Thyrotropin (TSH)-releasing hormone-responsive elements in the rat TSH beta gene have distinct biological and nuclear protein-binding properties.

Abstract

Two TRH-responsive elements have been identified in the rat TSH beta gene by deletion/mutation analysis of the 5'-flanking region of the gene and transfection of TSH beta-luciferase constructs into the GH3 pituitary cell line. Biological responsiveness was confirmed by inserting synthetic oligonucleotides next to the heterologous viral thymidine kinase (tk) promoter in tk luciferase (tkLUC) constructs. Both DNA regions, termed TSH A (at -274 to -258 bp) and TSH C (-402 to -385 bp), have a high level of sequence similarity to binding sites for the POU domain pituitary transcription factor Pit-1/GHF-1. In transfection assays, the TSH A region had no basal enhancer activity, but did confer 3- to 6-fold TRH- and PMA-stimulated transcriptional responses to the tk promoter. The TSH C region conferred basal enhancer activity (3- to 10-fold above control tkLUC) as well as a 2- to 3-fold TRH or PMA response. Combinations of TSH A and TSH C elements conferred both enhancer activity and a TRH- or PMA-stimulated response, but more than two copies of the regions resulted in no further stimulatory effect. Both TSH beta gene regions bound to nuclear proteins from GH3 cells, as determined by gel retardation analysis. The TSH A region DNA formed three prominent DNA-protein complexes, ranging from slowly to rapidly migrating bands and with calculated affinities of 32, 0.5, and 208 nM, respectively. The TSH C region formed two major complexes, which corresponded on the basis of mobility to the most slowly and rapidly migrating complexes formed by TSH A, but with calculated affinities of 3.1 and 33 nM. TSH C also formed a rapidly migrating minor complex unique for this gene region. The more rapidly migrating complexes appeared to be specific to nuclear proteins from GH3 cells. Treatment of cells with TRH did not significantly alter the affinity of protein binding. Mutation of TSH A and TSH C DNA by T to G substitutions abolished the ability of the DNA to confer a TRH response and severely inhibited the ability of the DNA to bind to GH3 nuclear proteins. Thus, transcriptional regulation of the rat TSH beta gene by TRH is correlated with the ability of the two TRH-sensitive elements to bind nuclear proteins. The differences noted in basal enhancer activity or the degree of TRH responsiveness may be related to some unique proteins bound to each DNA or to the differences in affinity of binding of the proteins common to both elements.

cAMP Response Element-binding Protein-binding Protein Mediates Thyrotropin-releasing Hormone Signaling on Thyrotropin Subunit Genes

“…1). We and others have also shown that Pit-1 plays an important role in TRH stimulation of the prolactin and TSH-␤ subunit genes (10,14,40,41). Recently, a mechanism was proposed whereby CBP bound to Pit-1 would mediate the effect of certain signaling pathways in the pituitary (19,20).…”

Pit-1/GHF-1 binds to TRH-sensitive regions of the rat thyrotropin .beta. gene

“…TSH/3 gene regions were labeled with [32P]dCTP and the Klenow fragment of Escherichia coli DNA polymerase. Protein interactions with TSH/3 gene regions were assayed using the nondenaturing Tris-acetate-EDTA gel system at 4 0 C as previously described (Shupnik et al, 1992). Each binding reaction of 10-15 mL contained 20 000 cpm of labeled double-stranded oligonucleotide representing specific gene sequences, 6.7 mM Tris-HC1 (pH 7.5), 1 mM dithiothreitol, 2 Mg of poly(dLdC)poly(dLdC) (Pharmacia, Piscataway, NJ), 100 mM KC1, and either 10 Mg of cellular nuclear protein extract or 3 mL of lysate (programmed or unprogrammed with Pit-1 mRNA) which contained approximately 5 Mg of protein and 5-7 fmol of Pit-1 as estimated by [35S] methionine incorporation.…”

“…Free and bound DNA was excised from the gels and quantitated directly by scintillation spectroscopy. For competition with homologous oligonucleotide, the affinity of binding was estimated by Scatchard analysis as described (Shupnik et al, 1992).…”

“…Within this TRH-responsive area of the gene are three DNA regions with sequence similarity to binding sites for the 0006-2960/93/0432-8932S04.00/0 pituitary-specific transcription factor Pit-l/GHF-1 (Bodner et al, 1988;Ingraham et al, 1988), hereafter referred to as Pit-1. Two of these regions, termed TSH A (-274 to -258 bp) and TSH C (-402 to -384 bp), can confer a TRH, cAMP, or kinase C-stimulated response to a heterologous promoter in transient expression assays in GH3 cells; the TSH C region can confer basal enhancer activity as well (Shupnik et al, 1992). The TSH B region does not confer a significant TRH response or enhancer activity.…”

“…The TSH B region does not confer a significant TRH response or enhancer activity. The gene regions defined by TSH A and TSH C appear to bind to some similar and some unique nuclear proteins, based on gel retardation assays and cross-competition experiments, although with somewhat different calculated affinities (Shupnik et al, 1992). At least one of these common DNA-protein complexes appears to be pituitary cell-specific.…”