Analysis of structure and expression of the Xenopus thyroid hormone receptor-beta gene to explain its autoinduction.
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.
Machuca
I
I
Laboratory of Developmental Biochemistry, National Institute for Medical Research, London, United Kingdom.
Esslemont
G
G
Fairclough
L
L
Tata
J R
JR
D000818:Animals; D017660:Autoreceptors / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D007814:Larva; D008675:Metamorphosis, Biological; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D000072482:Retinoic Acid Receptor alpha; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology*; D014982:Xenopus laevis / Q000235:genetics* / Q000254:growth & development
D017660:Autoreceptors; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D000072482:Retinoic Acid Receptor alpha; D014284:Triiodothyronine