AGING AND ANDROGEN RECEPTOR GENE REGULATION

在大鼠的一生中，雄性和雌性的肝脏 动物经历了三个阶段的雄激素反应，即， 青春期前(40天)雄激素不敏感，一种雄激素反应性 青壮年阶段与雄激素不敏感的衰老(&lt；750 天数)。这些与年龄相关的雄激素敏感性变化是 与肝脏雄激素受体(AR)表达的相关性 MRNA.因为AR基因的表达发生了如此显著的变化 在生命的三个阶段，大鼠的肝脏提供了一个独特的模型 探讨其时序调控的分子机制 吉恩。这种对该受体基因的年龄依赖性调节可能是 通过DNA-蛋白质相互作用的程序性改变来调节 是转录的关键。这项提案旨在承诺 对DNA-蛋白质相互作用的系统分析 肝脏AR基因表达的调控。为了实现 这一目标，将克隆AR基因的上游调节区 并以此为特征。克隆的基因片段将被用来鉴定 DNA酶I在体外足印中的特异性蛋白结合区研究 分析。DNA结合核因子的年龄特异性改变将 随后用带移分析来表征标记的 与人的足迹区域相对应的寡核苷酸双链 AR基因。细胞中DNA-蛋白质相互作用的存在将是 原代肝细胞的体内足迹和DNA酶证实 分离的肝细胞核的超敏反应分析。一旦这个时代- 特定的DNA-蛋白质相互作用被表征，它们的功能 相关将通过野生型的转基因试验得到证实。 和与异源植物连接的AR基因启动子的适当突变 记者基因。这些相互作用的生物学作用将是 的具体要求进一步验证 启动子-报告基因体外转录的顺式/反式相互作用 构造。这些研究的结果有望提供新的 对年龄依赖性变化的分子机制的认识 AR基因的表达。此外，受体失调也是 与年龄相关的系统性协调能力丧失的主要原因。这些 因此，研究将对全面的 了解衰老的分子基础。

During the life-span of the rat, the liver of both male and female animals goes through three phases of androgen responsiveness, i.e., prepubertal ( <40 days) androgen-insensitivity, an androgen responsive phase of young-adulthood and androgen-insensitivity of senescence ( <750 days). These age-dependent changes in androgen sensitivity are correlated with the hepatic expression of the androgen receptor (AR) mRNA. Because of such a marked alteration in the expression of AR gene during the three periods of life, the rat liver offers a unique model for exploring the molecular mechanism of the temporal regulation of this gene. Such an age-dependent regulation of this receptor gene may be mediated through a programmed alteration of DNA-protein interactions that are critical for transcription. This proposal is designed to undertake a systematic analysis of DNA-protein interactions that are involved in the regulation of AR gene expression in the liver. In order to achieve this goal, the upstream regulatory region of the AR gene will be cloned and characterized. Fragments of the cloned gene will be used to identify specific protein-binding regions by DNase I in vitro foot-printing analysis. Age-specific alterations of DNA-binding nuclear factors will be subsequently characterized by band-shift analysis of labeled oligonucleotide duplexes corresponding to the foot-print regions of the AR gene. Cellular existence of DNA-protein interactions will be substantiated by in vivo foot-printing of primary heptocytes and DNase I hypersensitivity analysis of isolated liver nuclei. Once the age- specific DNA-protein interactions are characterized, their functional relevance will be substantiated by transfection assay of the wild type and appropriate mutant of the AR gene promoters ligated to heterologous reporter genes. The biological role of these interactions will be further authenticated by examining the specific requirements of the cis/trans interactions for in vitro transcription of promoter-reporter constructs. Results of these studies are expected to provide new knowledge concerning the molecular mechanism of age-dependent changes in AR gene expression. Furthermore, receptor dysregulation is one of the major causes of the age-associated loss of systemic coordination. These studies will therefore, make an important contribution toward an overall understanding of the molecular basis of aging.