Pit-1 Exhibits a Unique Promoter Spacing Requirement for Activation and Synergism (∗)

• Published in: The Journal of biological chemistry Vol. 270; no. 9; pp. 4484 - 4491
• Main Authors: Smith, Kelly P., Liu, Bing, Scott, Clara, Sharp, Z.Dave
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.03.1995; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Base Sequence; Binding Sites; Cell Line; DNA-Binding Proteins - metabolism; Enhancer Elements, Genetic; Molecular Sequence Data; Oligodeoxyribonucleotides; Prolactin - genetics; Promoter Regions, Genetic; Rats; Transcription Factor Pit-1; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.9.4484

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.