Hesx1 homeodomain protein represses transcription as a monomer and antagonises transactivation of specific sites as a homodimer

The homeobox repressor Hesx1, expressed throughout Rathke's pouch and required for normal pituitary development, has been implicated in anterior pituitary pathogenesis in man. Prolonged expression of Hesx1 delays the appearance of anterior pituitary terminal differentiation markers in mice, particularly the gonadotroph hormones. We tested if Hesx1 could modulate gonadotrophin gene expression directly, and found that Hesx1 repressed both common alpha subunit (αGSU) and luteinising hormone β-subunit (LHβ) gene promoters. Repression mapped to the Pitx1 homeodomain protein transactivation site in the proximal αGSU promoter, but did not map to the equivalent site on LHβ. Hesx1 repression of the αGSU Pitx1 site was overridden by co-transfection of Pitx1. In contrast, Hesx1 antagonised Pitx1 transactivation of LHβ in a dose-dependent manner. This was due to monomeric binding of Hesx1 on αGSU and homodimerisation on LHβ. The homodimerisation site comprises the Pitx1 DNA binding site and a proximal binding site, and mutation of either inhibited homodimer formation. Conversion of the LHβ Pitx1 DNA binding site to an αGSU-type did not promote homodimer formation, arguing that Hesx1 has pronounced site selectivity. Furthermore, mutation of the proximal half of the homodimerisation site blocked Hesx1 antagonisation of Pitx1 transactivation. We conclude that Hesx1 monomers repress gene expression, and homodimers block specific transactivation sites.