The Pit-1 homeodomain and beta-domain interact with Ets-1 and modulate synergistic activation of the rat prolactin promoter

Pit-1/GHF-1 is a pituitary-specific, POU homeodomain transcription factor r equired for development of somatotroph, lactotroph, and thyrotroph cell lin eages and regulation of the temporal and spatial expression of the growth h ormone, prolactin (PRL), and thyrotropin-beta genes. Synergistic interactio n of Pit-1 with a member of the Ets family of transcription factors, Ets-1, has been shown to be an important mechanism regulating basal and Ras-induc ed lactotroph-specific rat (r) PRL promoter activity. Pit-1 beta/GHF-2, an alternatively spliced isoform containing a 26-amino acid insert (beta-domai n) within its transcription-activation domain, physically interacts with Et s-l but fails to synergize. By using a series of Pit-1 internal-deletion co nstructs in a transient transfection protocol to reconstitute rPRL promoter activity in HeLa cells, we have determined that the functional and physica l interaction of Pit-1 and Ets-1 is mediated via the POU homeodomain, which is common to both Pit-1 and Pit-1 beta. Although the Pit-1 homeodomain is both necessary and sufficient for direct binding to Ets-1 in a DNA-independ ent manner, an additional interaction surface was mapped to the beta-domain , specific to the Pit-1 beta isoform, Thus, the unique transcriptional prop erties of Pit-1 and Pit-1 beta on the rPRL promoter may be due to the forma tion of functionally distinct complexes of these two Pit-1 isoforms with Et s-1.