A conserved lysine in the thyroid hormone receptor-alpha1 DNA-binding domain, mutated in hepatocellular carcinoma, serves as a sensor for transcriptional regulation

Abstract

Nuclear receptors are hormone-regulated transcription factors that play key roles in normal physiology and development; conversely, mutant nuclear receptors are associated with a wide variety of neoplastic and endocrine disorders. Typically, these receptor mutants function as dominant negatives and can interfere with wild-type receptor activity. Dominant-negative thyroid hormone receptor (TR) mutations have been identified in over 60% of the human hepatocellular carcinomas analyzed. Most of these mutant TRs are defective for corepressor release or coactivator binding in vitro, accounting for their transcriptional defects in vivo. However, two HCC-TR mutants that function as dominant-negative receptors in cells display near-normal properties in vitro, raising questions about the molecular basis behind their transcriptional defects. We report here that a single amino acid substitution, located at the same position in the DNA-binding domain of both mutants, is responsible for their impaired transcriptional activation and dominant-negative properties. Significantly, this amino acid, K74 in TRalpha, is highly conserved in all known nuclear receptors and seems to function as an allosteric sensor that regulates the transcriptional activity of these receptors in response to binding to their DNA recognition sequences. We provide evidence that these two human hepatocellular carcinoma mutants have acquired dominant-negative function as a result of disruption of this allosteric sensing. Our results suggest a novel mechanism by which nuclear receptors can acquire transcriptional defects and contribute to neoplastic disease.

Figure 1. The TRα1 I and M mutants are defective for transcriptional activation in cells, but display relatively unimpaired biochemical properties in vitro

(A). Schematics of the wild-type and HCC mutant TRα1 proteins. The wild-type TRα1 protein is depicted as a horizontal bar, with the DNA binding domain (DBD) and hormone binding domain (HBD) indicated. The TRα1 I and M mutant proteins are represented below as horizontal lines with the location of the genetic lesions in each mutant indicated by codon number. (B). T3-induced activation of a DR4 luciferase reporter by mutant and wild-type TRs. Expression vectors for the mutant or wild-type TRα1 alleles were introduced by transient transfection into CV1 cells together with the DR-4-tk-Luc reporters indicated and a pCH110 lacZ internal control. After 24 h. 100 nM T3 hormone (or carrier only) was added to the cells, the cells were harvested 24 h. later, and relative luciferase activity (normalized to the β-galactosidase control) was calculated. The mean and standard deviation of 2 or more experiments are presented. (C). T3 binding, coactivator recruitment, corepressor release, and DNA binding by mutant and wild-type TRs in vitro. For the T3-binding assay, the percentage of TR resistant to elastase at different T3 concentrations, an measure of hormone binding, is shown; the average and SEM values of three independents experiments are presented. For the coactivator and corepressor binding assays, radiolabeled mutant or WT TRs were incubated with an immobilized GST-SRC1 or immobilized GST-NCoR construct at the T3 concentrations indicated. After washing, the percentage of receptor bound to the GST-coregulator (input = 100%) was determined. The average and SEM values of at least three independent experiments are shown. For assaying TRα1 binding to AGGTCA and ACGTCA DR4 response elements, the WT and mutant TRα1 proteins were mixed with RXRα and incubated with radiolabeled DR4 response element comprised of AGGTCA or ACGTCA repeats, as indicated. The resulting TRα1/DNA complexes (arrows) were resolved by native gel electrophoresis and visualized by phosphorimager analysis. The results in panel C are derived from experiments in (17).

Figure 2. The K74 substitution is responsible for the regulatory defects and dominant-negative properties of the TRα1-I and TRα1-M mutants on a positive response element

(A). Positive response gene regulation by TRα1-WT, TRα1-I, or mutants derived from TRα1–I. Expression vectors for the TRα1 alleles indicated were introduced by transient transfection into CV1 cells together with a DR-4-tk-Luc reporter and a pCH110 lacZ internal control. After 24 h. T3 hormone (or carrier only) was added to the cells to the concentrations indicated, the cells were harvested 24 h. later, and relative luciferase activity (normalized to the β-galactosidase control) was calculated. The mean and standard deviation of 2 or more experiments are presented. (B). Positive response gene regulation by TRα1-WT, TRα1-M, or mutants derived from TRα1–M. The same methodology was employed as in panel A. (C). Inhibition of wild-type TRα1 function by TRα1-I or mutants derived from TRα1-I. The same methodology was employed as in panel A, except utilizing a 5:1 mixture of TRα-1 mutant to TRα1-WT in each transfection. (D). Inhibition of wild-type TRα1 function by TRα1-M or mutants derived from TRα1-I. The same methodology was employed as in panel B, except utilizing a 5:1 mixture of TRα-1 mutant to TRα1-WT in each transfection.

