REGULATION OF THE HUMAN BETA-GLOBIN LOCUS IN VITRO

人类β-珠蛋白基因家族的调节转录需要 个体基因在不同阶段的选择性表达 红细胞发育 体内研究表明， DNA结合蛋白EKLF是形成开放染色质所必需的 β-珠蛋白启动子中的结构，并介导胎儿到成人的 红细胞生成过程中的珠蛋白转换。为了解 参与发育控制的珠蛋白基因转换的机制， 我们用生物化学的方法来组装克隆的β-珠蛋白基因 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 证明EKLF依赖的β-珠蛋白激活是在 染色质抑制模板只有在存在一个新定义的 共激活剂E-RC 1。 我们已经纯化和表征了E-RC 1，并显示 它是哺乳动物染色质SWI/SNF家族的独特成员 重塑复合体 与EKLF结合，E-RC 1特异性 激活成人β-珠蛋白转录，但不激活胎儿γ-珠蛋白转录， 染色质组装的γ-β-珠蛋白微位点， 连锁基因 另外，我们分离出了另一个重塑复合体 从胎儿红系细胞，激活γ-珠蛋白，但不β- 珠蛋白在染色质模板上的体外转录。 因此，两个 不同的重塑复合体被分离出来， 胎儿到成人珠蛋白基因转换的一个重要方面。 第一、 设计实验来分析E-RC 1的功能 除了EKLF外，还使用多种DNA结合蛋白来重塑 染色质结构和激活体外转录。 这将 解决染色质重塑复合物是否显示特异性 针对特定的DNA结合因子或蛋白质类。 第二、 E-RC 1重塑核小体结构的机制及其原因 靶向β-珠蛋白而不是γ-珠蛋白启动子， 分析了 第三，将检查其他重塑复合体的 在珠蛋白基因转换中的作用第四，胎儿的重塑复合体 特异性激活γ-珠蛋白基因的红细胞将 被描述。 β-珠蛋白基因家族是一个重要的 转录调控所需的许多水平的范例， 实现组织特异性和适当的细胞分化。 的 拟议的研究应提供对这些过程的深入了解， 更好地理解基因表达和细胞内 去分化发生在许多恶性肿瘤中。

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.