Overlapping Pit-1 and Sp1 binding sites are both essential to full rat growth hormone gene promoter activity despite mutually exclusive Pit-1 and Sp1 binding.

A DNA-binding factor present in a wide variety of cell types is shown to bind to a site (GHF2) within the rat growth hormone gene promoter between nucleotides -147 and -129. Biochemical and transcriptional properties identify this factor as Sp1. The degree of glycosylation of one of the forms of Sp1 is observed to vary in a cell type-specific fashion. The GHF2 binding site overlaps with the more distal (dGHF1) of the two binding sites for the pituitary-specific transcription factor Pit-1. DNase I footprinting and gel retardation analysis with partially purified Sp1 and Pit-1 show that Sp1 and Pit-1 binding to these sites is mutually exclusive. Despite the inability of these two transcription factors to be simultaneously bound to their overlapping sites in vitro, the structural integrity of both binding sites appears essential to the maximal expression of the rat growth hormone promoter after transfection into pituitary GC cells. These findings suggest that activation of the rat growth hormone promoter by Sp1 and Pit-1 may occur through a multistage mechanism.