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Silencing subdomains of v-ErbA interact cooperatively with corepressors: involvement of helices 5/6
K Busch1, B Martin, A Baniahmad
1Laboratoire de Biologie Moléculaire et de Génie Génétique, Institut de Chimie, Université de Liège, Belgium.
Abstract:
Members of the thyroid hormone receptor (TR) family act on vertebrate development and homeostasis by activating or repressing transcription of specific target genes in a ligand-dependent way. Repression by TR in the absence of ligand is mediated by an active silencing mechanism. The oncogene v-ErbA is a variant form of TR unable to bind hormone and thus acts as a constitutive repressor. Functional studies and mutation analysis revealed that the TR/v-ErbA silencing domain is composed of three silencing subdomains (SSD1-3) which, although nonfunctional individually, synergize such that silencing activity is restored when they are combined in a heteromeric complex. Here we demonstrate, using protein interaction assays in vitro and in vivo, that the inactive v-ErbA point mutant L489R within helix 5/6 in SSD2 fails to interact with the two corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptor). Furthermore, mutants in SSD1 and SSD3 exhibit a reduced corepressor recruitment corresponding to their weak residual silencing activity. In mammalian two-hybrid assays, only the combination of all three silencing subdomains, SSD1-3, leads to a cooperative binding to the corepressors N-CoR or SMRT comparable to that of the full-length v-ErbA repression domain. In conclusion, full silencing activity requires corepressor interaction with all three silencing subdomains, SSD1-3. Among these, SSD2 is a new target for N-CoR and SMRT and is essential for corepressor binding and function.
Insights
Thyroid hormone receptor (TR) silencing requires three subdomains (SSD1-3) to cooperate. The SSD2 subdomain is crucial for binding corepressors like N-CoR and SMRT, enabling gene repression.
Area of Science:
- Molecular Endocrinology
- Gene Regulation
- Oncogene Biology
Background:
- Thyroid hormone receptors (TRs) regulate vertebrate development and homeostasis through ligand-dependent gene transcription.
- TRs can actively repress gene transcription in the absence of ligand.
- The oncogenic v-ErbA protein is a TR variant that constitutively represses gene expression.
Purpose of the Study:
- To investigate the mechanism of transcriptional repression mediated by the TR/v-ErbA silencing domain.
- To identify the specific roles of the three silencing subdomains (SSD1-3) in corepressor interaction and silencing function.
Main Methods:
- Protein interaction assays (in vitro and in vivo) to assess binding between v-ErbA mutants and corepressors.
- Functional studies and mutation analysis of the v-ErbA silencing domain.
- Mammalian two-hybrid assays to evaluate cooperative binding to corepressors.
Main Results:
- A specific v-ErbA point mutant (L489R) in SSD2 failed to interact with corepressors N-CoR and SMRT.
- Mutants in SSD1 and SSD3 showed reduced corepressor binding, correlating with diminished silencing activity.
- Cooperative binding of all three subdomains (SSD1-3) was necessary for robust corepressor recruitment, mimicking full-length v-ErbA.
Conclusions:
- Transcriptional repression by v-ErbA requires the synergistic interaction of all three silencing subdomains (SSD1-3).
- SSD2 is identified as a novel and essential interaction site for N-CoR and SMRT corepressors.
- Effective gene silencing by TRs/v-ErbA depends on the cooperative engagement of multiple silencing subdomains with corepressor complexes.