Gal4 turnover and transcription activation

Abstract

Growing evidence supports the notion that proteasome-mediated destruction of transcriptional activators can be intimately coupled to their function. Recently, Nalley et al. challenged this view by reporting that the prototypical yeast activator Gal4 does not dynamically associate with chromatin, but rather 'locks in' to stable promoter complexes that are resistant to competition. Here we present evidence that the assay used to reach this conclusion is unsuitable, and that promoter-bound, active Gal4 is indeed susceptible to competition in vivo. Our data challenge the key evidence that Nalley et al. used to reach their conclusion, and indicate that Gal4 functions in vivo within the context of dynamic promoter complexes.

Activation of a Gal4 competitor with β-estradiol versus 4HT

a, Wild-type yeast were induced with 2% galactose for 60 minutes and β-estradiol or 4HT added. At indicated times, occupancy of endogenous Gal4 on the GAL1/10 promoter was determined by ChIP. ChIP signal is normalized to that at time zero. b, As in (a), except that experiment was performed in yeast expressing the Myc–G4–ER–VP16 competitor (supplied by T. Kodadek3). c, As in the 4HT experiment in (b), except that ChIP was used to monitor association of the Myc–G4–ER–VP16 competitor with the GAL1/10 promoter. The corresponding non-competitor controls are also shown. To calculate the percentage binding in this case, ChIP signals were normalized to those from a Myc–G4–ER–VP16 ChIP (60 minute time point) performed in the absence of endogenous Gal4, which corresponds to the total amount of competitor that can bind in this assay. d, ChIP signals from β-estradiol or 4HT experiments in (b) normalized to the relevant ‘no competitor’ control (a).