Abstract
The retinoid X receptor (RXR) isoform RXRgamma has limited tissue expression, including brain, skeletal muscle, and anterior pituitary gland. Within the anterior pituitary gland, RXRgamma expression is limited primarily to the thyrotropes. In this report, we have isolated approximately 3 kb of 5'-flanking DNA of the mouse RXRgamma1 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 alphaTSH cells, pituitary-derived GH3 somatomammotropes, and non-pituitary CV-1 cells. None of these cell lines has detectable RXRgamma1 mRNA. A previous report has identified a non-consensus direct repeat (DR-1) element in the RXRgamma2 gene promoter region that mediates stimulation of promoter activity by 9-cis-retinoic acid (9-cis-RA). Inspection of the RXRgamma1 promoter region revealed a non-consensus DR-1 element at -232 bp from the transcription start site. Interestingly, RXRgamma1 promoter activity was suppressed 50% by 9-cis-RA in the TtT-97 thyrotropes. Further experiments in non-pituitary cells showed that suppression of RXRgamma1 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 RXRgamma1 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 RXRgamma1 promoter region provides a tool for further investigation focusing on thyrotrope-specific gene expression as well as negative regulation of genes by retinoic acid.
Highlights
Tissue and cell-specific expression of many genes occurs at the level of gene transcription
The retinoid X receptor (RXR)␣ and RXR isoforms are widely distributed throughout the developing embryo and in the adult, while the RXR␥ isoform has restricted tissue distribution, which includes a high level of expression in the pituitary gland early in development [8]
To define the 5Ј extent of the RXR␥1 mRNA in thyrotropes, 5Ј-Rapid Amplification of cDNA Ends (RACE) was performed on TtT-97 poly(A)ϩ RNA
Summary
Tissue and cell-specific expression of many genes occurs at the level of gene transcription. The RXR␣ and RXR isoforms are widely distributed throughout the developing embryo and in the adult, while the RXR␥ isoform has restricted tissue distribution, which includes a high level of expression in the pituitary gland early in development [8]. Our group and others [9, 10] have shown that the RXR␥1 subtype is expressed in the anterior pituitary and has expression that is restricted to thyrotropes. The human RXR␥2 gene 5Ј-flanking region has been identified and contains a retinoid-responsive element, which confers positive regulation of promoter activity by retinoic acid [12]. An understanding of RXR␥1 promoter function in thyrotropes and other cell types will provide insight into mechanisms governing thyrotropespecific gene expression and phenotype, as well as the generalized mechanism of cell-specific gene expression
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