Regulation of gene transcription by the thyroid hormone receptors

Abstract: Thyroid hormone (TH) has important effects on postnatal development as well as on adult metabolic homeostasis. TH meditates its effects through different isoforms of the thyroid hormone receptors (TRs) encoded by the TRalpha and TRbeta genes, respectively. TRs belong to the nuclear hormone receptor superfamily. These receptors are ligand-modulated transcription factors that bind to specific DNA elements located in the regulatory regions of target genes. The nature of these elements determines the effect TR will have on transcription. On what is called a positive TH response element (pTRE), the un-liganded receptor binds to DNA and represses transcription. Hormone binding induces a conformational change in the receptor, allowing for activation of transcription. In contrast, on a negative TH response element (nTRE), activation of transcription is mediated by the un-liganded receptor, whereas the liganded receptor represses transcription. The mechanism behind the regulation of a pTRE is quite well understood, while the mechanism regulating nTREs is less elucidated. Interestingly, TH has been found to regulate more genes negatively than positively in vivo. In addition, studies of mutant mice that are unable to produce active TH but have intact TR expression, show a lethal phenotype, which contrasts to the viable phenotype of mice lacking all TR isoforms. This further underlines the physiological importance of TRs in the repression of transcription. The E2F-1 protein is one of the major regulators of cell cycle progression. In Paper I, we identified a putative nTRE in the E2F-1 promoter and showed that TR bound directly to this nTRE. This nTRE was sufficient for TRs to mediate both hormone-independent activation and hormone-dependent repression of transcription. Down-regulation of E2F-1 mRNA expression was observed after TH-treatment, both in P19 embryonic carcinoma cells and in oligodendrocyte precursor cells (OPCs), and correlated with an arrest in cell cycle progression. We further showed that both un-liganded TRalpha and TRbeta activated transcription from the E2F-1 promoter, that DNA binding was required for hormone-independent activation of transcription and that an intact ligand-binding domain was required for hormone-dependent repression of transcription. The results suggest that TH modulates the cell cycle through a direct repression of the E2F-1 gene. In Paper II, we identified a putative nTRE in the Necdin gene. Necdin is a growth suppressor that facilitates cell cycle exit and neuronal differentiation, and inhibits apoptosis. The Necdin nTRE sequence was similar to the E2F-1 nTRE and identical to the nTRE of the TSH gene, which also is negatively regulated by TH. We showed that TR, in the absence of TH, activated transcription of the Necdin promoter and that TH repressed this activation. We further showed that TR bound as a heterodimer with Retinoic-X-receptor (RXR) to the Necdin nTRE sequence and that RXR and the nuclear receptor corepressor (NCoR) enhanced the TR-mediated transcriptional activation of Necdin in the absence of TH. As Necdin is expressed in postmitotic regions in the mouse brain, and is down-regulated after birth coinciding in time with the crucial postnatal increase of TH, our results suggests that TRs are important to regulation of Necdin expression. To investigate whether TH mediates post-natal down-regulation of Necdin expression in the mouse brain, in vivo studies were performed. In Paper III we showed that TRs regulated expression of Necdin in a cell-type specific manner. In vivo gene transfer experiments showed that exogenous TRbeta, ? injected into the hypothalamus, was required for transactivation of the Necdin promoter in the absence of TH. In situ hybridization analysis of Necdin expression in the paraventricular nuclei of the hypothalamus in 2-day-old mice showed that TH did not repress Necdin expression. Finally, we showed that the transcription factor NSCL-2 counteracted the regulation of Necdin by TR in vitro. TH down-regulates the production of thyrotropin-releasing hormone (TRH) in a negative feedback mechanism. Both TRbeta1 and TRbeta2 down-regulate TRH in the presence of TH. However, only TR b 1 participates in activation of TRH transcription in the absence of TH. In Paper IV, we showed that the hsp90-associated co-chaperone protein, XAP2, bound to TRbeta1 and was involved in the hormone-independent activation of TRH expression. Furthermore, siRNA-mediated knock-down of XAP2 function in vivo abrogated TR-mediated activation of hypothalamic TRH transcription. In summary, we have identified nTRE sequences in the E2F-1 and Necdin promoters as being TR targets. These nTREs are required for hormone-independent activation of transcription by TR. In addition, factors, such as NCoR, RXR and XAP2, are required for efficient activation of negatively regulated TR target genes in the absence of TH.