Isolation and Functional Analysis of the Mouse RXRgamma 1 Gene Promoter in Anterior Pituitary Cells (original) (raw)
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Isolation and Functional Analysis of the Mouse RXRγ1 Gene Promoter in Anterior Pituitary Cells
Journal of Biological Chemistry, 2002
The retinoid X receptor (RXR) isoform RXR␥ has limited tissue expression, including brain, skeletal muscle, and anterior pituitary gland. Within the anterior pituitary gland, RXR␥ expression is limited primarily to the thyrotropes. In this report, we have isolated ϳ3 kb of 5-flanking DNA of the mouse RXR␥1 gene. We have identified the major transcription start site in the thyrotrope-derived TtT-97 cells. Transient transfection studies show that a 1.4-kb promoter fragment has full promoter activity in TtT-97 cells. This promoter has much less activity in thyrotrope-derived ␣TSH cells, pituitary-derived GH3 somatomammotropes, and non-pituitary CV-1 cells. None of these cell lines has detectable RXR␥1 mRNA. A previous report has identified a nonconsensus direct repeat (DR-1) element in the RXR␥2 gene promoter region that mediates stimulation of promoter activity by 9-cis-retinoic acid (9-cis-RA). Inspection of the RXR␥1 promoter region revealed a non-consensus DR-1 element at ؊232 bp from the transcription start site. Interestingly, RXR␥1 promoter activity was suppressed 50% by 9-cis-RA in the TtT-97 thyrotropes. Further experiments in non-pituitary cells showed that suppression of RXR␥1 promoter activity was RXR-dependent. Mutagenesis of the DR-1 element abrogated suppression of promoter activity by 9-cis-RA, suggesting that this negative regulation requires both RXR and this specific DR-1 element. In summary, we have isolated the mouse RXR␥1 gene promoter region and identified the major start site in thyrotropes. Promoter activity is uniquely suppressed by 9-cis-RA through a DR-1 element. Isolation and characterization of the mouse RXR␥1 promoter region provides a tool for further investigation focusing on thyrotrope-specific gene expression as well as negative regulation of genes by retinoic acid.
Characterization of three RXR genes that mediate the action of 9-cis retinoic acid
Genes & Development, 1992
An understanding of the differences and similarities of the retinoid X receptor (RXR) and retinoic acid receptor (RAR) systems requires knowledge of the diversity of their family members, their patterns of expression, and their pharmacological response to ligands. In this paper we report the isolation of a family of mouse RXR genes encoding three distinct receptors (RXRa, p, and y). They are closely related to each other in their DNAand ligand-binding domains but are quite divergent from the RAR subfamily in both structure and ligand specificity. Recently, we demonstrated that all-trans retinoic acid (RA) serves as a "pro-hormone" to the isomer 9-cis RA, which is a high-affinity ligand for the human RXRa. We extend those findings to show that 9-cis RA is also "retinoid X" for mouse RXRa, p, and y. Trans-activation analyses show that although all three RXRs respond to a variety of endogenous retinoids, 9-cis RA is their most potent ligand and is up to 40-fold more active than all-trans RA. Northern blot and in situ hybridization analyses define a broad spectrum of expression for the RXRs, which display unique patterns and only partially overlap themselves and the RARs. This study suggests that the RXR family plays critical roles in diverse aspects of development, from embryo implantation to organogenesis and central nervous system differentiation, as well as in adult physiology.
HAL (Le Centre pour la Communication Scientifique Directe), 1997
The thyroid hormone receptor-coding locus, c-erbA␣, generates several mRNAs originating from a single primary transcript that undergoes alternative splicing. We have identified for the first time two new transcripts, called TR⌬␣1 and TR⌬␣2 [mRNA for isoform ␣1 and ␣2 of the T 3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbA␣ gene. These two new transcripts exhibit tissue-specific patterns of expression in the mouse. These two patterns are in sharp contrast with the expression patterns of the full-length transcripts generated from the c-erbA␣ locus. TR⌬␣1 and TR⌬␣2 mR-NAs encode N-terminally truncated isoforms of T3R␣1 and T3R␣2, respectively. The protein product of TR⌬␣1 antagonizes the transcriptional activation elicited by T 3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3R␣1 and 9-cis-retinoic acid receptor-␣ but does not affect the retinoic aciddependent activating function of retinoic acid receptor-␣. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.
