MSX1 IS PRESENT IN THYROTROPIC CELLS AND BINDS TO A CONSENSUS SITE ONTHE GLYCOPROTEIN HORMONE ALPHA-SUBUNIT PROMOTER

Our studies are aimed at identifying the transcription factors that ac tivate the glycoprotein hormone alpha-subunit promoter. Therefore, we performed a Southwestern screening of a thyrotropic (alpha TSH) cDNA e xpression 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 Ms x1, which is a homeodomain-containing transcription factor that has be en found to play an important role in the development of limb buds and craniofacial structures. Northern blot analysis, using the cloned Msx 1 cDNA fragment as a probe, demonstrated that alpha-subunit-expressing thyrotrope cells (alpha TSH cells and TtT97 tumors) contained Msx1 RN A transcripts of 2.2 kb, while somatomammotrope (GH3) cells that do no t produce the alpha-subunit had barely detectable levels. The presence of Msx1 protein was demonstrated by Western blot analysis in alpha TS H cells. We also demonstrated that transcripts encoding the closely re lated Msx2 factor were not detectable by Northern blot analysis in eit her thyrotrope or somatomammotrope-derived cells. Subfragments of the region from -490 to -310 of the Lu-subunit promoter were used in a Sou thwestern blot assay using bacterially produced Msx1 and demonstrated that binding was localized specifically to the region from -449 to -42 1. Deoxyribonuclease I protection analysis, using purified Msx1 homeod omain, demonstrated structurally induced differences in DNA digestion patterns between -436 and -413, and sequence analysis of this region r evealed a direct repeat of the sequence GXAATTG, which is similar to t he Msx1 consensusbinding site. When nucleotides at both sites were mut ated, Msx1 binding was dramatically reduced, and the activity of an al pha-subunit promoter construct decreased by similar to 50% in transfec ted thyrotrope (alpha TSH) cells. These studies suggest that Msx1 may play a role in the expression of the alpha-subunit gene in thyrotrope cells.