Our studies are aimed at identifying the transcription factors that activate the glycoprotein hormone alpha-subunit promoter. Therefore, we performed a Southwestern screening of a thyrotropic (alphaTSH) cDNA expression library, using the region of the promoter from -490 to -310 that contains sequences critical for expression in thyrotrope cells. A clone was isolated corresponding to part of the coding sequence of Msx1, which is a homeodomain-containing transcription factor that has been found to play an important role in the development of limb buds and craniofacial structures. Northern blot analysis, using the cloned Msx1 cDNA fragment as a probe, demonstrated that alpha-subunit-expressing thyrotrope cells (alphaTSH cells and TtT97 tumors) contained Msx1 RNA transcripts of 2.2 kb, while somatomammotrope (GH3) cells that do not produce the alpha-subunit had barely detectable levels. The presence of Msx1 protein was demonstrated by Western blot analysis in alphaTSH cells. We also demonstrated that transcripts encoding the closely related Msx2 factor were not detectable by Northern blot analysis in either thyrotrope or somatomammotrope-derived cells. Subfragments of the region from -490 to -310 of the alpha-subunit promoter were used in a Southwestern blot assay using bacterially produced Msx1 and demonstrated that binding was localized specifically to the region from -449 to -421. Deoxyribonuclease I protection analysis, using purified Msx1 homeodomain, demonstrated structurally induced differences in DNA digestion patterns between -436 and -413, and sequence analysis of this region revealed a direct repeat of the sequence GXAATTG, which is similar to the Msx1 consensus-binding site. When nucleotides at both sites were mutated, Msx1 binding was dramatically reduced, and the activity of an alpha-subunit promoter construct decreased by approximately 50% in transfected thyrotrope (alphaTSH) cells. These studies suggest that Msx1 may play a role in the expression of the alpha-subunit gene in thyrotrope cells.

The glycoprotein hormone alpha-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between -4.6 and -3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to -341/+43 mouse alpha-subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact -4.6 kb/+43 construct. Deletion of sequences between -341 and -297 resuited in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a -341/+43 alpha-promoter in both alphaTSH and alphaT3 cells, but it did not enhance alpha-promoter activity significantly in CV-1 cells. Removal of sequences between -341 and -297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the alpha-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from -341 to -297 are critical for restricting expression to the anterior pituitary.