Growth hormone (GH) is essential for postnatal growth in the human. At the cellular level GH elicits its actions by initially binding to a cell surface protein termed the growth hormone receptor (GHR). A variety of factors influence the expression of the GHR; GHR is undetectable in fetal tissues while its expression increases dramatically during postnatal life. In addition, the level of expression of the GHR varies with the tissue examined. The factors controlling the temporal and tissue specific expression of the GHR are not known. The LONG TERM OBJECTIVE of this research proposal is to elucidate the factors controlling the expression of the GHR gene, and is based on the HYPOTHESIS that the major locus of control for the temporal and tissue specific expression of the GHR is at the level of GHR gene transcription. This proposal seeks SPECIFICALLY to elucidate the molecular mechanisms controlling the development-specific expression of the GHR gene. The experimental strategy detailed in this proposal is designed to characterize the promoter-regulatory region of the mouse growth hormone receptor gene by analysis of genomic clones, determination of the transcription start site(s), and mapping of the development-specific DNase I hypersensitivity sites in the 5' flanking region. The transcriptional activity of these cis-acting elements will be assayed by transient transfection assays using chimeric reporter gene constructs. To identify the cis-acting elements regulating the ontogeny of growth hormone receptor gene expression in the mouse, protein:DNA interaction profiles of DNA fragments containing regulatory sequences will be analyzed with nuclear extracts from fetal and adult tissues. Transfection of fetal and adult hepatocytes with reporter gene constructs containing these identified cis-acting elements will confirm the development-specific role of these DNA elements. Site-directed mutagenesis of these cis-acting elements will allow further detailed analysis of the DNA sequences controlling the ontogeny of GHR gene expression. These studies will (a) provide a detailed understanding of the molecular mechanisms involved in the expression of the GHR gene, enabling (b) the design of novel diagnostic tools and more effective treatment strategies for disorders of growth and development in the human, such as intra-uterine growth retardation and genetic short stature.
