Molecular Mechanisms of Globin Gene Expression

这个项目的长期目标是确定红系Kriippel样因子的机制 (EKLF)有助于13-珠蛋白基因表达的发育控制，更广泛地说，有助于 体内的红细胞生成。利用EKLF依赖的红细胞模型，研究结构决定因素 已发现可分离的染色质重塑和反式激活结构域。此外，这些 实验证明，在先前定义的体外重塑结构域之外的额外序列 是调节[3-珠蛋白启动子结构]所必需的。与使用瞬变记者的研究相反 一种足以诱导内源性13-珠蛋白基因表达的新的内部激活结构域 观察到野生型水平的表达。为了扩大这些观察，第一个具体目标将评估 已定义结构域调节局部和局部染色质重塑、转录和珠蛋白的能力 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域。其中两个小鼠品系将测试EK_LF域的假设 它可以介导染色质重塑，但缺乏反式激活潜能，足以招募远端 红系细胞中13-珠蛋白启动子的位点控制区增强子。在互补中 实验中，一个类似的敲入EKLF突变体编码了新的反式激活结构域，但缺乏一个 第二个先前描述的氨基末端反式激活区域将被测试其拯救正常的能力 红血球生成。确定需要额外的多肽序列来重塑 内源性13-珠蛋白启动子导致了一个工作假说，即其他尚未确定的因素 是这一过程所必需的。第二个特定目标的研究集中在鉴定和表征上 这些因素中。利用已有试剂的生化方法将被利用来鉴定 这个建筑群的组成部分。从长远来看，识别出的基因将通过衍生小鼠进行研究，在小鼠中 相应的基因组基因座被作为靶点。总而言之，这些研究将为关键的 EKLF对红细胞生成至关重要的功能。这一基础知识可能会扩大我们的 对珠蛋白基因表达中7-to[3-开关]调控的分子机制的理解 确定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.