Abstract
We investigated the molecular mechanisms underlying the transcriptional silencing and the hormone-induced activation of target genes by thyroid hormone receptor beta (TR-beta). We developed a cell-free transcription system containing HeLa cell nuclear extracts in which unliganded human TR-beta represses basal transcription from a promoter bearing thyroid hormone response elements. Binding of hormonal ligand to the receptor reverse this transcriptional silencing. Specific binding of TR-beta to the thyroid hormone response element at the target promoter is crucial for silencing. Studies employing TR-beta mutants indicate that the silencing activity is located within the C-terminal rather than the N-terminal domain of the receptor. Our studies reveal further that unliganded TR-beta inhibits the assembly of a functional transcription preinitiation complex (PIC) at the target promoter. We postulate that interaction with TR-beta impairs the function(s) of one or more assembling transcriptional complexes during the multistep assembly of a PIC. Consistent with this hypothesis, we observe that, in the absence of thyroid hormone, TR-beta or a heterodimer of TR-beta and retinoid-X-receptor undergoes direct protein-protein interactions with the transcription factor IIB-TATA binding protein complex, an early intermediate during PIC assembly. Binding of hormone to TR-beta dramatically reduces the interaction between the receptor and the transcription factor IIB-TATA binding protein complex. We propose that the role of ligand is to facilitate the assembly of functional PICs at the target promoter by reducing nonproductive interactions between TR-beta and the initiation factors.
Highlights
The nuclear receptors for thyroid hormone belong to the superfamily of ligand-inducible transcription factors that include receptors for steroid hormones, retinoids, and vitamin D3 [1,2,3]
Transcription of this template was carried out in HeLa cell nuclear extracts which served as a source of RNA polymerase II and several other general transcription factors that are required for basal level of RNA synthesis
When the transcription reaction was performed in the absence of any exogenously added thyroid hormone receptors (TRs), a significant level of basal transcription was observed from the test promoter
Summary
The nuclear receptors for thyroid hormone belong to the superfamily of ligand-inducible transcription factors that include receptors for steroid hormones, retinoids, and vitamin D3 [1,2,3]. Baniahmad et al [32] using transient transfection assays demonstrated that two different GRTH mutants displaying drastically reduced or no T3 binding activity, respectively, functioned as constitutive repressors of target genes with strong silencing activity. These results favor the hypothesis that the dominant negative GRTH mutant is not an intrinsic transcriptionally nonfunctional receptor. It rather is a receptor that has lost the ability to transactivate but fully retains an active and constitutive silencing function This viewpoint is consistent with the observation that a homozygous GRTH patient with two mutant (non-hormonebinding but active repressor) TR-f3 alleles displayed much more
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