DNAse I footprint analysis of nuclear proteins from pituitary and nonpituitary cells that specifically bind to the rat growth hormone promoter and 5'-regulatory region

Rat (rGH) is expressed exclusively in cells from the anterior lobe of the pituitary gland. Using DNAsel footprinting assays, we have examined both pituitary and nonpituitary cell nuclear extracts for proteins which bind specifically to the rGH promoter and 5'-flanking region. In agreement with previous studies, we have located binding sites between -96 and -65, and between -148 and -118 for proteins which have been termed GC1 and GC2, respectively. The GC2 footprint is found using extracts from both pituitary and nonpituitary cells, but GC1 is observed only in pituitary cells. We have also located a binding site for an additional pituitary-specific protein upstream from the GC1 binding site, between -241 and -220. The footprint for this protein, which we call GC3, is found with pituitary extracts, but not with extracts from nine other nonpituitary cell types. Although this pattern of activity is similar to that of GC1, competition experiments with synthetic oligonucleotides show that the two proteins are distinct. Deletion of the GC3 binding site has only a small effect on rGH promoter activity in transiently transfected pituitary cells and fibroblasts.     

Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element.

Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.