Chromatin loops at the beta globin locus

描述(由申请人提供)：我们建议使用一种新的策略来操纵细胞基因的表达，该策略涉及到染色质环的产生，重点是发育中的红细胞中的珠蛋白基因。珠蛋白基因座的位点控制区(LCR)是所有类似珠蛋白基因的表达和远距离功能所必需的。在整个发育过程中，LCR通过形成染色质环与活性珠蛋白基因启动子动态相互作用。在这里，我们将通过在体内内源性细胞基因上建立染色质环来检验环形成和基因转录之间的因果关系。具体地说，我们将研究1)LCR和指定的-珠蛋白基因之间的强制环形成是否会导致未成熟红系细胞的转录激活，2)诱导LCR与发育沉默的珠蛋白基因形成环是否可以导致其重新激活，3)是否可以通过强制环形成使LCR靠近而激活沉默的非珠蛋白基因。我们的方法利用人工锌指蛋白，旨在结合内源LCR和目标基因启动子上独特的预定序列。这将通过与Sangamo生物科学公司已经建立的合作来实现。该公司拥有大量的锌指蛋白档案和所需的生物信息学工具，可以组装多指锌指蛋白，以高选择性和亲和力与所需的目标序列结合。这些锌指蛋白将被融合到允许药物诱导的异二聚体的蛋白质部分，例如来自Argent(Tm)系统(ARIAD制药公司)的FKBP和FRB。我们已经获得的这个系统非常适合在FKBP和FRB承载蛋白之间产生高亲和力的相互作用，当接触到细胞渗透的无毒药物AP21967时。操纵细胞基因表达的替代方法通常包括引入转录激活物、抑制物或干扰这些转录因子的分子。这里提出的系统的一个潜在优势是，它不依赖于单一因素的影响，而是利用整个增强子或LCR的调节潜力。这项研究的长期目标是探索这一策略是否适合激活人类原代成年红系细胞中的β-珠蛋白基因。如果成功，这可能为治疗镰状细胞病和地中海贫血提供一种新的工具。

DESCRIPTION (provided by applicant): We propose to manipulate cellular gene expression using a novel strategy involving the generation of chromatin loops, focusing on the ¿-globin locus in developing erythrocytes. The locus control region (LCR) of the ¿-globin locus is required for the expression of all ¿-like globin genes and functions over great distances. Throughout development, the LCR interacts dynamically with the active globin gene promoters via the formation of chromatin loops. Here we will examine the cause-effect relationships between loop formation and gene transcription by establishing chromatin loops at endogenous cellular genes in vivo. Specifically, we will examine 1) whether forced loop formation between the LCR and a designated ?-globin gene leads to transcriptional activation in immature erythroid cells, 2) whether inducing the LCR to loop to a developmentally silenced globin gene can lead to its reactivation, 3) whether a silent non-globin gene can be activated by bringing the LCR into close proximity by forced loop formation. Our approach makes use of artificial zinc finger proteins designed to bind to unique, predetermined sequences at the endogenous LCR and the target gene promoters. This will be accomplished by an already established collaboration with Sangamo Biosciences. This company has in its possession a large archive of zinc finger proteins and the required bioinformatics tools to assemble polydactyl zinc finger proteins to bind to the desired target sequences with high selectivity and affinity. These zinc finger proteins will be fused to protein moieties that allow drug-induced heterodimerization such as FKBP and FRB from the Argent(tm) system (ARIAD Pharmaceuticals). This system, which we have acquired, is well suited to generate high affinity interactions between FKBP and FRB bearing proteins upon exposure to the cell permeable, non-toxic drug AP21967. Alternative approaches to manipulate cellular gene expression typically involve the introduction of transcriptional activators, repressors or molecules that interfere with such transcription factors. A potential advantage of the system proposed here is that it does not rely on the effects of a single factor but harnesses the regulatory potential of an entire enhancer or LCR. The long-term goal of this study is to explore whether this strategy is suitable to activate the ?-globin gene in human primary adult erythroid cells. If successful, this might provide a novel tool for the treatment of sickle cell disease and the ¿-thalassemias.