Abstract
We examine the generality of transcription factor-mediated chromatin remodeling by monitoring changes in chromatin structure in a yeast (Saccharomyces cerevisiae) episome outside of the context of a natural promoter. The episome has a well defined chromatin structure and a binding site for the transcription factor GAL4 but lacks a nearby functional TATA element or transcription start site, so that changes in chromatin structure are unlikely to be caused by transcription. To separate changes caused by binding and by activation domains, we use both GAL4 and a chimeric, hormone-dependent activator consisting of the GAL4 DNA-binding domain, an estrogen receptor (ER) hormone-binding domain, and a VP16 activation domain (Louvion, J.-F., Havaux-Copf, B. and Picard, D. (1993) Gene (Amst.) 131, 129-134). Both GAL4 and GAL4.ER.VP16 show very little perturbation of chromatin structure in their nonactivating configurations. Substantial additional perturbation occurs upon activation. This additional perturbation is marked by changes in micrococcal nuclease cleavage patterns, restriction endonuclease accessibility, and DNA topology and is not seen with the nonactivating derivative GAL4.ER. Remodeling by GAL4.ER.VP16 is detectable within 15 min following hormone addition and is complete within 45 min, suggesting that replication is not required. We conclude that activation domains can exert a major influence on chromatin remodeling by increasing binding affinity and/or by recruitment of other chromatin remodeling activities and that this remodeling can occur outside the context of a bona fide promoter.
Highlights
Transcriptional induction in eukaryotes requires binding of activators to promoter elements termed enhancers (called upstream activator sequences (UASs)1 in yeast)
Chromatin Perturbation by Activating and Nonactivating GAL41⁄7ER1⁄7VP16 and GAL4 Assessed by Micrococcal Nuclease Cleavage—Previously we showed that GAL4 can bind to a nucleosomal recognition site in yeast [10]
Chromatin Perturbation Caused by Activation Domain Unmasking—In this paper we have used the yeast episomes TA17⌬80 and TALS as reporters to monitor changes in chromatin structure caused by binding of GAL4 and GAL41⁄7ER1⁄7 VP16 in their activating and nonactivating configurations
Summary
(Received for publication, January 16, 1997, and in revised form, February 20, 1997). The yeast activator GAL4 can bind to promoter sites in both activating and nonactivating forms; this has allowed the detection of subtle effects on chromatin structure in the GAL1–10 promoter that require an unmasked activation domain [2, 5,6]. One of these nucleosomes contains a GAL4 binding site, which derives from the GAL3 promoter [12] We show that both GAL4 and GAL41⁄7ER1⁄7VP16 perturb TALS chromatin most strongly in their activating configurations, and we discuss possible mechanisms by which an unmasked transcriptional activation domain could remodel chromatin in vivo
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