Abstract
There are three known thyroid hormone receptor (TR) isoforms that arise from two distinct alpha and beta gene loci. TRalpha1 and TRbeta1 mRNAs are found in many tissues, whereas mRNA for the N-terminal TRbeta2 variant derived from the beta locus is readily detectable only in the pituitary gland and derived cell sources such as GH3 somatotropes and TtT-97 thyrotropes. We previously isolated the genomic region governing expression of the TRbeta2 isoform in thyrotropes and showed that transcription arose from multiple origins within a 400-base pair (bp) region. We now report that the region extending 500 bp upstream of the putative AUG codon (A is +1) contains six areas of interaction with the pituitary-specific transcription factor Pit-1. In addition there are separate areas that bind other factors present in thyrotrope cells. Promoter deletions revealed that removal of regions containing the Pit-1 sites at -456 to -432, -149 to -127, and -124 to -102 progressively decreased TRbeta2 promoter activity in thyrotropes. A more proximal footprinted area from -65 to -19, which accounted for the remaining promoter activity, contained sites that interacted with recombinant Pit-1; however, extracts of TtT-97 thyrotropes, which express Pit-1, footprinted this proximal region with a pattern of protection that differed from that produced by Pit-1. A comparative deletional analysis demonstrated that a shorter region extending only 204 bp from the AUG was sufficient to support TRbeta2 promoter activity in GH3 somatotropes. The more proximal Pit-1 sites, including the area from -53 to -19, whose pattern differed from Pit-1 in thyrotrope extracts, showed protection patterns with GH3 extracts that were indistinguishable from recombinant Pit-1. Site-directed mutagenesis that abrogated binding of both recombinant Pit-1 and Pit-1-containing nuclear extracts revealed that the two Pit-1 sites between -149 and -102 were important for TRbeta2 promoter activity with the more proximal being most critical. Finally, we showed that TRbeta2 promoter activity in alpha-TSH cells, which do not transcribe the endogenous TRbeta2 locus or produce Pit-1 protein, could be reconstituted to a level approaching that seen in expressing TtT-97 thyrotropes by cotransfecting a Pit-1 expression vector. Activation by Pit-1 was dependent on the same Pit-1 sites shown to be important for basal TRbeta2 promoter activity in thyrotropes as constructs lacking them by deletion or mutation were not stimulated by Pit-1.
Highlights
The effects of thyroid hormone (T3) are dependent on its interaction with high affinity nuclear receptor molecules that are related to those that mediate the effects of the steroid hormones, retinoids, and vitamin D [1]
We demonstrate that this genomic region supports promoter activity in GH3 somatotrope cells and that the activity in both cell types is dependent on the presence of areas that interact with Pit-1
Pit-1 binds to Multiple Sites within the thyroid hormone receptor (TR)2 Promoter Region Important for Expression in Thyrotropes—We previously reported that the TR2 genomic region upstream of the putative AUG codon when fused to a luciferase reporter directed luciferase expression in TtT-97 thyrotropic tumor cells that express endogenous TR2 mRNA [13]
Summary
The effects of thyroid hormone (T3) are dependent on its interaction with high affinity nuclear receptor molecules that are related to those that mediate the effects of the steroid hormones, retinoids, and vitamin D [1]. The  locus gives rise to two receptor isoforms (TR1 and TR2) as a result of transcription directed by two separate promoter regions and subsequent splicing of two different N termini onto the same DNA and hormone binding regions [2, 6]. Childs et al [10] subsequently demonstrated by in situ hybridization that transcripts encoding TR2 colocalized almost exclusively with cells in the pituitary gland that stained for thyroid-stimulating hormone (TSH) and growth hormone (GH). Differentiation of these two pituitary cell types has been shown to be dependent on the transcription factor Pit-1 [11, 12]. Pit-1 Regulates TR2 Promoter Activity that express neither TR2 nor Pit-1 establish that Pit-1 is capable of reconstituting TR2 promoter activity to a level approaching that observed with expressing TtT-97 thyrotropes and that the activation is dependent on the interaction of Pit-1 at specific sites
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