Somatotroph- and lactotroph-specific interactions with the homeobox protein binding sites in the rat growth hormone gene promoter

Abstract

Nuclear extracts prepared from growth hormone-secreting (GC) and prolactin-secreting (235-1) rat anterior pituitary cell lines were compared for their ability to bind to the DNA sequences conferring tissue-specificity to the expression of the rat growth hormone (rGH) gene promoter. Cell-specific differences in the interaction of Pit-1, a tissue-specific member of the POU-domain transcription factor family, with the pGHF1 binding site were detected by methylation interference experiments; otherwise the Pit-1 proteins present in GC cell and 235-1 cell extracts were similar. Two other protein/DNA complexes, GHF5 and GHF7, were detected by gel mobility shift assays and the binding of both complexes to the rGH promoter depended upon DNA sequences contained within the two binding sites for Pit-1. In contrast to Pit-1 which can bind to either of the two sites independently, a single Pit-1 binding site was insufficient for GHF5 and GHF7 binding; i.e. both Pit-1 binding sites within the rGH promoter were required. Whereas GHF5 was present in nuclear extracts of GC cells and a variety of cells not producing growth hormone, GHF7 binding activity was detected only in the GC cell line (and not in the 235-1 cell line). GHF7 binding activity was therefore more closely correlated with growth hormone gene transcription than was Pit-1. rGH promoters containing mutations which inhibited GHF5, GHF7 and Pit-1 binding were expressed less efficiently than the wild type promoter after transfection into GC cells. One promoter mutation to which the GHF7 complex but not the Pit-1 factor can bind was also transcription deficient demonstrating that Pit-1 binding, independent of GHF7 binding, was nevertheless important to the expression of the rat growth hormone promoter.