A role for A/T-rich sequences and Pit-1/GHF-1 in a distal enhancer located in 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 and required for efficient GH-N gene expression in the pituitary of transgenic mice was cloned as a 1.6-kb Bg/II (1.6G) fragment. The 1.6G fragment in the forward or reverse orientation increased -496GH-N promoter activity significantly in pituitary GC and GH3 cells after gene transfer. The 1.6G fragment 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'-end 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 nonpituitary (HeLa) cell nuclear protein. A major low-mobility complex was observed by electrophoretic mobility shift assay (EMSA) with GC cell nuclear protein, 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 elements, and their disruption, in the context of the 203-bp region fused to the TK promoter, reduced enhancer activity significantly in pituitary cells in culture. A mutation in this region was also shown to decrease enhancer activity in transgenic mice and correlated with a decrease in the 203-bp enhancer 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-rich sequences to the distal enhancer activity associated with the GH locus control region in vitro and in vivo.
Jin
Y
Y
Department of Physiology, University of Manitoba, Winnipeg, Canada.
Surabhi
R M
RM
Fresnoza
A
A
Lytras
A
A
Cattini
P A
PA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002822:Choriocarcinoma; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D017353:Gene Deletion; D006367:HeLa Cells; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D051381:Rats; D013937:Thymidine Kinase / Q000235:genetics; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000502:physiology*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D019382:Human Growth Hormone; D010446:Peptide Fragments; D013937:Thymidine Kinase; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494187:POU1F1 protein, human; C494185:Pou1f1 protein, mouse; C494186:Pou1f1 protein, rat