Molecular Mechanisms of Globin Gene Expression

本项目的长期目标是确定红系Kriippel样因子 （EKLF）特别有助于13-珠蛋白基因表达的发育控制，更一般地， 体内红细胞生成。利用EKLF依赖性成红细胞模型，研究了 的EKLF功能已经确定了可分离的染色质重塑和反式激活域。而且这些 实验证明在先前定义的体外重塑结构域之外的另外的序列 是调节β-珠蛋白启动子结构所必需的。与使用瞬时报告基因的研究相比， 分析，一种新的内部激活结构域，足以诱导内源性13-珠蛋白基因 观察到野生型水平的表达。为了扩展这些观察，第一个具体目标将评估 确定的结构域调节局部和区域染色质重塑、转录和球蛋白的能力 在一个完整的动物的背景下进行基因转换。这将通过衍生敲入小鼠品系来实现 表达不同的EK.LF结构域。这些小鼠品系中的两个将检验EK_LF结构域 它可以介导染色质重塑，但缺乏反式激活潜力，足以招募远端 在定形红系细胞中，基因座控制区增强子与13-珠蛋白启动子的连接。以互补 在实验中，类似地衍生的敲入EKLF突变体编码新的反式激活结构域，但缺乏 第二个先前描述的氨基末端反式激活区将测试其拯救正常人的能力。 红细胞生成确定需要额外的多肽序列来重塑细胞的结构。 内源性13-珠蛋白启动子导致了一个工作假设，即额外的尚未鉴定的因素 是这个过程所必需的。第二个具体目标的研究集中在识别和表征 这些因素。利用现有试剂的生物化学方法将被用来鉴定 这个复杂的组成部分。从长远来看，将通过衍生小鼠来研究所识别的基因， 靶向相应的基因组座位。这些研究将共同为关键问题提供重要见解 EKLF的功能是红细胞生成所必需的。这些基本知识可能会扩大我们的 了解调节珠蛋白基因表达中7-到[3-开关的分子机制， 鉴定用于治疗镰状细胞病的治疗靶点。

The long term objectives of this project are to determine the mechanisms by which erythroid Kriippel-like factor (EKLF) contributes specifically to the developmental control of 13-globin gene expression and more generally to erythropoiesis in vivo. Utilizing an EKLF-dependent erythroblast model, studies of the structural determinants of EKLF function have identified separable chromatin remodeling and transactivation domains. Moreover, these experiments demonstrate that additional sequences outside the previously defined in vitro remodeling domain are required for modulation of [3-globin promoter structure. In contrast to studies utilizing transient reporter assays, a novel internal activation domain, which is sufficient for induction of endogenous 13-globin gene expression to wild type levels was observed. To extend these observations, the first specific aim will assess the ability of the defined domains to modulate local and regional chromatin remodeling, transcription and globin gene switching in the context of an intact animal. This will be accomplished by deriving knock-in mouse strains that express various EK.LF domains. Two of these mouse lines will test the hypothesis that an EK_LFdomain which can mediated chromatin remodeling but lacks transactivation potential, is sufficient to recruit the distal locus control region enhancer to the 13-globin promoter in definitive erythroid cells. In complementary experiments, a similarly derived knock-in EKLF mutant encoding the novel transactivation domain but lacking a second previously described amino terminal transactivation region will be tested for its ability to rescue normal erythropoiesis. The determination that additional polypeptide sequences are required for remodeling of the endogenous 13-globin promoter has resulted in a working hypothesis that additional as yet unidentified factors are necessary for this process. Studies in the second specific aim focus on the identification and characterization of these factors. Biochemical approaches utilizing reagents already in hand will be exploited to identify the components of this complex. Long-term, the genes identified will be studied by deriving mice in which the corresponding genomic loci are targeted. Together, the studies will provide important insights into the critical functions of EKLF that are essential for erythropoiesis. This fundamental knowledge is likely to expand our understanding of the molecular mechanisms regulating the 7- to [3-switch in globin gene expression, potentially identifying therapeutic targets for the treatment of sickle cell disease.