A role for A/T-rich sequences and Pit-1/GHF-1 in a distal enhancer locatedin the human growth hormone locus control region with preferential pituitary activity in culture and transgenic mice

A region located remotely upstream of the human pituitary GH (GH-N) gene an d required for efficient GH-N gene expression in the pituitary of transgeni c mice was cloned as a 1.6-kb Bg/II (1.6G) fragment. The 1.6G fragment in t he forward or reverse orientation increased -496GH-N promoter activity sign ificantly in pituitary GC and GH3 cells after gene transfer. The 1.6G fragm ent was also able to stimulate activity from a minimal thymidine kinase (TK ) promoter which, unlike -496GH-N, lacked any Pit-1/GHF-1 element. Enhancer activity was localized by deletion analysis to a 203-bp region in the 3'-e nd of the 1.6G fragment and was characterized by the presence of a diffuse 136-bp nuclease-protected site, observed with pituitary (GC) but not nonpit uitary (HeLa) cell nuclear protein. A major low-mobility complex was observ ed by electrophoretic mobility shift assay (EMSA) with GC cell nuclear prot ein, and the pattern was distinct from that seen with a HeLa cell extract. The nuclease-protected region contains three A/T-rich Pit-1/GHF-1-like elem ents, and their disruption, in the context of the 203-bp region fused to th e TK promoter, reduced enhancer activity significantly in pituitary cells i n; culture. A mutation in this region was also shown to decrease enhancer a ctivity in transgenic mice and correlated with a decrease in the 203-bp enh ancer region complex observed by EMSA. The participation of Pit-1/GHF-1 in this complex is indicated by competition studies with Pit-1/GHF-1 elements and antibodies, and direct binding of Pit-1/GHF-1 to the A/T-rich sequences was shown by EMSA using recombinant protein. These studies link the A/T-ri ch sequences to the distal enhancer activity associated with the GH locus c ontrol region in vitro and in vivo.