Transcriptional activation by artificial recruitment in yeast is influenced by promoter architecture and downstream sequences

Abstract

The idea that recruitment of the transcriptional machinery to a promoter suffices for gene activation is based partly on the results of "artificial recruitment" experiments performed in vivo. Artificial recruitment can be effected by a "nonclassical" activator comprising a DNAbinding domain fused to a component of the transcriptional machinery. Here we show that activation by artificial recruitment in yeast can be sensitive to any of three factors: position of the activator-binding elements, sequence of the promoter, and coding sequences downstream of the promoter. In contrast, classical activators worked efficiently at all promoters tested. In all cases the "artificial recruitment" fusions synergized well with classical activators. A classical activator evidently differs from a nonclassical activator in that the former can touch multiple sites on the transcriptional machinery, and we propose that that difference accounts for the broader spectrum of activity of the typical classical activator. A similar conclusion is reached from studies in mammalian cells in the accompanying paper [Nevado, J., Gaudreau, L

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

“…This may well account in part for the generally low transcription levels induced by artificially recruited TBP and also for part of its differential activity at different promoters (10,22,36,39,79). However, it does not readily explain the ability of RAP1 to synergize with GAL4-TBP at the modified HIS4 promoter of Fig.…”

“…4, nor does it account for the inverse correlation observed between the requirement for chromatin remodeling and the ability of artificially recruited TBP to function at the promoters examined here. Another explanation (not mutually exclusive with the preceding) for differential activity of nonclassical activators at different promoters considered by Gaudreau et al (22) is that some promoters may require recruitment of different components of the transcriptional machinery than others. This is entirely in accord with the interpretation offered here, if the transcription machinery is allowed to include chromatinmodifying activities, as is in fact considered in the discussion of Gaudreau et al (22).…”

“…A third possible explanation for differential activity of artificially recruited TBP (or other components of the general transcription machinery), as discussed by Gaudreau et al (22), is that nonclassical activators such as GAL4-TBP may suffer steric constraints that classical activators can overcome by interacting with multiple targets within the general transcriptional machinery, thus affording them greater flexibility. This may well account in part for the generally low transcription levels induced by artificially recruited TBP and also for part of its differential activity at different promoters (10,22,36,39,79).…”

“…Thus, our findings suggest that in spite of the histone acetyltransferase activity of yeast TAF II 130 (50), TFIID is considerably poorer at remodeling chromatin than are true activators. Previous studies have also reported variable transcriptional activation by artificially recruited TBP at different promoters in yeast, but chromatin structure has not generally been examined (10,22,36,39,79). Similarly, experiments done in mammalian cells have been performed with transiently transfected DNA, which is not incorporated into organized chromatin in the same fashion as genomic DNA (2,48,57,80).…”

“…This could occur through recruitment of chromatin-modifying complexes such as the SWI-SNF complex, or complexes capable of modifying histones, such as the GCN5-containing SAGA complex (14,26,45,55,56,65,72,83). The strong and general capability of artificially recruited GAL11 to activate transcription and to remodel the PHO5 promoter (18,21,22) suggests that such chromatin-modifying activity can be recruited along with the holoenzyme (76). Indeed, we have shown that activation of GAL10-MEL1 by GAL4-GAL11 is almost completely abolished in swi1⌬ yeast (65).…”