scholarly journals An Inhibitory Region of the DNA-Binding Domain of Thyroid Hormone Receptor Blocks Hormone-Dependent Transactivation

1998 ◽  
Vol 12 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Ying Liu ◽  
Akira Takeshita ◽  
Takashi Nagaya ◽  
Aria Baniahmad ◽  
William W. Chin ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Dna Binding ◽  
Ligand Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Chimeric Receptor ◽  
Receptor System ◽  
Inhibitory Region

Abstract We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor β ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-β (TRβ), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRβ derepressed transcription to basal level. Using this system and a battery of TRβ mutants, we found that TRβ/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRβ ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRβ DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRβ from achieving transcriptional activation above basal level in this chimeric receptor system.

scholarly journals A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

1995 ◽  
Vol 15 (8) ◽  
pp. 4507-4517 ◽  
Author(s):  
E Hadzic ◽  
V Desai-Yajnik ◽  
E Helmer ◽  
S Guo ◽  
S Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Thyroid Hormone ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Alpha Helix ◽  
Binding Domain ◽  
Terminal Region ◽  
Binding Studies

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site

1993 ◽  
Vol 13 (5) ◽  
pp. 3042-3049
Author(s):  
G Lopez ◽  
F Schaufele ◽  
P Webb ◽  
J M Holloway ◽  
J D Baxter ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Transcriptional Activation ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Plasmid Sequence ◽  
Regulatory Effects

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.

scholarly journals Genetic dissection of thyroid hormone receptor beta: identification of mutations that separate hormone binding and transcriptional activation.

1995 ◽  
Vol 15 (3) ◽  
pp. 1499-1512 ◽  
Author(s):  
R Uppaluri ◽  
H C Towle
Keyword(s):  
Thyroid Hormone ◽  
Dna Binding ◽  
Ligand Binding ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Receptor Activation ◽  
Hormone Binding ◽  
D Region

The thyroid hormone receptors (TR) are members of the nuclear receptor family of ligand-mediated transcription factors. The large region of TR that lies C-terminal to its DNA-binding domain subserves functions of ligand binding, dimerization, and transactivation. Little is known regarding the structural or functional determinants of these processes. We have utilized genetic screening in the yeast Saccharomyces cerevisiae to identify residues involved in these functions. Random mutations of the rat TR beta 1 isoform between amino acid residues 179 and 456 were screened, and mutants with reduced hormone-dependent activation of reporter gene activity were isolated. In this paper we describe the characterization of a class of mutants that exhibit a dissociation between hormone binding and transcriptional activation. These mutants retained hormone binding (> 15% of the wild-type level) yet failed to transactivate a reporter gene. A number of these mutations occurred within the D region, which links the DNA-binding and ligand-binding domains of the receptor. One subset of these mutations abrogated DNA binding, supporting a role of the D region in this process. The remainder retain DNA binding and thus highlight residues critical for receptor activation. In addition, an unexpected group of "superactivator" mutations that led to enhanced hormone-dependent activation in S. cerevisiae were found. These mutations localized to the carboxy-terminal portion of the receptor in a region which contains elements conserved across the superfamily of nuclear receptors. The hormone-dependent phenotype of these superactivator mutations suggests an important role of this segment in ligand-mediated transcriptional activation.

scholarly journals Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site.

1993 ◽  
Vol 13 (5) ◽  
pp. 3042-3049 ◽  
Author(s):  
G Lopez ◽  
F Schaufele ◽  
P Webb ◽  
J M Holloway ◽  
J D Baxter ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Transcriptional Activation ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Plasmid Sequence ◽  
Regulatory Effects

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.

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