FUNCTION OF BETA GLOBIN DNA BINDING PROTEINS

β 珠蛋白 DNA 结合蛋白的功能

• 批准号: 2179510
• 负责人: Beverly Marie Emerson
• 金额: $ 31.74万
• 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/2179510
• 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-珠蛋白基因簇的调控需要长程 远端 DNA 序列之间的相互作用以组织特异性激活 染色体位点并差异调节个体基因 使用红细胞发育。 一个控制元件可同时发挥两种功能 这些能力中的一个是β-ε增强子。 该区域残留 成人β(A)-和胚胎ε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-ε增强子还具有Locus 控制区域（LCR）活动，因为需要建立一个活动的 与 β(A)-珠蛋白​​基因或基因连接时的染色体结构域 簇。 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的调节，β-ε增强子 代表了分析长链 DNA 的优秀模型 互动。 研究人员之前证明，βA-珠蛋白 转录完全依赖于 3' β-ε 增强子， 使用重构为染色质的基因，在 2 kb 的距离上起作用 或存在红细胞蛋白的合成细胞核。 在这个 建议，研究人员希望继续组织的研究 通过详细检查特定的增强子功能 它发生的机制。 该方法是利用体外 转录和染色质重建系统以重现关键 增强子调节的βA-珠蛋白基因表达的方面。 第一的， 实验旨在定义赋予DNA所需的结构 体外β-ε增强剂活性。 具体来说，是否 核小体组装或 DNA 结构蛋白对于 生成此拓扑。 二、最小转录蛋白 增强子依赖性βA-珠蛋白表达所需的将是 通过使用纯化或重组红细胞 DNA 结合来鉴定 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。 三、能力 将检查激活或抑制启动子的增强子 通过生物化学和结构研究的结合。 这些包括 使用纯化组分的加成顺序转录实验 以及增强子依赖性和非依赖性 betaA- 的质粒足迹 珠蛋白基因。 第四，研究者将检查增强剂是否 调控涉及 DNA 循环或使用电子追踪蛋白质 显微镜和“蛋白质阻断”体外转录实验。 最后，由 β-ε 增强子进行的启动子转换将是 在含有连接的 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，但对远程遗传效应 特别是与健康相关的问题。 这些包括 增强子在控制中的作用已被证明但知之甚少 免疫球蛋白和T细胞受体基因重排和 观察发现，在多种淋巴瘤和白血病中，癌基因 放松管制与异常易位相关 活性增强子附近。