Analysis of structure and expression of the Xenopus thyroid hormone receptor-beta gene to explain its autoinduction.

Abstract

Transcription of both Xenopus thyroid hormone receptor (TR) genes, xTR alpha and -beta, is strongly up-regulated by their own ligand T3 during natural or T3-induced metamorphosis of tadpoles and in some Xenopus cell lines. To explain this autoinduction, we analyzed the sequence of 1.6 kilobases of xTR beta promoter for putative T3-responsive elements. Two direct repeat +4 AGGTCA hexamer motifs (DR+4), an imperfect distal (-793/-778) and a perfect proximal (-5/11) site, a DR+1 site, and some possible half-sites were located in the 1.6-kilobase promoter. Transfection of Xenopus XTC-2 cells (which express xTR alpha and -beta) and XL-2 cells (which predominantly express TR alpha) with chloramphenicol acetyltransferase reporter constructs of deletion mutants and promoter fragments showed that the distal and proximal DR+4 sites responded to T3, although other flanking sequences may also play a role. The thyroid hormone-responsive element half-site present as DR+1 in the up-stream sequence at -1260/-950, when cloned in front of a heterologous promoter, functions independently. T3 enhanced transcription from the two DR+4-containing fragments when present together by only 2- to 3-fold due to a high basal activity. Overexpression of unliganded xTR alpha and xTR beta in XTC-2 cells repressed basal activity, which was then enhanced 7- to 4-fold by T3, respectively; with XL-2 cells cotransfected with xTR beta, T3 inducibility increased to 16-fold. Electrophoretic mobility shift assays with recombinant Xenopus TR alpha, TR beta, retinoid-X receptor-alpha (RXR alpha) and RXR gamma proteins showed that TR-RXR heterodimers, but not TR or RXR monomers or homodimers, strongly bound the natural and synthetic distal and proximal DR+4 elements in a ligand-independent manner. TR/RXR heterodimers exhibited the highest binding affinity for a 28-mer oligonucleotide probe for the -5/11 proximal DR+4 site, with only slight binding to DR+1 (retinoid-X-responsive element-like) site. The xTR beta promoter binding to XTC-2 cell nuclear extract suggested the in vivo relevance of the findings with recombinant TR/RXR heterodimers. It is concluded that xTR alpha and -beta proteins are capable of regulating the expression of xTR beta gene, which can explain its autoinduction seen during T3-induced metamorphosis.

Thyroid hormone receptor α is a molecular switch of cardiac function between fetal and postnatal life

“…Activation of the expression of the TR␤ gene by TR␣ holoreceptors has not been previously documented in the mouse, although it has been described in the metamorphosing tadpole (26). Together with the fact that the expression of the TR␤ gene is increased in TR␣ 0/0 mutants, it strongly suggests that this expression in the fetal heart is directly controlled by the TR␣ receptor.…”

Induction of Larval Tissue Resorption in Xenopus laevis Tadpoles by the Thyroid Hormone Receptor Agonist GC-1

“…Our tail culture results are also most consistent with at least a partial requirement for activation for xTR␣ to induce sufficient xTR␤ levels for a response in larval tissues like the tail. Indeed, the xTR␤ gene contains a strong thyroid hormone response element near its transcription start site (35,36), and TR␤ can be induced in tadpoles even in the presence of protein synthesis inhibitors (10). However, there are other possibilities to account for the differences in tissue sensitivity to T3 and GC-1.…”

A Thyroid Hormone Antagonist That Inhibits Thyroid Hormone Action in Vivo

“…The two thyroid hormone receptor isotypes (TR␣ and TR␤) (14), their heterodimer partners (RXR␣, ␤, and ␥) (15,16), and the receptorassociated co-repressors and co-activators 2 are structurally and functionally well conserved with the mammalian counterparts. The TRs recognize thyroid hormone response elements in native target genes that contain canonical direct repeats similar to AGGTCA separated by 4 base pairs (17)(18)(19). Tadpoles readily take up thyroid hormones from the aqueous rearing solution and respond in a dose-dependent and highly stereotypic manner.…”