Abstract
Estrogen-dependent recruitment of coactivators by estrogen receptor alpha (ERα) represents a crucial step in the transcriptional activation of target genes. However, studies of the function of individual coactivators has been hindered by the presence of endogenous coactivators, many of which are potentially recruited in the presence of agonist via a common mechanism. To circumvent this problem, we have generated second-site suppressor mutations in the nuclear receptor interaction domain of p160 coactivators which rescue their binding to a transcriptionally defective ERα that is refractory to wild-type coactivators. Analysis of these altered-specificity receptor-coactivator combinations, in the absence of interference from endogenous coregulators, indicated that estrogen-dependent transcription from reporter genes is critically dependent on direct recruitment of a p160 coactivator in mammalian cells and that the three p160 family members serve functionally redundant roles. Furthermore, our results suggest that such a change-of-specificity mutation may act as a transposable protein-protein interaction module which provides a novel tool with which to dissect the functional roles of other nuclear receptor coregulators at the cellular level.
ACKNOWLEDGMENTS
We thank Hinrich Gronemeyer, Don Chen, Philip James, Borja Belandia, and David Heery for plasmid and yeast strain gifts; Geoff Greene for the H222 antibody; I. Goldsmith and staff for oligonucleotides; and G. Clark and staff for DNA sequencing. We also thank Caroline Hill, Jesper Svejstrup, Roger White, and members of the Molecular Endocrinology Laboratory for discussions and comments on the manuscript.
This work was supported by the Imperial Cancer Research Fund.