Pit-1 exhibits a unique promoter spacing requirement for activation and synergism

The developmentally regulated Pit-1 transcription factor is involved in the activation of prolactin, growth hormone, and TSHβ expression. Using templates with spacing mutations to program an in vitro transcription system, the activity of a single Pit-1 proximal binding site within the rat prolactin promoter was shown to have a unique bimodal distance requirement. Transcription activity rapidly decreased with each 5-base pair (bp) addition to the spacing between the binding site and the TATA box. When positioned 20 bp upstream from its normal -36 position in the prolactin promoter, the activity of the Pit-1 binding site is reduced to basal levels. Placement of the site at a position 30 bp upstream resulted in a return of Pit-1-mediated activation. Using transient transfection assays in GH₃ cells, the prime bimodal sites are also a requirement for optimum expression of chimeric prolactin-luciferase reporter constructs. Interestingly, optimal synergism of transcription in vivo by the prolactin distal enhancer, containing four Pit- 1 binding sites and an estrogen-responsive element, is also sensitive to the placement of the proximal Pit-1 binding site. These data have important implications for Pit-1 activator function in pituitary cells and for general models of transcription synergism.