FETAL/POSTNATAL CONTROL OF GH RECEPTOR EXPRESSION

生长激素（GH）是人类出生后生长所必需的。 在细胞水平上，GH通过最初结合 生长激素受体（GHR）。 多种因素影响GHR的表达; GHR是 在胎儿组织中检测不到，而其表达增加 在出生后的生活中。 此外， GHR的表达随所检查的组织而变化。的因素 控制GHR的时间和组织特异性表达 不知道。 本研究提案的长期目标 目的是阐明GHR表达的调控因素 基因，并基于假设，主要基因座的 控制GHR的时间和组织特异性表达 是在GHR基因转录水平上。 该提案旨在明确阐明分子 控制发育特异性表达的机制 GHR基因。 本提案中详细介绍的实验策略是 设计来表征启动子调控区的 小鼠生长激素受体基因通过基因组克隆分析， 转录起始位点的确定，以及 5'端发育特异性DNA酶I超敏位点 侧翼区 这些顺式作用的转录活性 元件将通过瞬时转染测定进行测定， 嵌合报告基因构建体。 为了确定顺式作用 生长激素受体基因个体发育调控元件 在小鼠中的表达，DNA的蛋白质：DNA相互作用谱 含有调节序列的片段将用 胎儿和成人组织的核提取物。 转染 具有报告基因构建体的胎儿和成人肝细胞 含有这些已鉴定的顺式作用元件将证实 这些DNA元件的发育特异性作用。定点 这些顺式作用元件的诱变将允许进一步的 详细分析了控制个体发育的DNA序列， GHR基因表达。 这些研究将（a）提供对 参与GHR基因表达的分子机制， 使得（B）能够设计新颖的诊断工具等 有效治疗生长障碍的策略， 在人类中的发育，如子宫内生长迟缓 和遗传性身材矮小

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.