Molecular Mechanisms of Globin Gene Expression

描述(由申请人提供)：本项目的长期目标是确定红系Kruppel样因子(EKLF)对β-珠蛋白基因表达的发育控制以及更广泛地促进体内红细胞生成的特殊作用机制。利用EKLF依赖的红细胞模型，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反式激活区域。此外，这些实验表明，在先前定义的体外重塑结构域之外的额外序列是调节β-珠蛋白启动子结构所必需的。与使用瞬时报告分析的研究相比，观察到了一个新的内部激活结构域，足以诱导内源性β-珠蛋白基因表达达到野生型水平。为了扩展这些观察，第一个特定的目标是评估所定义的结构域在完整动物的背景下调节局部和局部染色质重塑、转录和珠蛋白基因切换的能力。这将通过衍生表达各种EKLF结构域的敲入小鼠品系来实现。其中两个小鼠品系将测试一种假设，即EKLF结构域可以介导染色质重塑，但缺乏反式激活潜力，足以将远端位点控制区增强子招募到确定的红系细胞中的β-珠蛋白启动子。在补充性实验中，将测试编码新的反式激活区域但缺少先前描述的第二个氨基末端反式激活区域的类似敲入EKLF突变体的拯救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，这一确定导致了一个工作假说，即这一过程需要额外的未知因素。第二个具体目标的研究侧重于这些因素的识别和表征。利用已有试剂的生化方法将被用来鉴定这个复合体的成分。从长远来看，识别出的基因将通过衍生相应基因组位点为靶点的小鼠进行研究。总之，这些研究将为EKLF的关键功能提供重要的见解，这些功能对红细胞生成至关重要。这一基础知识可能会扩大我们对调节珠蛋白基因表达中伽马到贝塔开关的分子机制的理解，有可能确定治疗镰状细胞疾病和B地中海贫血的靶点。

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.