The objective of this proposal is to provide further insight into regulation of the rat growth hormone (GH) gene expression by cyclic AMP. This basic research will aid in understanding of the multifactorial regulation of GH gene expression. cAMP regulation of the rat growth hormone gene expression requires the first 104 base pairs of 5'-flanking DNA [Copp, R.P. and Samuels, H.H. (1989) Molecular Endocrinology 3, 790-796]. Preliminary data suggests that much of this cAMP effect may require 5'-flanking sequences extending to only -49 of the rat GH gene. Thus, the cell-specific element (-95/-65) normally thought to be required for efficient expression may not be required for some of the cAMP effect. This proposed work thus extends these studies to determine the precise sequences involved in the cAMP regulation. The effects of cAMP on transcription of GH and fusion genes containing various 5'-flanking DNA segments of the rat GH gene linked to coding sequences for chloramphenicol acetyltransferase (CAT) or in other expression vectors will be analyzed after DNA transfection into rat anterior pituitary cell lines. Further examination will include analysis of effects of cAMP effectors on nuclear protein binding to potential regulatory regions as seen by techniques such as gel shift and DNase I footprinting as well as competition for regulatory factors by specific DNA sequences of other genes known to be involved in cAMP regulation. If DNA binding analysis does not show cAMP-induced changes in protein bound to locations determined by functional analysis then an examination will be made of whether protein phosphorylation is required for activation of GH gene transcription. The interactions between cAMP effects and those of factors, such as thyroid hormone and retinoic acid, which transcriptionally regulate the rat GH gene will be analyzed in detail. In addition, effects of growth hormone releasing factor and cAMP on regulation of GH expression will be compared using recently described growth hormone releasing factor-responsive rat anterior pituitary cell lines. Finally, as time permits, any sequence- specific DNA binding factors involved in the cAMP regulation will be characterized and their cDNA cloned. Thus, a detailed analysis of the transcriptional regulation of the GH gene and of the nuclear proteins involved in this regulation by cAMP or growth hormone releasing factor has been proposed.
