Locus control region function on inactive x-chromosomes

描述(研究人员摘要)：人类B-珠蛋白基因座控制区 (LCR)是一种复杂而强大的DNA调控元件，位于8-22kBP之间 胚胎E-珠蛋白基因的上游。LCR由五个亚区组成 这表明红系细胞对脱氧核糖核酸酶I具有很强的敏感性 (过敏部位HS1至HS5)。之前的许多研究结果提供了 有力的证据支持LCR能够授予高 人类珠蛋白基因的水平和组织特异性表达 转基因小鼠的位置非依赖和拷贝数依赖的方式。这 活动可能基于LCR固有的两个活动。首先是LCR 能够主导地打开染色质结构而与位置无关 在转基因小鼠中整合的珠蛋白基因座。第二，LCR调解 珠蛋白基因的高效表达可能是通过定向通讯实现的 带有单独的珠蛋白基因启动子。然而，之前的研究中没有一项是， 解决了LCR在定义的异色环境中的功能 老鼠的基因组。为了清楚地证明LCR具有内在的和 显性染色质开放活性对分析LCR功能具有重要意义。 一个封闭和压抑的染色质环境的背景。这样做的目的是 建议将完整的人类p-珠蛋白基因整合到老鼠体内。 次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因(HPRT)在酵母中的表达 基于人工染色体的靶标构建及LCR活性分析 在活跃和非活跃的X染色体中。通过在LCR两侧使用/loxP 位点，Cre/lox介导的重组将用于分析染色质 LCR存在和不存在时的结构和珠蛋白基因表达。 此外，这个模型系统将被用来研究LCR对 靶基因HPRT在非活性X染色体上的染色质结构。 这些研究可以为深入了解某些特定的 不活跃的X染色体上的结构域可以逃脱失活。

DESCRIPTION (Investigator's abstract): The human B-globin locus control region (LCR) is a complex and powerful DNA regulatory element located from 8 to 22 kbp upstream of the embryonic E-globin gene. The LCR is composed of five subregions that reveal strong sensitivity to deoxyribonuclease I in erythroid cells (hypersensitive sites HS1 to HS5). The results of many previous studies provide strong evidence supporting the hypothesis that the LCR is able to confer high level, tissue-specific expression to the human globin genes in a position-independent and copy-number dependent manner in transgenic mice. This activity is likely based on two activities intrinsic to the LCR. First the LCR is able to dominantly open the chromatin structure regardless of the position of the integrated globin locus in transgenic mice. Second, the LCR mediates high-level expression of the globin genes probably by direction communication with individual globin gene promoters. None of the previous studies, however, addressed the function of the LCR in a defined heterochromatic environment in the mouse genome. To clearly demonstrate that the LCR has intrinsic and dominant chromatin opening activity it is important to analyze LCR function in the context of a closed and repressive chromatin environment. The aim of this proposal is to integrate the complete human p-globin locus into the mouse hypoxanthine-guanine phosphoribosyltransferase gene (Hprt) using a yeast artificial chromosome based target construct and to then analyze LCR activity within the active and inactive X chromosome. By flanking the LCR with /loxP sites, Cre/lox mediated recombination will be used to analyze chromatin structure and globin gene expression in the presence and absence of the LCR. Furthermore, this model system will be used to examine the effect of the LCR on chromatin structure of the targeted Hprt gene on the inactive X chromosome. These studies could provide insight into the mechanisms by which certain domains on the inactive X chromosome escape inactivation.