Figure 3. The A264V substitution is responsible for the delayed release of corepressor by the TRα1-I mutant

Radiolabeled TRα1-K74E or TRα1-A264V mutant proteins were assayed for the ability to bind to GST-NCoR under different T3 conditions, as in Figure 1C. The mean and standard error of 2 independent experiments are presented. Error bars smaller than the graph symbols may not be visible.

Figure 4. The K74 substitution also accounts for the regulatory defects and dominant negative properties of the TRα1-I and TRα1-M mutants on an AP-1 negative-response element

(A). Negative response gene regulation by TRα1-WT, TRα1-I, or TRα1-K74E. Expression vectors for the TRα1 alleles indicated were introduced by transient transfection into CV1 cells together with an AP-1 Col-Luc reporter and a pCH110 lacZ internal control. After 24 h. T3 hormone (or carrier only) was added to the cells to the concentrations indicated, the cells were harvested 24 h. later, and relative luciferase activity (normalized to the β-galactosidase control) was calculated. The mean and standard deviation of 2 or more experiments are presented. (B). Negative response gene regulation by TRα1-WT, TRα1-M, or TRα1-K74R. The same methodology was employed as in panel A. (C). Inhibition of wild-type TRα1 function by TRα1-I or TRα1-K74E. The same methodology was employed as in panel A, except utilizing a 5:1 mixture of TRα-1 mutant to TRα1-WT in each transfection. (D). Inhibition of wild-type TRα1 function by TRα1-M or TRα1-K74R. The same methodology was employed as in panel B, except utilizing a 5:1 mixture of TRα-1 mutant to TRα1-WT in each transfection. (E). Positive response gene regulation by TRα1-WT or an artificial TRα1-K74A mutant. A DR4-Luc reporter was used employing the same methodology as in Figure 2A. The mean and standard deviation of 2 or more experiments are presented. (F). Negative response gene regulation by TRα1-WT or an artificial TRα1–K74A mutant. An AP-1 Col-Luc reporter was used employing the same methodology as in Figure 4A. The mean and standard deviation of 2 or more experiments are presented.

Figure 5. The wild-type TRα1 DBD inhibits positive response gene regulation; the K74E mutant, or deletions of the DBD, reverse this inhibition

The Gal4DBD-domain was fused to TRα1-WT full-length, TRα1-I full-length, TRα1-K74E full-length, or TRα1-WT with the DNA binding domain deleted (TRα1-ΔABC), and the constructs were introduced into CV-1 cells together with a Gal17-mer luciferase reporter and the pCH110 lacZ internal control. T3 was added as indicated 24 h. after transfection, the cells were harvested an addition 24 h. later, and relative luciferase was determined as in Figure 2. The GalDBD-domain alone was also tested.

Figure 6. A conserved lysine in the P-box of multiple nuclear receptors makes important contacts within the response element half-site

(A). The amino acid sequence of the P box of the DNA binding domain of different nuclear receptors. Sequences include TR, retinoic acid receptor (RAR), vitamin D3 receptor (VDR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), androgen receptor (AR), estrogen receptor (ER), COUP-TFI, Drosophila Tailless (Tailless), and a mammalian Tailless ortholog (Tlx). (B). The crystal structure of a portion of the DNA binding domain of TRα1 bound to a DNA response element. The TR protein backbone is presented as a ribbon/helix/strand schematic; the DNA and K74 are shown as space fill (47). The half-site sequence of the DNA is indicated to the right; the contact between the second base pair in the DNA and K74 is highlighted in blue and yellow. A “lever-arm” identified in glucorticoid receptors is shown in the TR structure as green (40).