Effects of ligand and thyroid hormone receptor isoforms on hepatic gene expression profiles of thyroid hormone receptor knockout mice

Abstract

Little is known about the overall patterns of thyroid hormone (Th)-mediated gene regulation by the main Th receptor (Tr) isoforms, Tr-alpha and Tr-beta, in vivo. We used 48 complementary DNA microarrays to examine hepatic gene expression profiles of wild-type and Thra and Thrb knockout mice under different Th conditions: no treatment, treatment with 3,3',5-triiodothyronine (T(3)), Th-deprivation using propylthiouracil (PTU), and treatment with a combination of PTU and T(3). Hierarchical clustering analyses showed that positively regulated genes fit into three main expression patterns. In addition, only a subpopulation of target genes repressed basal transcription in the absence of ligand. Interestingly, Thra and Thrb knockout mice showed similar gene expression patterns to wild-type mice, suggesting that these isoforms co-regulate most hepatic target genes. Differences in the gene expression patterns of Thra/Thrb double-knockout mice and Th-deprived wild-type mice show that absence of receptor and of hormone can have different effects. This large-scale study of hormonal regulation reveals the functions of Th and of Tr isoforms in the regulation of gene expression patterns.

Figure 1

Experimental design. Hepatic RNA was harvested from mice of the genotypes indicated under various conditions of thyroid hormone (Th) treatment. Each RNA was compared to the same reference RNA pool, which was prepared from euthyroid wild-type (WT) mice. Each experiment was repeated using three or four mice. DKO, double knockout of the thyroid hormone receptor (Tr) genes Thra and Thrb; KO, knockout; PTU, propylthiouracil; T3, 3,3′,5-triiodothyronine; UN, untreated.

Figure 2

Hierarchical clustering of genes that are positively regulated by thyroid hormone in thyroid-hormone-deprived and thyroid-hormone-treated wild-type and knockout mice. Samples are clustered and are presented in the same order as in Fig. 1. Expression ratios are colour-coded according to the scale at the bottom of the figure. Clusters of interest are numbered and are indicated by the black bars on the right. The conditions used are indicated by the colour coding at the top of each column as follows: turquoise, untreated; brown, treated with propylthiouracil (PTU); yellow, treated with 3,3′,5-triiodo-thyronine (T₃) and PTU; purple, treated with T₃ only. Genotypes are indicated above the colour codes. D, double knockout of the thyroid hormone receptor (Tr) genes Thra and Thrb; KO, knockout; WT, wild type.

Figure 3

Hierarchical clustering of genes that are negatively regulated by thyroid hormone in thyroid hormone-deprived and thyroid-hormone-treated wild-type and knockout mice. Expression ratios are colour-coded according to the scale at the bottom of the figure. Clusters of interest are numbered and are indicated by the black bars on the right. The conditions used are indicated by the colour coding at the top of each column as follows: turquoise, untreated; brown, treated with propylthiouracil (PTU); yellow, treated with 3,3′,5-triiodothyronine (T₃) and PTU; purple, treated with T₃ only. Genotypes are indicated above the colour codes. D, double knockout of the thyroid hormone receptor (Tr) genes Thra and Thrb; KO, knockout; WT, wild type.

Figure 4

Comparison of T₃-regulated gene expression in wild-type and Thrb knockout mice. The magnitudes of changes in gene expression in response to thyroid hormone (Th) in Th-deprived (propylthiouracil-treated) mice are usually greater in wild-type mice than in mice in which the Thrb gene is knocked out. Individual genes are arranged along the x axis according to the ranked order of decreases and increases in gene expression in wild-type mice. The y axis shows the log ratio of the transcription signals in T₃-treated and euthyroid mice (T₃/euthyroid). KO, knockout; T₃, 3,3′,5-triiodothyronine; Thrb, thyroid-hormone-receptor-β.

Figure 5

Model of gene expression patterns of positively and negatively regulated target genes. The model is based on clusters 1–5 in Figs 2,3. EU, euthyroid mice; T3, 3,3′,5-triiodothyronine; Th, thyroid hormone.