FUNCTION OF BETA-GLOBIN DNA BINDING PROTEINS

描述(改编自申请人的摘要)：抄本 β-珠蛋白基因簇的调节依赖于远端 可能激活非常大的核小体结构的序列 染色体区域。这些序列被称为远端控制区(DCR)， 以高度组织特异性的方式发挥作用，并可提供 红系特异性基因在这种细胞类型中不正常表达。 一旦染色体区域被激活，单个珠蛋白基因就会 在红系发育过程中的差异转录。这个 激活区域内单个基因的调节依赖于 多个蛋白质在局部作用，影响基因特异性的表达 促进剂和增强剂。申请者此前曾分析过 通过确定如何调控β-珠蛋白启动子的发育 结合到这个控制区的蛋白质会改变核小体结构并 启动转录。 在这项建议中，申请者希望继续研究如何 通过分析β-珠蛋白基因的作用机制，研究其在发育中的调控作用 两个远端调控元件，DCR和3‘增强子的作用。第一, 实验旨在通过研究一种新的增强子 与启动子和增强子结合的红系特异性因子 其可以调节这两个元素之间的相互作用。的影响 该因子对增强子活性的影响将通过体外转录进行分析。 使用核小体重组的β-珠蛋白基因模板 在自由DNA上不需要增强子。第二，DCR的作用机制 将通过监测其对β-珠蛋白表达的影响进行评估 同时利用游离DNA和核小体重组的染色质结构 体外试验。第三，增强子和DCR的功能将是 在整个β-珠蛋白基因簇的背景下使用 宇宙飞船DNA克隆。利用大的染色体区域可以揭示 可能无法观察到的远程转录调控的几个方面 用体外分离的基因。最后，含有β-珠蛋白的宇宙 星团将与非洲爪哇卵重组为合成核 萃取物。在红系蛋白存在的情况下进行重组 胚胎发育的不同阶段可以使人接近于核 在特定时间内存在的β-珠蛋白星团的结构 红系发育。不同珠蛋白的转录能力 然后，这些合成细胞器中的基因将在体外进行检测，以 分析涉及血红蛋白基因转换的机制。就这样， 申请者希望开始复制红系特有的规定 在一个大的染色体区域内的一个基因家族，并分析 核的远端控制区和其他参数的作用 组织在这一过程中的作用。如果成功，这种实验性的方法 将对其他基因有广泛的应用，并可能应用于 对任何由基因表达异常引起的疾病的研究 转录水平。

DESCRIPTION (Adapted from the applicant's abstract): Transcriptional regulation of the beta-globin gene cluster is dependent upon distal sequences that may activate the nucleosomal structure of very large chromosomal domains. These sequences, termed distal control regions (DCR), function in a highly tissue-specific manner and can confer erythroid-specificity upon genes not normally expressed in this cell type. Once the chromosomal domain is activated, individual globin genes are differentially transcribed during the course of erythroid development. The regulation of individual genes within the activated domain depends upon multiple proteins that act locally to affect expression from gene-specific promoters and enhancers. The applicants have previously analyzed the developmental regulation of the beta-globin promoter by determining how proteins that bind to this control region alter nucleosomal structure and activate transcription. In this proposal, the applicants wish to continue the studies on how the beta-globin gene is developmentally regulated by analyzing the mechanism of action of two distal control elements, the DCR and the 3' enhancer. First, experiments are designed to examine enhancer function by studying an erythroid-specific factor that binds to both the promoter and enhancer and which may mediate interaction between these two elements. The effect of this factor on enhancer activity will be analyzed by in vitro transcription using nucleosome-reconstituted beta-globin gene templates since the enhancer is not required on free DNA. Second, the mechanism of DCR action will be assessed by monitoring its effects on beta-globin expression and chromatin structure using both free DNA and nucleosome reconstitutes in in vitro assays. Third, the function of the enhancer and the DCR will be examined in the context of the entire beta-globin gene cluster using a cosmid DNA clone. The use of large chromosomal regions could reveal aspects of long-range transcriptional regulation that may not be observable with isolated genes in vitro. Finally, cosmids containing the beta-globin cluster will be reconstituted into synthetic nuclei with Xenopus egg extracts. Reconstitution in the presence of erythroid proteins from different stages of embryogenesis may enable one to approximate the nuclear structure of the beta-globin cluster as it exists during specific times in erythroid development. The transcriptional capacity of different globin genes within these synthetic organelles will then be examined in vitro to analyze the mechanisms involved in hemoglobin gene switching. In this way, the applicants hope to begin to reproduce the erythroid-specific regulation of a family of genes within a large chromosomal domain and analyze the function of distal control regions and other parameters of nuclear organization in this process. If successful, this experimental approach will have wide-spread application for other genes and may be applied to the study of any disease that results from abnormal gene expression at the level of transcription.