The POU-protein Pit-1 is an important developmentally regulated transcription factor that activates both the prolactin and growth hormone genes. However, the exact mechanism(s) involved in final determination of lactotroph and somatotroph phenotype is unknown. Based on recent evidence, it is proposed that Pit-1 has a unique spacing requirement which is met precisely in the prolactin and growth hormone promoters. This hypothesis will be tested using cell transfection experiments using prolactin promoters with 5 bp spacing alterations of a high-affinity Pit-1 binding site. Additionally, it is hypothesized that transcriptional synergism mediated by upstream Pit-1 binding sites is accomplished by a channeling model. This hypothesis will also be tested by cell transfection assays using promoters with 5 bp spacing alterations of a high-affinity Pit-1 binding site linked to three upstream Pit-1 binding sites. Since nothing is known concerning the mechanism of Pit-1 transcriptional activation, it is important to determine which step in the assembly of an RNA polymerase II preinitiation complex that Pit-1 is influencing. A goal of this project is to use isolated preinitiation complexes in an in vitro transcription system to identify the general transcription factors(s) that Pit-1 recruits to the template. These data are crucial to understanding the mechanism of action of Pit-1 in transcriptional activation. Based on the above spacing requirement of Pit-1 and the fact that the prolactin and growth hormone fit these requirements perfectly, it is hypothesized that lactotrophic and somatotrophic adaptors, which work in the context of the prolactin and growth hormone promoters, have an important role in cell-type-specific expression of both genes. This hypothesis will be tested in a transgenic model using the beta- galactosidase reporter gene linked to prolactin and growth hormone promoter/enhancers which have the position of the proximal prolactin and growth hormone Pit-1 binding site switched. In addition, the Pit-1 target molecule will be identified and cloned using protein-affinity chromatography and screening of cDNA expression libraries. These studies will provide fundamental information concerning transcription activation and synergism and will address the fundamentally important issue of lactotroph and somatotroph differentiation. These investigations should provide the basis for a new line of investigation into additional players which orchestrate pituitary organogenesis.
