GENETIC ANALYSIS OF THE CHICKEN BETA-GLOBIN LOCUS

该方案的目的是利用同源重组(HR)策略来阐明鸡β-珠蛋白基因座是如何在染色质结构、复制时间和起始用途以及转录水平上进行调控的。我们最近的工作表明，小鼠和人类β-珠蛋白基因座控制区(LCRs)的缺失并不会使该基因座沉默，并在小鼠或人类中产生等同于西班牙裔地中海贫血的表型。在这个方案中，我们将在鸡β-珠蛋白基因座上进行类似的实验，其中基因座区域结构和5‘边界元素(绝缘体)的存在已经得到了很好的表征。当足够的顺式调控元件被删除以创建沉默的鸡基因座时，我们将使用加回法或敲入法在转录、染色质结构和DNA复制水平上确定激活该基因座所涉及的最小序列。这些实验将提供关于先前定义的调控元件(如增强子、LCR和绝缘体)的功能的重要概念，以及识别鸡β-珠蛋白基因座中对在多个水平上控制该基因座重要的额外序列。将这些序列包含在基因治疗载体中可能会克服目前病毒基因治疗策略的一些局限性，如低表达水平和随着时间的推移而失去表达，从而有助于未来对血红蛋白疾病和地中海贫血的基因治疗。这一建议的具体目的是：1.利用HR介导的DT40缺失和随后在红系细胞中的检测，研究鸡β珠蛋白基因座(β/epsilon Enhancer/LCR)基因间调控序列的作用。2.研究鸡β-珠蛋白基因座上游超敏位点(HSS)(5‘HS1-4)的作用，包括绝缘子边界区(5’HS4)。这些HSS将在重组熟练的鸡B细胞(DT40)和红系细胞中被单独和联合删除，并确定它们在基因座染色质结构和转录启动和维持中的作用。3.我们将在DT40细胞中使用HR介导的缺失来确定与鸡β-珠蛋白基因座DNA复制启动有关的必要序列，以及上游或其他调控序列在DNA复制启动和时间选择中的潜在作用。4.一旦我们在AIMS 1-4中构建了染色质结构、转录和复制沉默的鸡基因座，我们将使用Addback或Huckin方法重新激活该基因座，并定义可能具有活性的最小和一致序列以及不相关的序列。我们还将使用和加回或敲门的方法来确定鸡肉调控元件激活沉默的小鼠和人类β-珠蛋白基因的能力。

The goal of this proposal is to use homologous recombination (HR) strategies to elucidate how the chicken beta-globin locus is regulated at the level of chromatin structure, replication timing and origin use, and transcription. Our recent work suggests that the deletion of the mouse and human beta-globin locus control regions (LCRs) does not silence the locus and create the equivalent of a Hispanic thalassemia phenotype in mouse or human. In this proposal, we will perform similar experiments in the chicken beta-globin locus, where locus domain structure, and the presence of a 5' boundary element (insulator) have been well characterized. When sufficient cis regulatory elements have been deleted to created a silenced chicken locus, we will use an addback or knock in approach to determine the minimal sequences involved in activating this loci at the level of transcription, chromatin structure, and DNA replication. These experiments will provide important concepts regarding the function of previously define regulatory elements such as enhancers, LCRs, and insulators as well as identify additional sequences in the chicken beta-globin locus that are important for control of the locus at multiple levels. Inclusion of these sequences in gene therapy vectors may overcome some of the limitations of current viral gene therapy strategies such as low expression levels and loss of expression over time, and thereby aid future efforts towards gene therapy of the hemoglobinopathies and thalassemias. The specific aims of this proposal are: 1. The role of intergenic regulatory sequences in the chicken beta globin locus (beta/epsilon enhancer/LCR) will be investigated using HR mediated deletion in DT40 and subsequent assay in erythroid cells. 2. The role of the upstream hypersensitive sites (HSS) (5'HS1-4) in the chicken beta-globin locus will be investigated including the insulator boundary region (5'HS4). These HSS will be deleted singly and in combination in recombination proficient chicken B cells (DT40) and erythroid cells and their role in the initiation and maintenance of locus chromatin structure and transcription determined. 3. We will use HR mediated deletion in DT40 cells to define essential sequences involved in initiation of DNA replication in the chicken beta- globin locus and the potential role of upstream or other regulatory sequences in DNA replication initiation and timing. 4. Once we have constructed chicken loci silenced for chromatin structure, transcription, and replication in aims 1-4, we will use addback or knockin approaches to reactivate the locus and define minimal and consensus, and unrelated sequences that may have activity. We will also use and addback or knockin approach to determine the ability of chicken regulatory elements to activate silenced mouse and human beta-globin loci.