Molecular Mechanisms of Globin Gene Expression

球蛋白基因表达的分子机制

• 批准号: 7528441
• 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: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7528441
• 关键词: DNA binding protein; chromatin; cooperative study; gene expression; globin; laboratory mouse; sickle cell anemia

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.

该项目的长期目标是确定红系 Kriippel 样因子的机制 (EKLF) 特别有助于 13 珠蛋白基因表达的发育控制，更普遍的是 体内红细胞生成。利用 EKLF 依赖性成红细胞模型，研究结构决定因素 EKLF 功能的研究已经确定了可分离的染色质重塑和反式激活域。而且，这些 实验表明，先前定义的体外重塑域之外的其他序列 是调节[3-珠蛋白启动子结构所必需的。与利用瞬时报告基因的研究相比 检测，一种新的内部激活结构域，足以诱导内源 13-珠蛋白基因 观察到野生型水平的表达。为了扩展这些观察结果，第一个具体目标将评估 定义的结构域调节局部和区域染色质重塑、转录和珠蛋白的能力 在完整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 表达各种 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，但缺乏反式激活潜力，足以招募远端 定型红系细胞中 13 珠蛋白启动子的基因座控制区增强子。互补中 实验中，类似衍生的敲入 EKLF 突变体编码新的反式激活结构域，但缺乏 将测试先前描述的第二个氨基末端反式激活区域其拯救正常的能力 红细胞生成。确定重构需要额外的多肽序列 内源性 13 珠蛋白启动子产生了一个工作假设，即其他尚未识别的因素 对于这个过程是必要的。第二个具体目标的研究重点是识别和表征 这些因素。将利用现有试剂的生化方法来识别 这个复合体的组成部分。从长远来看，将通过衍生小鼠来研究所识别的基因，其中 相应的基因组位点被靶向。总之，这些研究将为关键的问题提供重要的见解。 EKLF 的功能对于红细胞生成至关重要。这些基础知识可能会扩展我们的 了解调节球蛋白基因表达中 7- 至 [3-开关的分子机制，可能 确定治疗镰状细胞病的治疗靶点。