FETAL/POSTNATAL CONTROL OF GH RECEPTOR EXPRESSION

生长激素(GH)对人类出生后的生长发育是必不可少的。 在细胞水平上，生长激素通过最初的结合而引起其作用 一种称为生长激素受体(GHR)的细胞表面蛋白。 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 在出生后的生活中戏剧性地。此外，这一水平 GHR的表达因所检查的组织而异。影响因素 控制GHR的时间和组织特异性表达 都是未知的。这项研究计划的长期目标是 目的是阐明控制GHR表达的因素 基因，并且基于这样的假设，即 GHR在时间和组织中的特异性表达调控 处于GHR基因转录水平。 这项提议专门寻求阐明分子 控制特定于发育的表达的机制 Ghr基因。本提案中详述的实验策略是 旨在表征启动子-调节区的特征 小鼠生长激素受体基因的基因组克隆分析， 转录起始点的确定(S)，并定位 5‘端发育特异的DNase I超敏位点 侧翼区域。这些顺式作用的转录活性 元素将通过瞬时转染试验进行分析，方法是使用 嵌合报告基因结构。识别顺位代理 生长激素受体基因个体发育的调控元件 在小鼠中的表达，蛋白质：DNA的DNA相互作用图谱 含有调控序列的片段将用 胎儿和成人组织的核提取液。转染腺病毒载体 具有报告基因结构的胎儿和成人肝细胞 包含这些已识别的顺式作用元件将证实 这些DNA元素在发育中的特定作用。站点定向 这些顺式作用元件的突变将允许进一步 个体发育控制的DNA序列的详细分析 生长激素受体基因的表达。 这些研究将(A)提供对 GHR基因表达的分子机制， 使(B)设计新的诊断工具和更多 生长和发育障碍的有效治疗策略 人类的发育，如胎儿宫内发育迟缓 和遗传矮小。

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.