In vitro transcriptional studies of the roles of the thyroid hormone (T3) response elements and minimal promoters in T3-stimulated gene transcription.
Thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that are encoded by two different genes, TR alpha and TR beta, and bind to thyroid hormone response elements (TREs) in the promoters of thyroid hormone (T3)-regulated genes. Retinoid X receptors (RXRs), major members of the thyroid hormone receptor auxiliary proteins, have recently been shown to enhance the binding of TRs to TREs. We previously showed that TRs extracted from rat pituitary GH3 cells retain ligand (T3) and DNA binding specificity and stimulate rat growth hormone (rGH) promoter activity in a cell-free in vitro transcription system. In this report, we have studied further how T3 activates endogenous TRs and stimulates transcription from different TRE-containing promoters. We found that T3 (10(-8) M) selectively stimulates transcription from rGH-TRE- and TREpal-, but not ME-TRE- and F2-TRE-, containing templates in which these TREs are linked in front of the rGH minimal promoter containing only the TATA box binding protein, but not any other proximal binding protein, sequence. In contrast, only the TREpal/AdML template, in which TREpal oligonucleotide was linked in front of the adenovirus major late gene (AdML) minimal promoter, was stimulated by T3. Electrophoretic mobility shift assay (EMSA) demonstrates that endogenous TR complexes specifically bind to either natural or idealized TRE (rGH-TRE, TREpal, ME-TRE, and F2-TRE) oligonucleotides. To further understand these receptor-DNA complexes formed on various TREs, isoform-specific anti-receptor antisera (TR alpha, TR beta 1, TR beta 2, and RXR beta) were added in the EMSA. These antisera differentially supershifted TR.DNA complexes formed on the TREs. These data suggest either that endogenous TR isoforms and RXR beta may form different complexes on the various TREs or that TR.RXR complexes have distinct conformations when bound to the various TREs. Taken together, these data suggest that particular TREs in which specific TR.RXR complexes are formed and different minimal promoters may provide specificity in T3-mediated transcriptional stimulation of gene expression.
Suen, CS; Yen, PM; Chin, WW
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