Mutations in the AF-2/hormone-binding domain of the chimeric activator GAL4 center dot estrogen receptor center dot VP16 inhibit hormone-dependent transcriptional activation and chromatin remodeling in yeast

GAL4.estrogen receptor.VP16 (GAL4.ER.VP16), which contains the GAL4 DNA-bin ding domain, the human EB hormone binding (AF-2) domain, and the VP16 activ ation domain, functions as a hormone-dependent transcriptional activator in yeast (Louvion, J.-F., Havaux-Copf, B., and Picard, D, (1993) Gene (Amst.) 131, 129-134). Previously, we showed that this activator can remodel chrom atin in yeast in a hormone-dependent manner, in this work, we show that a w eakened VP16 activation domain in GAL4.ER.VP16 still allows hormone-depende nt chromatin remodeling, but mutations in the AF-2 domain that abolish acti vity in the native ER, also eliminate the ability of GAL4.ER.VP16 to activa te transcription and to remodel chromatin. These findings suggest that an i mportant role of the AF-2 domain in the native ER is to mask the activation potential of the AF-1 activation domain in the unliganded state; upon liga nd activation, a conformational change releases AF-2-mediated repression an d transcriptional activation ensues. We also show that the AF-2 domain, alt hough inactive at simple promoters on its own in yeast, can enhance transcr iption by the MCM1 activator in hormone-dependent manner, consistent with i ts having a role in activation as well as repression in the native ER.