GENETIC ANALYSIS OF THE CHICKEN BETA-GLOBIN LOCUS

本提案的目标是使用同源重组（HR）策略来阐明鸡β-珠蛋白基因座是如何在染色质结构，复制时间和起点使用以及转录水平上进行调节的。 我们最近的工作表明，小鼠和人类β-珠蛋白基因座控制区（LCR）的缺失不会使基因座沉默，并在小鼠或人类中产生等同于西班牙地中海贫血表型的基因。在这个提议中，我们将在鸡β-珠蛋白基因座中进行类似的实验，其中基因座结构域结构和5'边界元件（绝缘体）的存在已经被很好地表征。 当足够的顺式调控元件已被删除，以创建一个沉默的鸡基因座，我们将使用addback或敲入的方法来确定参与激活这个基因座在转录水平，染色质结构，和DNA复制的最小序列。这些实验将提供关于先前定义的调控元件（如增强子、LCR和绝缘子）的功能的重要概念，以及鉴定鸡β-珠蛋白基因座中对于在多个水平上控制基因座重要的额外序列。 在基因治疗载体中包含这些序列可以克服当前病毒基因治疗策略的一些局限性，例如低表达水平和随时间推移的表达丧失，从而有助于未来对血红蛋白病和地中海贫血的基因治疗的努力。 该提案的具体目标是：1.将使用DT 40中HR介导的缺失和红系细胞中的后续测定来研究鸡β珠蛋白基因座（β/β-珠蛋白增强子/LCR）中基因间调控序列的作用。 2.将研究鸡β-珠蛋白基因座中上游超敏位点（HSS）（5 'HS 1 -4）的作用，包括绝缘子边界区（5' HS 4）。这些HSS将在重组熟练的鸡B细胞（DT 40）和红系细胞中单独和组合地缺失，并确定它们在位点染色质结构和转录的起始和维持中的作用。 3.我们将在DT 40细胞中使用HR介导的缺失来定义参与鸡β-珠蛋白基因座中DNA复制起始的必需序列以及上游或其他调控序列在DNA复制起始和定时中的潜在作用。4.一旦我们在目标1-4中构建了对染色质结构、转录和复制沉默的鸡基因座，我们将使用加回或敲入方法来重新激活基因座并定义可能具有活性的最小和共有以及不相关的序列。 我们还将使用加回或敲入方法来确定鸡调节元件激活沉默的小鼠和人β-珠蛋白基因座的能力。

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.