Molecular Mechanisms of Globin Gene Expression

球蛋白基因表达的分子机制

• 批准号: 6508219
• 负责人: John Michael Cunningham
• 金额: $ 30万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6508219
• 关键词: RNase protection assay; chromatin; clinical research; complementary DNA; developmental genetics; erythroid stem cell; erythropoiesis; gene expression; gene targeting; genetic enhancer element; genetic promoter element; genetic regulation; genetically modified animals; globin; laboratory mouse; mass spectrometry; mutant; protein purification; protein structure function; tissue /cell culture; transcription factor; yeast two hybrid system

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.

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）特异性促进β-珠蛋白基因表达的发育控制以及更普遍地促进体内红细胞生成的机制。利用EKLF依赖性成红细胞模型，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结构域。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。与利用瞬时报告基因测定的研究相反，观察到一种新的内部激活结构域，其足以将内源性β-珠蛋白基因表达诱导至野生型水平。为了扩展这些观察结果，第一个具体目标将评估所定义的结构域在完整动物的背景下调节局部和区域染色质重塑、转录和球蛋白基因转换的能力。这将通过衍生表达各种EKLF结构域的敲入小鼠品系来实现。这些小鼠系中的两个将测试这样的假设，即可以介导染色质重塑但缺乏反式激活潜力的EKLF结构域足以在定形红系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，将测试类似衍生的敲入EKLF突变体的拯救正常红细胞生成的能力，所述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。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，即额外的尚未鉴定的因子是该过程所必需的。第二个具体目标的研究重点是确定和表征这些因素。利用现有试剂的生物化学方法将被用来鉴定这种复合物的成分。从长远来看，将通过衍生小鼠来研究所鉴定的基因，在小鼠中靶向相应的基因组位点。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γ-至β-开关的分子机制的理解，可能确定用于治疗镰状细胞病和B-地中海贫血的治疗靶点。