David F. Gordon

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

The molecular determinants governing cell-specific expression of the thyrotropin (TSH) β-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHβ promoter (−133 to −88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSHβ promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.

GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.

ABSTRACT The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

Analysis of Pit-1 in regulating mouse TSH β promoter activity in thyrotropes

Abstract TSHβ gene expression is restricted to pituitary thyrotropes. Since Pit-1 is present in these cells, we characterized Pit-1 RNA and protein in thyrotropes, and tested its function in regulating TSHβ promoter activity. We demonstrate that both TtT-97 thyrotropic tumors and pituitaries contain four Pit-1 transcripts of 3.2, 2.6, 2.4, and 1.9 kb, respectively. Only two transcripts of 2.7 and 2.1 kb were detected in αTSH cells, a thyrotrope derived cell that no longer expresses TSHβ. Western analysis revealed Pit-1 protein in TtT-97 cells but not in αTSH cells. DNase I protection assays localized Pit-1 binding to three areas of the mouse TSHβ promoter. However, basal TSHβ promoter activity was minimally stimulated when αTSH cells or TtT-97 thyrotropes were co-transfected with mouse Pit-1 and a mTSHβ luciferase construct. These studies suggest that Pit-1 is not limiting for cell-specific expression of the TSHβ gene in thyrotrope-derived cells and implies that additional thyrotropic factors are likely required.

Determinants of thyrotrope-specific thyrotropin beta promoter activation. Cooperation of Pit-1 with another factor.

Thyrotropin (TSH) β is a subunit of TSH, the expression of which is limited to the thyrotrope cells of the anterior pituitary gland. We have utilized the thyrotrope-derived TtT-97 thyrotropic tumors to investigate tissue-specific expression of the TSHβ promoter. TSHβ promoter activity in thyrotropes is conferred by sequences between −270 and −80 of the 5′-flanking region. We have recently reported that the proximal region from −133 to −100 (P1) is required for promoter expression in thyrotropes. This region interacts with the pituitary-specific transcription factor Pit-1. While Pit-1 appears necessary for TSHβ promoter activity in thyrotropes, this transcription factor is not alone sufficient for promoter activity in pituitary-derived cells. In this report, we have generated a series of promoter mutations in the P1 region to identify additional protein-DNA interactions and determine their functional significance. We have found that Pit-1 interacts with the distal portion of the P1 region, and a second protein interacts with the proximal segment of this region. Each protein is able to independently interact with the TSHβ promoter, but neither alone can maintain promoter activity. Both proteins appear to be necessary for full promoter activity in thyrotropes. Southwestern analysis with the proximal segment of the P1 region (−117 to −88) reveals interaction with a 50-kDa protein. Interestingly, this protein is not found in the pituitary-derived GH3 cells and may represent a thyrotrope-specific transcription factor. Further characterization of this newly identified DNA-binding protein will further our understanding of the tissue-specific expression of the TSHβ gene.

Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSHβ gene

Previous studies showed that Pit-1 functionally cooperates with GATA-2 to stimulate transcription of the TSH beta gene. Pit-1 and GATA-2 are uniquely coexpressed in pituitary thyrotropes and activate transcription by binding to a composite promoter element. To define the domains of Pit-1 important for functional cooperativity with GATA-2, we cotransfected a set of Pit-1 deletions with an mTSH beta-luciferase reporter. Plasmids were titrated to express equivalent amounts of protein. A mutant containing a deletion of the hinge region between the POU and homeodomains retained the ability to fully synergize with GATA-2. In contrast, mutants containing deletions of amino acids 2-80 or 72-125 demonstrated 56 or 34% of the synergy found with the full-length protein, suggesting that these regions contributed to cooperativity. Mutants with deletions of the POU-specific or homeodomain further reduced the effect signifying the requirement for DNA binding. GST interaction studies demonstrated that only the homeodomain of Pit-1 interacted with GATA-2. Finally, several mutations between the Pit-1 and GATA-2 sites on the TSH beta promoter reduced binding for each factor and greatly reduced ternary complex formation. Thus multiple domains of Pit-1 are required for full synergy with GATA-2 and sequences between the two binding sites contribute to co-occupancy with both factors on the proximal TSH beta promoter.

