The Pit-1 Homeodomain and β-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 required for development of somatotroph, lactotroph, and thyrotroph cell lineages and regulation of the temporal and spatial expression of the growth hormone, prolactin (PRL), and thyrotropin-beta genes. Synergistic interaction 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-induced lactotroph-specific rat (r) PRL promoter activity. Pit-1beta/GHF-2, an alternatively spliced isoform containing a 26-amino acid insert (beta-domain) within its transcription-activation domain, physically interacts with Ets-1 but fails to synergize. By using a series of Pit-1 internal-deletion constructs in a transient transfection protocol to reconstitute rPRL promoter activity in HeLa cells, we have determined that the functional and physical interaction of Pit-1 and Ets-1 is mediated via the POU homeodomain, which is common to both Pit-1 and Pit-1beta. Although the Pit-1 homeodomain is both necessary and sufficient for direct binding to Ets-1 in a DNA-independent manner, an additional interaction surface was mapped to the beta-domain, specific to the Pit-1beta isoform. Thus, the unique transcriptional properties of Pit-1 and Pit-1beta on the rPRL promoter may be due to the formation of functionally distinct complexes of these two Pit-1 isoforms with Ets-1.