The 26-amino acid beta-motif of the Pit-1beta transcription factor is a dominant and independent repressor domain.

The POU-homeodomain transcription factor Pit-1 governs the pituitary cell-specific expression of Pit-1, GH, prolactin (PRL), and TSHbeta genes. Alternative splicing generates Pit-1beta, which contains a 26-amino acid beta-domain inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain (TAD). Pit-1beta represses GH, PRL, and TSHbeta promoters in a pituitary-specific manner, because Pit-1beta activates these same promoters in HeLa nonpituitary cells. Here we comprehensively analyze the role of beta-domain sequence, position, and context, to elucidate the mechanism of beta-dependent repression. Repositioning the beta-motif to the Pit-1 amino terminus, hinge, linker, and carboxyl terminus did not affect its ability to repress basal rat (r) PRL promoter activity in GH4 pituitary cells, but all lost the ability to repress Ras-induced rPRL promoter activity. To determine whether beta-domain repression is independent of Pit-1 protein and DNA binding sites, we generated Gal4-Pit-1TAD, Gal4-Pit-1betaTAD, and Gal4-beta-domain fusions and demonstrated that the beta-motif is sufficient to actively repress VP16-mediated transcription of a heterologous promoter. Moreover, beta-domain point mutants had the same effect whether fused to Gal4 or within the context of intact Pit-1beta. Surprisingly, Gal4-beta repression lost histone deacetylase sensitivity and pituitary specificity. Taken together, these results reveal that the beta-motif is a context-independent, modular, transferable, and dominant repressor domain, yet the beta-domain repressor activity within Pit-1beta contains cell type, promoter, and Pit-1 protein context dependence.