FUNCTION OF BETA GLOBIN DNA BINDING PROTEINS

描述(改编自申请者摘要)：抄本 对鸡β-珠蛋白基因簇的调控需要长距离的 远端DNA序列之间的相互作用以组织特异性激活 染色体定位与个体基因的差异调控 利用红系发育。一个在两者中都起作用的控制元素 这些能力中有一种是β-epsilon增强剂。这一区域残留物 成体β(A)-和胚胎epsilon珠蛋白基因和 在不同的时间分别激活红细胞中的每个启动子 成熟。此外，β-epsilon增强子也有Locus 控制区域(LCR)活动，因为它需要建立活动 与β(A)-珠蛋白基因或基因连锁时的染色体结构域 集群。鉴于组织特异性的广泛重要性 染色体结构域激活与增强子依赖的转录 β-epsilon增强子在多种不同基因系统中的调控 代表了一种分析远程DNA的优秀模型 互动。研究人员此前证明了贝塔A-珠蛋白 转录完全依赖于3‘β-epsilon增强子， 作用距离为2kb，使用重组为染色质的基因 或在红系蛋白存在的情况下合成细胞核。在这 建议，研究人员希望继续研究组织-- 通过对特定的增强子功能进行详细检查 它发生的机制。这种方法是在体外使用 转录和染色质重组系统复制关键 增强子调控的βA-珠蛋白基因表达的一些方面。第一, 实验被设计用来定义授予的DNA结构 β-epsilon增强子的体外活性。具体地说，是否 核小体组装或DNA结构蛋白是必需的 生成此拓扑。第二，最低转录蛋白 依赖于增强子的BetaA-珠蛋白表达所需的 用纯化或重组红系DNA结合进行鉴定 蛋白质和一般起始因子以及未知蛋白质 通过红系提取物的层析获得。第三，能力 将检查用于激活或抑制启动子的增强剂的数量 通过生化和结构研究相结合。这些措施包括 使用纯化组分的加法顺序转录实验 和依赖于增强子的和非依赖的βA的质粒足迹 珠蛋白基因。第四，调查员将检查增强剂是否 调节涉及使用电子的DNA环或蛋白质跟踪 显微镜和“蛋白质阻断”体外转录实验。 最后，β-epsilon增强子的启动子转换将是 在包含连接的BetaA和epsilon珠蛋白的DNA模板中进行检查 基因。βA-珠蛋白增强子的体外功能分析 系统应提供有用的信息，不仅适用于 其他组织特异性基因，但对长程遗传效应是 特别是与健康相关的问题。其中包括 展示了增强剂在控制中的作用，但知之甚少 免疫球蛋白和T细胞受体基因重排及其意义 观察发现，在各种淋巴瘤和白血病中，癌基因 放松管制与反常的移位到 活性促进剂的附近。

DESCRIPTION (Adapted from Applicant's Abstract): Transcriptional regulation of the chick beta-globin gene cluster requires long-range interaction between distal DNA sequences to tissue-specifically activate the chromosomal locus and to differentially regulate individual genes using erythroid development. One control element that functions in both of these capacities is the beta-epsilon enhancer. This region residues between the adult beta(A)-and embryonic epsilon globin genes and separately activates each promoter at distinct times in red cell maturation. In addition, the beta-epsilon enhancer also has Locus Control Region (LCR) activity since it is required to establish an active chromosomal domain when linked to the beta(A)-globin gene or gene cluster. In view of the widespread importance of tissue-specific chromosomal domain activation and enhancer-dependent transcriptional regulation in many different gene system, the beta-epsilon enhancer represents an excellent model in which to analyze long-range DNA interactions. The investigator previously demonstrated that betaA-globin transcription is completely dependent upon the 3' beta-epsilon enhancer, acting at a distance of 2 kb, using genes reconstituted into chromatin or synthetic nuclei in the presence of erythroid proteins. In this proposal, the investigator wishes to continue the studies of tissue- specific enhancer function by conducting a detailed examination of the mechanism by which it occurs. The approach is to use in vitro transcription and chromatin reconstitution systems to reproduce critical aspects of enhancer-regulated betaA-globin gene expression. First, experiments are designed to define the DNA structure required to confer beta- epsilon enhancer activity in vitro. Specifically, whether nucleosome assembly or DNA architectural proteins are necessary to generate this topology. Second, the minimum transcription proteins required for enhancer-dependent betaA-globin expression will be identified by using purified or recombinant erythroid DNA binding proteins and general initiation factors as well as unknown proteins obtained by chromatography of erythroid extracts. Third, the ability of enhancers to either activate or depress promoters will be examined by a combination of biochemical and structural studies. These include order- of-addition transcription experiments using purified components and plasmid footprinting of enhancer-dependent and -independent betaA- globin genes. Fourth, the investigator will examine whether enhancer regulation involves DNA looping or protein tracking using electron microscopy and "protein blockage" in vitro transcription experiments. Finally, promoter switching by the beta- epsilon enhancer will be examined in DNA templates containing linked betaA and epsilon globin genes. The analysis of betaA-globin enhancer function in this in vitro system should provide useful information that is applicable not only to other tissue-specific genes but to long-range genetic effects that are particularly relevant to health related issues. These include the demonstrated, but poorly understood, role of enhancers in controlling immunoglobulin and T cells receptor gene rearrangement and the observation that in a variety of lymphomas and leukemias, oncogene deregulation is associated with an aberrant translocation into the vicinity of active enhancers.