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

说明(申请人提供)：血红蛋白是一种由两个α和两个β-珠蛋白链组成的异四聚体。这些蛋白质由两个不同的基因座编码。许多人类遗传病与人类β-珠蛋白基因座的突变有关，并导致轻度或严重形式的贫血。严重病例的治疗选择有限，而且往往伴随着有害的副作用。导致血红蛋白疾病的最常见的突变会减少成人β-珠蛋白基因的表达。预计，了解珠蛋白基因在分化和发育过程中是如何调控的，将导致涉及基因和干细胞治疗的新形式的治疗。人类β-珠蛋白基因座由五个基因组成，这些基因在发育过程中只在红系细胞中顺序表达。这些基因的高水平表达是由一个基因位点控制区介导的，该区域是位于基因上游的一个强大而复杂的DNA调控元件。该基因座控制区的显著之处在于，它能够在转基因检测中赋予珠蛋白基因不依赖于位置的高水平表达。这种活性在基因治疗实验中很重要，基因治疗实验的目的是在红系细胞中表达治疗性珠蛋白基因的生理水平。最近的证据表明，基因座控制区由几个含有许多转录因子结合位点的核心区组成，它招募在β-珠蛋白基因座上建立可访问的染色质结构域所需的活动。此外，最近的一些报道表明，RNA聚合酶II被招募到位点控制区核心元件。我们假设LCR是转录复合体招募的主要附着部位，转录复合体以发育阶段特异性的方式传递到珠蛋白基因。我们将使用生化、分子细胞生物学和遗传学实验来检验这一模型。此外，我们还将研究螺旋-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，这些蛋白质拮抗地调节成人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。我们将建立表达这些蛋白的显性负突变的转基因小鼠，并分析这些蛋白的表达对小鼠发育过程中珠蛋白基因调控的影响。

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.