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An artificial thyroid hormone receptor mutant without DNA binding can have dominant negative effect
Abstract:
The syndrome of resistance to thyroid hormone (RTH) encompasses a heterogeneous group of conditions which are caused by mutations of thyroid hormone receptor beta 1 (TR beta 1). Mutations usually cluster in two regions of the ligand-binding domain. The mutant receptors can inhibit normal receptor activity in a dominant negative manner, consistent with the dominant mode of inheritance of RTH. Recent evidence suggested that this dominant negative effect (DNE) of the RTH mutants involves competition for DNA binding and emphasized the essential role of intact DNA binding activity for mutants in order to exert DNE. However, we found that a Cys73Ser substitution in the DNA-binding domain (DBD) of wild-type produces a TR which can inhibit the transcriptional activation by TR alpha 1, either in the presence or absence of T3, on three different TRE-containing reporter genes, in transient co-transfection studies. Co-expression of TRv alpha 2, a TR alpha splicing variant, can enhance this DNE. However, DNE was not observed on the negatively-regulated TSH alpha Luc reporter gene when wild-type and DBD mutant were co-transfected at equimolar ratios. The DNE of DBD mutant is not reversed by co-transfection with excess retinoid X receptor alpha. DBD mutant alone can also inhibit the transactivation from a TK-luciferase reporter gene either linked with rat malic enzyme thyroid response element, or not. These observations parallel those we previously observed using TRv alpha 2. Our results indicate that a DBD mutant can have DNE, possibly through a mechanism similar to that of TRv alpha 2, which may involve interference with basal transcription factors. The clinical significance of these DBD mutants is currently unclear, but it is logical to expect such mutants do occur in nature.
Insights
Thyroid hormone receptor (TR) mutations causing resistance to thyroid hormone (RTH) can inhibit normal TR activity. This study shows a DNA-binding domain mutant TR can also exert a dominant negative effect, independent of DNA binding.
Area of Science:
- Endocrinology
- Molecular Biology
- Genetics
Background:
- Resistance to thyroid hormone (RTH) is a syndrome caused by mutations in the thyroid hormone receptor beta 1 (TR beta 1).
- Mutant TRs often inhibit normal receptor activity through a dominant negative effect (DNE), consistent with RTH's dominant inheritance.
- Previous research suggested intact DNA-binding activity is crucial for this DNE.
Purpose of the Study:
- To investigate whether a mutation in the DNA-binding domain (DBD) of a TR can exert a dominant negative effect (DNE).
- To explore the mechanism of DNE for DBD mutants and compare it to known mechanisms.
Main Methods:
- Transient co-transfection studies were performed using wild-type and mutant TRs with reporter genes containing thyroid hormone response elements (TREs).
- The effects of co-expressing TR alpha 1, TRv alpha 2, and retinoid X receptor alpha were examined.
- Reporter gene assays were used to measure transcriptional activity.
Main Results:
- A Cys73Ser substitution in the TR's DBD resulted in a mutant TR that inhibited transcriptional activation by TR alpha 1, with or without T3.
- This DNE was observed on TRE-containing reporter genes but not on a negatively-regulated TSH alpha Luc reporter gene at equimolar ratios.
- The DNE of the DBD mutant was not reversed by excess retinoid X receptor alpha and could inhibit transcription independently of TREs.
Conclusions:
- A TR mutant with a substitution in the DNA-binding domain can exert a dominant negative effect.
- This DNE may occur through a mechanism involving interference with basal transcription factors, similar to TRv alpha 2.
- The clinical relevance of such DBD mutants in RTH remains to be determined but is plausible.