Co-operative binding of the glucocorticoid receptor DNA binding domain is one of at least two mechanisms for synergism.

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor binding sequences with adjacent binding sites either for other transcription factors or for further steroid receptors. Analysis of the human glucocorticoid receptor revealed that the DNA-binding domain of the receptor is sufficient to mediate co-operative binding to adjacent receptor binding sites. This is a novel feature of the domain in addition to its DNA-binding, trans-activating and trans-repressing properties. Chimaeric proteins containing the N- or C-terminal receptor halves fused to the GAL4 DNA-binding domain do not co-operate in DNA-binding, however they do functionally synergize. Thus, at least two mechanisms contribute to the synergism of the human glucocorticoid receptor bound to two adjacent receptor binding sites.

Multiple domains of the glucocorticoid receptor involved in synergism with the CACCC box factor(s).

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor-binding sequences with adjacent transcription factor-binding sites. To analyze the mechanism of synergy we tested different human glucocorticoid receptor mutants for synergistic function with another transcription factor in comparison with intrinsic trans-activation obtained with a single receptor binding site (glucocorticoid response element). Multiple domains were found to be involved in synergistic activity of the glucocorticoid receptor with the CACCC box factor. Deletions within the N-terminal receptor half affected simultaneously intrinsic trans-activation and synergism. However, deletion of the hormone-binding domain mainly impaired synergism rather than intrinsic trans-activation, clearly showing that this domain synergizes by a mechanism independent of intrinsic activation. A chimeric protein where the DNA-binding domain of the glucocorticoid receptor was replaced by that of the yeast GAL4 protein also showed functional synergism. These data suggest that some of the receptor domains outside the DNA-binding domain synergize by their intrinsic trans-activating property, but the hormone-binding domain contributes to synergism by a different mechanism.

Enhancement of human estrogen receptor activity by SPT6: a potential coactivator.

The conserved nature of the transcriptional machinery between yeast and higher eukaryotes makes the yeast system suitable to genetically dissect the signal transduction pathway of steroid hormone receptors. This report describes the yeast protein, SPT6, which modulates the transcriptional activity of the human estrogen receptor (hER) by affecting the C-terminal activation domain. It is demonstrated that SPT6 is able to potentiate hER activity in yeast and also in mammalian cells in vivo. SPT6 interacts specifically with the hormone-binding domain of hER in vivo. The in vivo studies are substantiated by specific protein-protein interactions between SPT6 and the hormone-binding domain of hER in vitro. Therefore, the data suggest that the SPT6 protein may be involved in signal transmission of ER by acting as a coactivator.

The yeast SIN3 gene product negatively regulates the activity of the human progesterone receptor and positively regulates the activities of GAL4 and the HAP1 activator.

The activation of gene transcription in eukaryotic organisms is regulated by sequence-specific DNA-binding proteins as well as by non-DNA-binding proteins. In this report we describe the modulatory functions of a non-DNA-binding protein, SIN3 (also known as SDI1, UME4, RPD1, and GAM2) on the transactivation properties of the human progesterone receptor (hPR), GAL4, and the HAP1 activator in yeast. Our data suggest that SIN3 is a dual function protein. It negatively regulates the transcriptional activities of hPR-A and hPR-B by affecting the N-terminal activation domain (AF1). SIN3 positively regulates the transcriptional activities of GAL4 and the HAP1 activator. However, it has no effect on the transcriptional activities of the human glucocorticoid receptor (hGR) and GCN4. The SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif. Deletion analysis of the SIN3 PAH motifs shows that the PAH3 motif is essential for SIN3-mediated regulation of hPR, GAL4, and the HAP1 activator. In contrast, the PAH1, PAH2, and PAH4 motifs are not required for SIN3-mediated regulation of these activators. Additionally, we examined the mechanism(s) by which the SIN3 protein modulate the activities of various activators. We are unable to demonstrate the direct interaction of SIN3 protein with these activators using the yeast two-hybrid system or co-immunoprecipitation. These data suggest that SIN3 regulates the transactivation functions of hPR, GAL4, and the HAP1 activator by an indirect mechanism.