Structure and Function of the Human Beta-Globin Locus Control Region

描述（由申请人提供）：血红蛋白是由两条α-珠蛋白链和两条β-珠蛋白链组成的异源四聚体。这些蛋白质由两个不同的基因座编码。许多人类遗传性疾病与人类β-珠蛋白基因座的突变有关，并导致轻度或重度贫血。严重病例的治疗选择是有限的，往往伴随着有害的副作用。导致血红蛋白病的最常见突变减少成人β-珠蛋白基因的表达。预计有关珠蛋白基因在分化和发育过程中如何调节的知识将导致涉及基因和干细胞疗法的新形式的治疗。人β-珠蛋白基因座由在发育期间仅在红系细胞中顺序表达的五个基因组成。这些基因的高水平表达是由基因座控制区介导的，基因座控制区是位于基因上游的强大而复杂的DNA调控元件。基因座控制区是显著的，因为它能够在转基因测定中赋予珠蛋白基因位置非依赖性和高水平表达。这种活性在基因治疗实验中是重要的，其目的是在红系细胞中表达生理水平的治疗性珠蛋白基因。最近的证据表明，基因座控制区，其中包括几个核心区域窝藏许多转录因子结合位点，招聘活动所需的β-珠蛋白基因座中建立可访问的染色质结构域。此外，最近的一些报道表明，RNA聚合酶II被募集到基因座控制区的核心元件。我们假设LCR代表了转录复合物募集的主要附着位点，这些转录复合物以发育阶段特异性的方式传递到珠蛋白基因。我们将使用生物化学、分子细胞生物学和遗传学实验来测试这个模型。此外，我们将研究螺旋-环-螺旋蛋白USF和TFII-I在β-珠蛋白基因调控中的作用。我们的数据表明，这些蛋白质拮抗调节成人β-珠蛋白基因的表达，并可能参与珠蛋白基因的阶段特异性表达。我们将产生转基因小鼠表达这些蛋白质的显性负突变体，并分析这些蛋白质的表达在小鼠发育过程中珠蛋白基因调控的后果。

DESCRIPTION (provided by applicant): Hemoglobin is a heterotetramer composed of two alpha and two beta-globin chains. The proteins are encoded by two different gene loci. Many human genetic diseases are associated with mutations in the human beta-globin gene locus and lead to mild or severe forms of anemia. Treatment options for severe cases are limited and often accompanied by deleterious side effects. The most common mutations that cause hemoglobinopathies reduce expression of the adult beta-globin gene. It is anticipated that knowledge about how the globin genes are regulated during differentiation and development will lead to new forms of treatment involving gene and stem cell therapies. The human beta-globin gene locus consists of five genes that are sequentially expressed during development exclusively in erythroid cells. High-level expression of these genes is mediated by a locus control region, a powerful and complex DNA regulatory element located far upstream of the genes. The locus control region is remarkable in that it is able to confer position- independent and high-level expression to globin genes in transgenic assays. This activity is important in gene therapy experiments, which are aimed at expressing physiological levels of a therapeutic globin gene in erythroid cells. Recent evidence suggests that the locus control region, which consists of several core regions harboring many transcription factor binding sites, recruits activities that are required for establishing accessible chromatin domains in the beta-globin locus. Moreover, several recent reports demonstrate that RNA polymerase II is recruited to locus control region core elements. We hypothesize that the LCR represents the primary attachment site for recruitment of transcription complexes, which are delivered to the globin genes in a developmental stage specific manner. We will use biochemical, molecular cell biological and genetic experiments to test this model. In addition, we will investigate the role of helix-loop-helix proteins USF and TFII-I in beta-globin gene regulation. Our data suggest that these proteins antagonistically regulate expression of the adult beta-globin gene and may participate in the stage-specific expression of the globin genes. We will generate transgenic mice expressing dominant negative mutants of these proteins and analyze the consequence of expression of these proteins on globin gene regulation during mouse development.