REGULATION OF THE HUMAN BETA-GLOBIN LOCUS IN VITRO

人类β-珠蛋白基因家族的转录调控需要 单个基因在不同阶段的选择性表达 红系发育。体内研究表明，红系 DNA结合蛋白，EKLF，是产生开放染色质所必需的 β-珠蛋白启动子的结构与胎儿到成人的调节 红血球生成过程中的珠蛋白转换。为了更好地理解 涉及发育控制的珠蛋白基因切换的机制， 我们使用了一种生化方法来组装克隆的β-珠蛋白基因。 到特定的染色质结构，然后分析它们的 体外转录活性。在目前的项目中，我们 染色质抑制的模板仅在存在新定义的 共激活剂，E-RC1。我们已经提纯和表征了E-RC1，并展示了 重塑复合体。与EKLF、E-RC1特别结合 激活成人β-，但不激活胎儿γ-珠蛋白转录 染色质组装的伽马-β-珠蛋白微斑 相互关联的基因。此外，我们还分离出了另一个重塑复合体 来自胎儿红系细胞，它能激活伽马珠蛋白，但不能激活β-珠蛋白- 因此，两个 不同的重塑复合体被分离出来，并显示为概括 第一, 设计了实验来分析E-RC1的功能 染色质结构和体外激活转录。这将是 染色质重塑复合体是否表现出特异性 第二, E-RC1重塑核小体结构的机制及其原因 是针对β-珠蛋白，而不是伽马珠蛋白启动子 分析过了。第三，将检查其他改建综合体的 在珠蛋白基因转换中的作用。第四，胎儿的重塑情结 特异性激活伽马珠蛋白基因的红系细胞将 被刻画出来。β-珠蛋白基因家族是一种重要的 转录调控所需的多个水平的范例 实现组织特异性和适当的细胞分化。这个 更好地理解基因表达和细胞的变化 许多恶性肿瘤都会发生去分化现象。

Regulated transcription of the human beta-globin gene family requires the selective expression of individual genes at distinct stages of erythroid development. In vivo studies have shown that the erythroid DNA-binding protein, EKLF, is essential to generate an open chromatin structure in the beta-globin promoter and mediate the fetal-to-adult globin switch during erythropoiesis. In order to understand the mechanisms involved in developmentally-controlled globin gene switching, we have used a biochemical approach to assemble cloned beta-globin genes into specific chromatin structures and then analyze their transcriptional activity in vitro. In the current project, we demonstrate that EKLF-dependent beta-globin activation is achieved on chromatin-repressed templates only in the presence of a newly defined co-activator, E-RC1. We have purified and characterized E-RC1 and shown it to be a distinct member of the mammalian SWI/SNF family of chromatin remodeling complexes. In combination with EKLF, E-RC1 specifically activates adult beta-but not fetal gamma-globin transcription in a chromatin-assembled gamma-beta-globin minilocus containing the two linked genes. In addition, we have isolated another remodeling complex from fetal erythroid cells that activates gamma-globin but not beta- globin transcription in vitro on chromatin templates. Thus, two different remodeling complexes are isolated and shown to recapitulate an important aspect of fetal-to-adult globin gene switching. First, experiments are designed to analyze the ability of E-RC1 to function with a variety of DNA-binding proteins, in addition to EKLF, to remodel chromatin structure and activate transcription in vitro. This will address whether chromatin remodeling complexes display specificity towards particular DNA-binding factors or classes of proteins. Second, the mechanism by which E-RC1 remodels nucleosomal structure and why it is targeted to the beta-globin but not gamma-globin promoter will be analyzed. Third, other remodeling complexes will be examined for their role in globin gene switching. Fourth, the remodeling complex from fetal erythroid cells that specifically activates the gamma-globin gene will be characterized. The beta-globin gene family serves as an important paradigm for the many levels of transcriptional regulation required to achieve tissue-specificity and proper cellular differentiation. The proposed studies should provide insight into these processes and a greater understanding of how changes in gene expression and cellular dedifferentiation occur in many malignancies.