BUCHHOLZ, D.R.*; HSIA, V.; FU, L.; SHI, Y.-B.; National Institutes of Health, Bethesda: In-vivo molecular mechanisms of thyroid hormone receptor during frog metamorphosis

Frog metamorphosis is an excellent model to study in-vivo roles of thyroid hormone receptor (TR). Exogenous thyroid hormone (T3) acts as an “on” switch in premetamorphic tadpoles to initiate hormone-induced changes in gene expression and morphology. We examined molecular mechanisms of TR action using dominant negative receptors (dnTR) ubiquitously expressed in transgenic Xenopus laevis.First, we used RT-PCR to show that the TH direct-response genes, TR-beta, the basic leucine zipper transcription factor TH/bZIP, and the matrix metalloproteinase stromelysin-3, were up-regulated in response to T3 in wild type but not dnTR transgenic animals. Second, T3-induced morphological changes, such as limb growth, head and intestine remodeling, and gill resorption, failed to occur in TH-treated tadpoles expressing dnTR. Third, we used the chromatin immunoprecipitation assay to show that the receptors were constitutively bound to TR-beta and TH/bZIP promoters. Also, we showed that NcoR and SMRT, co-repressor complexes that bind to TR and repress transcription in the absence of TH, dissociated from TR-beta and TH/bZIP promoters in the presence of TH in wild type and not transgenic tadpoles. In addition, increased histone acetylation, which signifies open, transcriptionally active chromatin, was observed at the promoters in the presence of TH only in non-transgenic animals. These results show that TR mediates the metamorphic effects of thyroid hormone. More importantly, they outline molecular mechanisms for gene repression by unliganded TR in vivo, namely TR binds target promoters in chromatin constitutively and recruits co-repressors to deacetylate histones.