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）：该项目的长期目标是确定红斑基因表达的发育控制，更普遍地对体内红斑基因表达的发育控制特别贡献了红斑kruppel样因子（EKLF）的机制。利用EKLF依赖性的红细胞模型，对EKLF功能的结构决定因素的研究已经鉴定出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，在调节β-珠蛋白启动子结构的调节需要之前定义的体外重塑结构域之外的其他序列。与利用瞬态报道测定法的研究相反，一种新型的内部激活结构域，足以诱导内源性β-珠蛋白基因表达到野生型水平。为了扩展这些观察结果，第一个具体目标将评估定义的域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。这将通过得出表达各种EKLF域的敲入小鼠应变来实现。这些小鼠系中的两种将检验以下假设：可以介导染色质重塑但缺乏反式激活潜力的EKLF结构域足以募集远端基因座控制区域增强子在确定性红细胞中的β-珠蛋白启动子中的远端基因座控制区。在补充实验中，编码新型反式激活域但缺乏先前描述的氨基末端反式激活区域的类似衍生的敲入EKLF突变体，该突变体的能力将被测试。确定内源性β-珠蛋白启动子重塑需要其他多肽序列，这导致了一个有效的假设，即此过程对于此过程是必需的。第二个特定目的的研究集中于这些因素的识别和表征。利用已手中的试剂的生化方法将被利用以识别该复合物的组成部分。长期，将通过得出相应基因组基因座的小鼠来研究所鉴定的基因。总之，这些研究将提供对EKLF的关键功能的重要见解，而EKLF对于促红细胞生成至关重要。这种基本知识很可能会扩大我们对环球蛋白基因表达中γ-开关的分子机制的理解，这可能鉴定出治疗镰状细胞疾病和B-心理症的治疗靶标。