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Use of suppressor mutants to probe the function of estrogen receptor-p160 coactivator interactions
1Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
Abstract:
Estrogen-dependent recruitment of coactivators by estrogen receptor alpha (ERalpha) 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 ERalpha 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.
Insights
Estrogen receptor alpha (ERalpha) function in gene transcription relies on coactivators. This study developed a novel method to isolate individual coactivator roles, revealing p160 family members are essential and redundant for estrogen-dependent transcription.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- Estrogen receptor alpha (ERalpha) mediates gene transcription through coactivator recruitment.
- Studying individual coactivator functions is challenging due to endogenous coactivator interference.
- Existing methods lack specificity for dissecting coactivator roles in ERalpha signaling.
Purpose of the Study:
- To develop a novel system to study individual coactivator functions in ERalpha-mediated transcription.
- To investigate the specific roles of p160 coactivators in estrogen-dependent gene activation.
- To determine if p160 family members have redundant or distinct functions.
Main Methods:
- Generated second-site suppressor mutations in p160 coactivators.
- Created a transcriptionally defective ERalpha variant refractory to wild-type coactivators.
- Utilized altered-specificity receptor-coactivator combinations to analyze reporter gene transcription.
- Assessed estrogen-dependent transcription in the absence of endogenous coregulator interference.
Main Results:
- Estrogen-dependent transcription critically requires direct p160 coactivator recruitment.
- The three p160 family members (e.g., SRC-1, GRIP1, ACTR) exhibit functionally redundant roles.
- Altered-specificity mutations serve as transposable modules for studying coregulator functions.
Conclusions:
- Direct recruitment of p160 coactivators is essential for ERalpha-mediated transcription in mammalian cells.
- p160 coactivators play redundant roles in estrogen signaling.
- The developed mutation strategy offers a novel tool for dissecting nuclear receptor coregulator functions.