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Functional inhibition of retinoic acid response by dominant negative retinoic acid receptor mutants
K Damm1, R A Heyman, K Umesono
1Gene Expression Laboratory, Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA 92037.
Abstract:
The diverse effects of retinoids on the development, growth, and homeostasis of vertebrate organisms are mediated in part by three distinct isoforms of retinoic acid receptors (RARs). These proteins, which are structurally and functionally closely related to thyroid hormone receptors and the oncogene product v-ErbA, regulate patterns of gene expression in target tissues. One approach to study the distinct effects of retinoic acid in cells is to subvert this activity of endogenous receptors by expression of dominant negative receptor derivatives. We demonstrate here that RAR alpha, RAR beta, and RAR gamma can be converted into potent negative transcriptional regulators that block wild-type RAR function. Furthermore, these mutant RARs, but not the wild-type receptors, actively repress the basal transcription level of target promoters. When expressed in transgenic mice, the most potent of these inhibitory receptor mutants is apparently able to disturb developmental processes by inducing a cleft palate in transgenic offspring.
Insights
Dominant negative retinoic acid receptors (RARs) were created to block normal RAR function. These mutants repressed gene transcription and, in mice, caused developmental defects like cleft palate.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Retinoids are crucial for vertebrate development, growth, and homeostasis.
- Three retinoic acid receptor (RAR) isoforms mediate retinoid signaling by regulating gene expression.
- Thyroid hormone receptors and v-ErbA share structural and functional similarities with RARs.
Purpose of the Study:
- To investigate the potential of dominant-negative RAR derivatives to inhibit endogenous RAR function.
- To characterize the transcriptional regulatory properties of mutant RARs.
- To assess the in vivo developmental effects of expressing dominant-negative RARs in transgenic mice.
Main Methods:
- Expression of dominant-negative RAR alpha, beta, and gamma derivatives in cells.
- Assays to measure the inhibition of wild-type RAR transcriptional activity.
- Analysis of basal transcription repression by mutant RARs.
- Generation and analysis of transgenic mice expressing dominant-negative RAR mutants.
Main Results:
- RAR alpha, beta, and gamma were successfully converted into potent dominant-negative transcriptional regulators.
- Mutant RARs effectively blocked wild-type RAR function.
- Mutant RARs, unlike wild-type receptors, actively repressed basal transcription of target promoters.
- Expression of the most potent inhibitory RAR mutant in transgenic mice induced cleft palate in offspring.
Conclusions:
- Dominant-negative RARs can effectively antagonize endogenous RAR signaling pathways.
- Mutant RARs possess intrinsic repressor activity on target gene promoters.
- In vivo expression of dominant-negative RARs can disrupt normal vertebrate development, leading to specific malformations.