Human progesterone A-receptors can be synthesized intracellularly and are biologically functional

In order to investigate the origin and functional independence of the human progesterone receptor A binding protein, we have expressed a truncated human progesterone receptor cDNA in both gene transfer and in vitro translation assays. Proteins identical in size and antigenicity to the A-receptors found naturally in human progesterone target cells are synthesized from this cDNA that lacks the putative B receptor initiator methionine codon of the complete cDNA. The functional independence of A-receptors is suggested by their ability to bind hormone and to stimulate transcription from the progestin responsive mouse mammary tumor virus promoter.

Cloning of the Mouse Somatostatin Receptor Subtype 5 Gene: Promoter Structure and Function

Somatostatin is a peptide hormone whose actions are mediated by five somatostatin receptor subtypes (sst1–5). In the pituitary, somatostatin inhibits TSH release from thyrotropes and GH release from somatotropes. We have shown that sst5 transcripts and protein are induced by thyroid hormone in TtT-97 thyrotropic tumors. To map sequences responsible for promoter activity in pituitary cells, we cloned the mouse sst5 coding region of 362 amino acids and 12 kb of upstream DNA. Initial transfection studies in TtT-97 or GH3 cells mapped high levels of basal promoter activity to a 5.6-kb fragment upstream of the translational start, whereas shorter genomic fragments had low activity. To identify the transcriptional start site we used 5′ RACE with TtT-97 poly A+ RNA and a sst5 antisense coding region primer. Sequence comparison between the complementary DNA and the gene revealed that the mouse sst5 gene contains 3 exons and 2 introns. The entire coding region was contained in exon 3. Two differently sized RACE pr...

Activation of the glycoprotein hormone α-subunit gene promoter in thyrotropes

Pituitary expression of the glycoprotein hormone alpha-subunit gene localizes to thyrotropes and gonadotropes. Factors that activate the alpha-subunit promoter exclusively in thyrotropes have not been described. In the current studies, alpha-subunit promoter activity was compared in alphaTSH thyrotropic and alphaT3 gonadotropic cells. Deletional analysis revealed a more important contribution of the -453/-381 region in thyrotropic cells and the -341/-297 region in gonadotropic cells, while other deletions had similar effects. Mutational analysis revealed some regions that were functionally similar, while others were active only in thyrotropic cells, such as the regions - 434/ - 421, - 398/ - 385, - 376/- 364 and - 363/- 351, that decreased activity by approximately 2-fold each. Combined mutation of the regions - 434/ - 421, - 398/ - 385 and - 376/ - 364 decreased activity by 5-fold in thyrotropic cells and not at all in gonadotropic cells. Cotransfection with Ptx1 in CV-1 cells resulted in a strong stimulation of alpha-subunit promoter activity, that was significantly diminished with mutation of the - 398/ - 385 region that disrupts the Ptx1 binding site, suggesting that this factor may play a role in thyrotrope-specific activation. This analysis provides basic information important to identify common and unique factors contributing to the cell-specific expression of the alpha-subunit gene.

AN UPSTREAM REGULATOR OF THE GLYCOPROTEIN HORMONE ALPHA -SUBUNIT GENE MEDIATES PITUITARY CELL TYPE ACTIVATION AND REPRESSION BY DIFFERENT MECHANISMS

Targeting of α-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived α-TSH and α-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in α-TSH and α-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal α-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in α-TSH and α-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence.

Effect of thyroid hormone on T3-receptor mRNA levels and growth of thyrotropic tumors

Thyrotropic tumors (TtT97) contain mRNA transcripts for three T3-receptor (TR) isoforms, alpha 1, beta 1 and beta 2, and a non-receptor alpha 2-variant. We administered T4 (5 mg/l of drinking water) for one month to TtT97-bearing mice, to examine its effect on tumor growth and tumor TR isoform steady-state mRNA levels. Baseline mice were killed at the start of the experiment, and placebo mice were maintained hypothyroid. The treated tumors were 30-35% smaller than the baseline tumors (p = NS), while the placebo tumors were 2- to 7-fold larger than the baseline tumors (p < 0.05). TR beta 1 mRNA increased 5- to 6-fold, while TR beta 2 mRNA decreased by 76%. TR alpha 1 and the alpha 2-variant decreased by 52% and 70%, respectively. Therefore, the tumors decreased their growth rate in response to T4 administration, and increased the ratio of TR beta 1 to TR beta 2 mRNA. This raises the intriguing possibility of a correlation between the relative abundance of the TR beta isoforms and tumor growth.