Charakterisierung von Alien Isoformen in Vertebraten

Alien protein isoforms have been described to be involved in a number of biological processes. Alienalpha is a corepressor of the thyroid hormone receptor mediating transcriptional repression in a ligand-sensitive manner. Furthermore, Alienalpha is a corepressor for the orphan receptor DAX1 and the vitamin-D3 receptor. Alienbetta/CSN2 is part of the COP9-signalosome complex that acts in protein phosphorylation, protein degradation and cell cycle regulation. The major goal of this work was to characterize the Alienalpha and Alienbetta isoforms. Little was known about their expression pattern and the regulation of their expression had not been addressed. It was determined in this work that the expression pattern of Alien is rather ubiquitous in rat tissues. Interestingly, a putative novel Alien protein isoform and an additional alien messenger specific for adrenal gland were identified. Furthermore, it was shown in vivo and in vitro, by in situ hybridization, Northern and Western blotting that Alien expression is regulated by thyroid hormone in the rat brain and brain-derived cell lines. Subsequently, hints for a second T3-independent mechanism of regulation of Alien expression depending on cell confluence or quiescence were discovered. The comparison of Alien isoforms in functional aspects identified Rb and E2F as novel Alien-interacting proteins with similar binding characteristics in vitro and in yeast but functional differences in vivo. Alienbetta interfered with Rb-mediated superactivation of Sp1-driven transcription, whereas Aliena exerted strong repression on E2F transactivation. Common traits for both Alienalpha and Alienbetta are their silencing potential, interaction with TR and activation of AP1-driven transcription. Phosphorylation studies raised the possibility of regulation by non-hormonal signaling since Alienalpha and Alienbetta are phosphorylated in vivo. In gel kinase assays suggested the existence of two different Alien-phosphorylating kinases. Further experiments identified MLK2 and the cell cycle kinase p34cdc2 as such kinases, suggesting a possible function of Alien in cell cycle regulation. Taken together, the expression of Alien is regulated by thyroid hormone, and by cell density; the isoforms can be phosphorylated and can act either as transcriptional repressors or as activators. Additional data indicate a role of Alien isoforms in cell cycle regulation through p34cdc2 phosphorylation and isoform-specific interference with Rb and E2F

hSrb7, an essential human Mediator component, acts as a coactivator for the thyroid hormone receptor

Nuclear hormone receptors interact with the basal-transcriptional complex and/or coactivators to regulate transcriptional activation. These activator-target interactions recruit the transcriptional machinery to the promoter and may also stimulate transcriptional events subsequent to the binding of the machinery to the promoter or enhancer element. We describe a novel functional interaction of the nuclear thyroid receptor (TR), with a human Mediator component (hSrb7), and a human TFIIH component (hMo15). In mammalian two-hybrid experiments as well as in GST-pull down assays, hSrb7 interacts with TR but not with other nuclear receptors such as the retinoic acid receptor (RAR) or the vitamin D receptor (VDR). Whereas hMo15 also interacts with VDR and RAR in mammalian two-hybrid assays, no association of hSrb7 with VDR or RAR is found. Accordingly, cotransfection of TR and hSrb7 increases thyroid hormone (T3)-dependent transcription in an AF-2-dependent manner, while hSrb7 causes no stimulation of vitamin D- or retinoic acid-mediated transactivation. These results reveal a novel co-activator role for hSrb7 and hMo15 on TR transcriptional responses, and demonstrate that different receptors can selectively target different co-activators or general transcription factors to stimulate transcription.This work has been supported by the grant BMC2001-2275 from the Dirección General de Enseñanza Superior e Investigación of the Ministerio de Ciencia y Tecnologı́a of Spain. Dr. J. Nevado is a recipient of a Research Contract from ISCIII (FIS 99/3077)

Alien/CSN2 gene expression is regulated by thyroid hormone in rat brain

AbstractAlien has been described as a corepressor for the thyroid hormone receptor (TR). Corepressors are coregulators that mediate gene silencing of DNA-bound transcriptional repressors. We describe here that Alien gene expression in vivo is regulated by thyroid hormone both in the rat brain and in cultured cells. In situ hybridization revealed that Alien is widely expressed in the mouse embryo and also throughout the rat brain. Hypothyroid animals exhibit lower expression of both Alien mRNAs and protein levels as compared with normal animals. Accordingly, we show that Alien gene is inducible after thyroid hormone treatment both in vivo and in cell culture. In cultured cells, the hormonal induction is mediated by either TRα or TRβ, while cells lacking detectable amounts of functional TR lack hormonal induction of Alien. We have detected two Alien-specific mRNAs by Northern experiments and two Alien-specific proteins in vivo and in cell lines by Western analysis, one of the two forms representing the CSN2 subunit of the COP9 signalosome. Interestingly, both Alien mRNAs and both detected proteins are regulated by thyroid hormone in vivo and in cell lines. Furthermore, we provide evidence for the existence of at least two Alien genes in rodents. Taken together, we conclude that Alien gene expression is under control of TR and thyroid hormone. This suggests a negative feedback mechanism between TR and its own corepressor. Thus, the reduction of corepressor levels may represent a control mechanism of TR-mediated gene silencing

The thyroid hormone receptors as tumor suppressors

AbstractIn addition to the well-known role of the thyroid hormone receptors (TRs) in growth, development and metabolism, there is increasing evidence that they have profound effects on cell proliferation and malignant transformation. TRs repress transcriptional induction of</jats:p