger protein. Regions of sequence conservation relevant to this study (CR) are shown as are the regions expressed as LexA DBD fusions. B) Yeast strain BY4741 was transformed with the pSH1834 LexA responsive reporter plasmid and vectors expressing the LexA DBD, or the LexA DBD fused to the indicated portions of E1A. Extracts were prepared from transformed yeast and assayed for β-galactosidase activity as described previously [29]. Experiments were performed in triplicate with s.d's indicated.

Figure 2

Influence of the SAGA, ADA and SLIK complexes on E1A dependent transcriptional activation. The indicated yeast deletion strains isogenic to BY4741 (refer to Additional file 2) were transformed with the pSH1834 LexA responsive reporter plasmid and vectors expressing the LexA DBD, or the LexA DBD fused to the indicated portions of E1A. Extracts were prepared from transformed yeast and assayed for β-galactosidase activity as described previously [29] and detailed in the Methods. Experiments were performed in triplicate with s.d's indicated. The asterisks indicate which complexes are expected to be influenced by the individual gene disruptions, as several of these genes encode factors that are components of more than one complex. For example, Spt3 is a component of the SAGA and SLIK complexes, but not the ADA complex.

Figure 3

Influence of Mediator on E1A dependent transcriptional activation in yeast. Experiments were performed as in Figure 2. The asterisks indicate which modules are expected to be influenced by the individual gene disruptions.

Figure 4

Influence of the SWI/SNF complex on E1A dependent transcriptional activation in yeast. Experiments were performed as in Figure 2.

Figure 5

Influence of Bre1, Rad6, COMPASS/Set1C complex and Dot1 complex on E1A dependent transcriptional activation in yeast. Experiments were performed as in Figure 2. A) Bre1, Rad6 and related deletion strains. B) COMPASS/Set1C and Dot1 deletion strains.

Figure 6

Influence of the ISW1 complex and Spt4 on E1A dependent transcriptional activation in yeast. Experiments were performed as in Figure 2.