Molecular Mechanisms of Globin Gene Expression

描述（由申请人提供）：该项目的长期目标是确定红细胞克虏伯样因子（EKLF）在体内对β -珠蛋白基因表达的发育控制和更普遍的红细胞生成的机制。利用EKLF依赖的红母细胞模型，对EKLF功能的结构决定因素的研究已经确定了可分离的染色质重塑和转激活域。此外，这些实验表明，β -珠蛋白启动子结构的调节需要先前定义的体外重塑域之外的其他序列。与利用瞬时报告基因法的研究相反，研究人员发现了一个新的内部激活结构域，足以将内源性β -珠蛋白基因表达诱导到野生型水平。为了扩展这些观察结果，第一个具体目标将评估在完整动物的背景下，所定义的结构域调节局部和区域染色质重塑、转录和珠蛋白基因开关的能力。这将通过获得表达各种EKLF结构域的敲入小鼠品系来实现。其中两种小鼠系将验证EKLF结构域的假设，该结构域可以介导染色质重塑，但缺乏反激活潜力，足以在确定的红细胞中将远端基因座控制区增强子募集到β -珠蛋白启动子。在补充实验中，一个类似衍生的敲入EKLF突变体编码新的反活化结构域，但缺乏第二个先前描述的氨基末端反活化区域，将测试其恢复正常红细胞生成的能力。内源性β -珠蛋白启动子的重塑需要额外的多肽序列的确定，导致了一个工作假设，即这个过程需要额外的尚未确定的因素。第二个具体目标的研究侧重于这些因素的识别和表征。利用现有试剂的生化方法将被用来鉴定这种复合物的成分。从长远来看，鉴定出的基因将通过培养具有相应基因组位点的小鼠进行研究。总之，这些研究将为EKLF对红细胞生成至关重要的关键功能提供重要的见解。这一基础知识很可能扩展我们对调节珠蛋白基因表达中γ -到β -开关的分子机制的理解，潜在地确定治疗镰状细胞病和b -地中海贫血的治疗靶点。

DESCRIPTION (provided by applicant): The long term objectives of this project are to determine the mechanisms by which erythroid Kruppel-like factor (EKLF) contributes specifically to the developmental control of beta-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 beta-globin promoter structure. In contrast to studies utilizing transient reporter assays, a novel internal activation domain, which is sufficient for induction of endogenous beta-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 EKLF domains. Two of these mouse lines will test the hypothesis that an EKLF domain which can mediated chromatin remodeling but lacks transactivation potential, is sufficient to recruit the distal locus control region enhancer to the beta-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 beta-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 gamma- to beta-switch in globin gene expression, potentially identifying therapeutic targets for the treatment of sickle cell disease and B-thalassemia.