Locus control region function on inactive x-chromosomes

描述（研究者摘要）：人B-珠蛋白基因座控制区 (LCR)是一个复杂而强大的DNA调控元件，位于8 - 22 kbp之间 胚胎E-珠蛋白基因的上游。LCR由五个次区域组成 其揭示了红系细胞中对脱氧核糖核酸酶I强敏感性 （超敏位点HS 1至HS 5）。许多以前的研究结果提供了 强有力的证据支持这一假设，即LCR能够赋予高 水平，组织特异性表达的人珠蛋白基因， 在转基因小鼠中以位置非依赖性和拷贝数依赖性的方式表达。这 活性可能基于LCR固有的两种活性。首先是LCR 无论位置如何，都能够主导打开染色质结构 在转基因小鼠中整合珠蛋白基因座。第二，LCR介导 珠蛋白基因的高水平表达可能是通过直接通讯 具有单独的珠蛋白基因启动子。然而，以前的研究都没有， 解决了LCR在一个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具有固有的 显性染色质开放活性，重要的是分析LCR功能， 一个封闭和压抑的染色质环境的背景。的目的 一个建议是将完整的人β-珠蛋白基因座整合到小鼠中 使用酵母的次黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因（Hprt 基于人工染色体的靶构建体，然后分析LCR活性 位于活跃和不活跃的X染色体内。通过用/loxP侧接LCR 位点，Cre/lox介导的重组将用于分析染色质 结构和珠蛋白基因表达的LCR的存在和不存在。 此外，该模型系统将用于检查LCR对 非活性X染色体上靶向Hprt基因的染色质结构。 这些研究可以深入了解某些人 失活X染色体上的结构域逃脱失活。

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.