FUNCTION OF BETA GLOBIN DNA BINDING PROTEINS

β 珠蛋白 DNA 结合蛋白的功能

• 批准号: 2684863
• 负责人: Beverly Marie Emerson
• 金额: $ 37.01万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684863
• 关键词: DNA footprinting; chickens; chromatin; developmental genetics; electron microscopy; erythropoiesis; gene induction /repression; genetic enhancer element; genetic promoter element; globin; molecular cloning; nucleic acid sequence; nucleosomes; plasmids; protein structure function; recombinant proteins; transcription factor

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.

描述（改编自申请人的摘要）：转录 鸡β-珠蛋白基因簇的调节需要长距离的 远端DNA序列之间的相互作用以组织特异性激活 染色体基因座和差异调节单个基因 使用红细胞发育。 一个控制元件同时在两者中发挥作用 其中之一就是β-淀粉样蛋白增强剂。 该区域残留 在成人β（A）-和胚胎β-珠蛋白基因之间， 在红细胞中的不同时间分别激活每个启动子 成熟 此外，β-内酰胺酶增强剂还具有基因座 控制区（LCR）活动，因为需要建立一个有效的 当与β（A）-珠蛋白基因或基因连接时， 集群 鉴于组织特异性的广泛重要性， 染色体结构域激活和增强子依赖性转录 在许多不同的基因系统中，β-淀粉样蛋白增强子 代表了一个分析长距离DNA的极好模型 交互. 研究者先前证明β A-珠蛋白 转录完全依赖于3 ′ β-κ B增强子， 作用于2 kb的距离，使用重组到染色质中的基因 或在红系蛋白存在下的合成核。 在这 研究人员希望继续研究组织- 特异性增强子的功能，进行详细的检查， 它发生的机制。 方法是在体外使用 转录和染色质重建系统， 增强子调控的β A-珠蛋白基因表达的方面。 第一、 实验的目的是确定所需的DNA结构， 体外β-淀粉样蛋白增强剂活性。 具体而言，无论 核小体组装或DNA结构蛋白是必要的， 生成这个拓扑。 第二，最小转录蛋白 增强子依赖性β A-珠蛋白表达所需的 通过使用纯化或重组红系DNA结合鉴定 蛋白质和一般起始因子以及未知蛋白质 通过红细胞提取物的色谱法获得。 第三，能力 将检查增强子激活或抑制启动子的能力 通过生物化学和结构研究的结合。 这些包括 使用纯化组分的加和顺序转录实验 和增强子依赖性和非依赖性β A- 珠蛋白基因 第四，研究人员将检查增强剂是否 调控涉及DNA环或蛋白质跟踪使用电子 显微镜和"蛋白质阻断"体外转录实验。 最后，启动子转换的β-淀粉样蛋白增强子将是 在含有连接的β A和β珠蛋白的DNA模板中检查 基因. β A-珠蛋白增强子在此体外作用的分析 系统应提供有用信息，不仅适用于 其他组织特异性基因，但长期的遗传效应， 特别是与健康相关的问题。 其中包括 已经证明，但知之甚少的是，增强子在控制 免疫球蛋白和T细胞受体基因重排以及 观察到在各种淋巴瘤和白血病中，癌基因 失调与异常易位到 活性增强剂附近。