TRANSCRIPTION FACTOR GATA-1 IN TERMINAL ERYTHROID DEVELOPMENT

终末期红细胞发育中的转录因子 GATA-1

• 批准号: 6336640
• 负责人: STUART H ORKIN
• 金额: $ 30.53万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336640
• 关键词: cell differentiation; cell growth regulation; chimeric proteins; clone cells; developmental genetics; embryonic stem cell; erythroid stem cell; erythropoiesis; estrogen receptors; gene expression; gene induction /repression; genetic promoter element; genetic regulation; protein structure function; transcription factor

The transcription factor GATA-1 is essential for normal erythroid development. Study of GATA-1-mouse embryonic stem (ES) cells previously showed that adult (definitive) erythroid precursors arrest at the proerythroblast stage and undergo apoptosis. How GATA-1 functions to control terminal erythroid maturation is unknown. Although various domains of the protein have been defined in heterologous cell transfection systems or in vitro, those relevant to function in an erythroid environment are uncertain, largely due to the absence of a suitable assay for GATA-1 function. In an effort to address these aspects, a novel erythroid cell line (G1E) was generated directly from in vitro differentiation GATA-1-ES cells. This proerythroblast-like cell line lacks GATA-1, but retains the ability to complete terminal erythroid maturation upon reintroduction of functional GATA-1 cDNA by retroviral infection. Using this stringent assay, a structure-function analysis of GATA-1, and its related family members (GATA-2 and GATA-3), will be performed. Particular attention will be directed to comparing the abilities of these factors (GATA-1, 2, 3) to trigger terminal erythroid maturation, the role (if any) of amino- and carboxyl-terminal activation domains for in vivo GATA-1 function, and the role and specificity of the DNA-binding domain for function in an erythroid cell environment. With G1E cells harboring conditionally active GATA-factors (GATA-estrogen receptor fusions), a systematic search for RNAs induced (or repressed) under the direct control of these factors during terminal erythroid maturation will be performed. These "difference" transcripts will ultimately provide the reagents with which to understand why deficiency of GATA-1 leads to developmental arrest. Finally, the functional redundancy of GATA-1 and GATA-2 in the early phase of erythroid development, which is suggested by the phenotype of GATA-1-erythroid precursors, will be tested formally by the generation of targeted ES cells lacking both GATA-1 and GATA-2 function. Ultimately, these studies will reveal how GATA-1, and other GATA-factors, function within erythroid cells to program their differentiation.

转录因子GATA-1是正常红系所必需的 发展。GATA-1小鼠胚胎干细胞的研究进展 显示成人(明确的)红系前体细胞在 原红细胞处于成熟阶段，并经历细胞凋亡。GATA-1如何发挥作用 对照终末红系成熟尚不清楚。尽管有各种各样的 在异源细胞中已经确定了该蛋白的结构域 转导系统或体外，那些与功能相关的 红系环境是不确定的，主要是由于缺乏 适用于GATA-1功能的测定。为了解决这些问题 Aspects，一种新的红系细胞系(G1E)是从 体外分化GATA-1-ES细胞。这个原红细胞样细胞 LINE缺乏GATA-1，但保留了完成末期红系的能力 逆转录病毒重新导入功能性GATA-1基因后的成熟 感染。使用这一严格的分析方法，对其结构-功能分析 GATA-1及其相关家族成员(GATA-2和GATA-3)将被 已执行。我们将特别注意比较 这些因子(GATA-1、2、3)触发终末期红系的能力 成熟度，氨基和羧基末端激活的作用(如果有的话) 体内GATA-1功能域，以及GATA-1的作用和特异性 在红系细胞环境中起作用的DNA结合域。使用 G1E细胞含有有条件激活的GATA-因子(GATA-雌激素 受体融合)，系统地寻找诱导(或抑制)的RNA 在这些因素的直接控制下，在红系末期 将进行成熟。这些“不同”的成绩单将 最终提供试剂来了解为什么缺乏 GATA-1导致发育停滞。最后，功能 GATA-1和GATA-2基因在红系早期的冗余 GATA-1-红系表型提示的发育 前体，将通过目标ES的生成进行正式测试 同时缺乏GATA-1和GATA-2功能的细胞。最终，这些研究 将揭示GATA-1和其他GATA因子如何在红系中发挥作用 对细胞的分化进行编程。