Retinoid-dependent in vitro transcription mediated by the RXR/RAR heterodimer
Genes & Development, 1994
The effects of retinoids on gene regulation are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Here, we provide the first biochemical evidence that, in vitro, ligand governs the transcriptional activity of RXRa/RARa by inducing conformational changes in the ligand-binding domains. Using limited proteolytic digestion we show that binding of the cognate ligand causes a conformational change in the carboxy-terminal part of the receptor. We also show that recombinant RXRa/RARa is partially active in the absence of exogenously added ligand. Trans-activation depends critically on the ligand-dependent transcriptional activation function AF-2 of RARer. Full activation by recombinant RXRoL/RAR~, however, requires the addition of either all-trans RA, 9-cis RA, or other RAR-specific agonists, whereas an RARa-specific antagonist abolishes trans-activation. Intriguingly, the ligand-dependent AF-2 of RXR does not contribute to the level of transcription from the RARI32 promoter in vitro even when the cognate ligand (9-cis RA1 is bound. Thus, the major role of RXR in trans-activation of the RARI32 promoter is to serve as an auxiliary factor required for the binding of RAR which, in turn, is directly responsible for transcriptional activity.
Retinoic X receptor subtypes exert differential effects on the regulation of Trh transcription
Molecular and Cellular Endocrinology, 2013
How Retinoid X receptors (RXR) and thyroid hormone receptors (TR) interact on negative TREs and whether RXR subtype specificity is determinant in such regulations is unknown. In a set of functional studies, we analyzed RXR subtype effects in T 3 -dependent repression of hypothalamic thyrotropinreleasing hormone (Trh). Two-hybrid screening of a hypothalamic paraventricular nucleus cDNA bank revealed specific, T 3 -dependent interaction of TRs with RXRb. In vivo chromatin immuno-precipitation showed recruitment of RXRs to the TRE-site 4 region of the Trh promoter in the absence of T 3 . In vivo overexpression of RXRa in the mouse hypothalamus heightened T 3 -independent Trh transcription, whereas RXRb overexpression abrogated this activity. Loss of function of RXRa and b by shRNAs induced inverse regulations. Thus, RXRa and RXRb display specific roles in modulating T 3 -dependent regulation of Trh. These results provide insight into the actions of these different TR heterodimerization partners within the context of a negatively regulated gene.
Journal of Molecular Endocrinology, 2002
Thyroid hormone receptors (TRs) often modulate transcriptional activity of target genes by heterodimerization with the 9-cis retinoic acid receptor (RXR). On positive thyroid response elements (TREs), RXR favors binding of the TR-RXR complex to DNA and stimulates transcription. RXR action on negative TREs is unclear. Furthermore, the single half-site configuration of many negative TREs does not favor the binding of a classic TR-RXR heterodimer. In a comparative study using CV-1 cells (relatively RXR- and TR-deficient) and JEG-3 cells (relatively TR-deficient), we demonstrate the importance of RXR in the negative transcriptional regulation of genes of the hypothalamo-pituitary axis by tri-iodothyronine. While RXR has variable effects on ligand-independent activation produced by TRs, it was required for efficient ligand-dependent repression of the TRH gene for TRalpha1 and TRbeta1 and of the TSH genes by all TRs. Using different RXR constructs we also observed the importance of the C-...
Journal of Biological Chemistry, 1997
The retinoid X receptor (RXR), a member of the superfamily of hormone nuclear receptors, is a ligand-inducible transcription factor that is activated by the vitamin A derivative 9-cis-retinoic acid. We previously showed that RXR self-associates into tetramers with a high affinity and that ligand binding induces rapid dissociation of receptor tetramers to smaller species. Here, the RXR region that is responsible for mediating tetramer formation is identified. It is shown that this interface, which we term the "tetramerization domain," critically contains two consecutive phenylalanine residues located at the C-terminal region of the receptor. Mutation of these residues is sufficient to disrupt RXR tetramers without affecting the overall fold of the protein or interfering with ligand binding, dimer formation, or DNA binding by the receptor. Nevertheless, the tetramer-impaired mutant was found to be transcriptionally defective. The newly characterized tetramerization domain and the previously identified main dimerization interface of RXR act autonomously to affect separate intersubunit interactions that, overall, lead to formation of tetramers. Protein-protein interactions mediated by the tetramerization domain, but not those that involve the dimerization interface, are disrupted following ligand binding by RXR. Overall, these data attest to the specificity of the interaction and implicate the tetramerization interface in playing a direct role in regulating transcriptional activation by RXR.