In situ distinction between steroid receptor binding and transactivation at a target gene.

Abstract

We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor. The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence. This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously. The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of (3,8,15,20,30,41). Structurally, these molecules are similar, consisting of well-defined DNA and hormone binding domains and other less well defined activation domains that presumably mediate the interaction of receptors with the transcription apparatus (12,15,39). The mechanisms of activation of these transactivators are related, since ligand binding is required for converting these receptors from an inactive to an active form in vivo (8,15,20 A second model of activation suggests that a receptor is always bound to its response element and that the role of hormone is to convert it into a transcriptionally active form, possibly by displacing an inhibitory protein or directing an allosteric or covalent modification. This is analogous to the mechanism by which galactose regulates the binding of the GAL80 inhibitor to the GAL4 transactivator (23). In support of this model are the in vitro observations that retinoic acid receptor, thyroid hormone receptor, and estrogen receptor bind to DNA constitutively in the absence of hormone (4,18,19,26,39). Recent reports have suggested that the estrogen receptor is constitutively bound to its response element in vivo (40). Furthermore, it has been suggested that the bound receptor may possess ligand-independent activating and repressor activities. In fact, since progesterone receptor can be converted to an active form by mild heat treatment, it has been suggested that at body temperature, all of the progesterone receptor could be activated and constitutively bound to DNA (42).Finally, questions remain as to whether ligand-induced DNA-binding capacity can be separated from transactivation activity for a steroid receptor. Clearly there is an element of confusion as to the form of the receptor prior to transformation and the role of ligand in this process. It is quite possible that a single model will not explain the mode of action of all receptors and that the steps of activation must be elucidated for each receptor. In this study, we use a reconstituted estrogen-responsive system in Saccharomyces cerevisiae to examine the interaction of estrogen receptor with its response element in situ and the role of ligand in this association. The yeast system has been shown by several groups to be a representative model for steroid hormone action (27)(28)(29)34). S. cerevisiae was chosen for this study because of the absolute requirement for estrogen for gene activation by this receptor in ...

Chromatin Remodeling by Transcriptional Activation Domains in a Yeast Episome

“…1 and 2). This suggests that the ER hormone-binding domain can mask activator function even when bound at a promoter, as also proposed for intact ER expressed in yeast (59) and mammalian cells (60). Remarkably, this occurs with a heterologous activation domain.…”

Identification of a negative regulatory function for steroid receptors.

“…Opposing this model is the fact that only a limited subset of genes have been reported to be derepressed in a mutant SSN6 background (25). We We have demonstrated previously that the hormone estradiol is responsible for a two-step activation process in yeast (19). The first step included the dissociation of heat-shock proteins from the receptor and the subsequent conversion of the receptor to a DNA-binding form.…”

The cooperation of B-Myb with the coactivator p300 is orchestrated by cyclins A and D1

“…The concommittant increase of cyclin A/Cdk2 leads to phosphorylation of B-Myb followed by preferential acetylation of phosphorylated B-Myb and stimulation of its transactivation potential. The recent finding that cyclin A/Cdk2-induced phosphorylation of B-Myb disrupts the interaction of B-Myb with the corepressor N-CoR can easily be accommodated in this model (Li and McDonnell, 2002). Thus, activation of B-Myb by cyclin A/Cdk2 apparently not only involves in increase in the interaction with a stimulatory factor (p300) but also a decrease of an inhibitory interaction (with N-CoR).…”