The Rat Growth Hormone Proximal Silencer Contains a Novel DNA‐Binding Site for Multiple Nuclear Proteins that Represses Basal Promoter Activity

Abstract

Cell-type-specific expression of the rat growth hormone (rGH) gene is determined by the interaction of both positive as well as negative regulatory proteins with cis-acting elements located upstream of the rGH mRNA start site. We have recently shown that the rat liver transcription factor NF1-L binds to the proximal rGH silencer (called silencer-1) to repress its transcriptional activity. However, this single factor proved to be insufficient by itself to confer cell-specific gene repression. We therefore attempted to identify other regulatory proteins interacting with silencer 1, which might be needed to achieve full cell-specific repression of that gene. A common recognition site for three yet uncharacterized nuclear proteins (designated as SBP1, SBP2 and SBP3) which bind a DNA sequence adjacent to the NF1-L-binding site in the rGH silencer-1 element were identified. UV crosslinking of DNA/protein complexes and nuclear protein fractionation/renaturation from SDS/polyacrylamide gels further indicated that the molecular masses for SBP1-3 are 41, 26 and 17 kDa respectively, the major species being the 26-kDa protein (SBP2) which account for 83% of the shifted SBP double-stranded oligonucleotide in gel mobility-shift assays. For this reason, most of this study focussed on the characterization of SBP2. We demonstrated that binding of NF1-L and SBP2 to their respective recognition sequence is a mutually exclusive event. Although an SBP-binding activity has been found in every non-pituitary tissue or cell line tested, no such activity could be detected in either rat pituitaries or rat pituitary GH4C1 cells. Insertion of the SBP element upstream of the basal promoter of the mouse p12 heterologous gene resulted in a consistent decrease in chloramphenicol acetyl transferase reporter gene expression following transient transfections in non-pituitary cells only, suggesting that the related SBP1-3 proteins might be involved in generally repressing gene transcription in a cell-specific manner.