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: 6202235
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202235
• 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.

转录因子加塔-1对正常红细胞系是必需的 发展 加塔-1小鼠胚胎干细胞的前期研究 表明，成人（永久）红系前体细胞阻滞在 原成红细胞期并经历细胞凋亡。 加塔-1如何发挥作用 控制终末红细胞成熟是未知的。 尽管各种 蛋白质的结构域已经在异源细胞中被定义， 转染系统或体外，那些相关的功能， 红细胞环境是不确定的，主要是由于缺乏一个 用于加塔-1功能合适测定。 为了解决这些问题， 在某些方面，直接从人红细胞系（G1 E）产生新的红细胞系（G1 E）。 体外诱导加塔-1-ES细胞分化。 这种类原成红细胞的细胞 细胞系缺乏加塔-1，但保留完成终末红细胞的能力 通过逆转录病毒重新引入功能性加塔-1 cDNA后的成熟 感染 使用这种严格的分析，结构-功能分析， 加塔-1及其相关家族成员（加塔-2和加塔-3）将被 执行。 将特别注意比较 这些因子（加塔-1、2、3）触发终末红细胞系的能力 成熟，氨基和羧基末端活化的作用（如果有的话） 体内加塔-1功能的结构域，以及这些结构域的作用和特异性。 在红系细胞环境中发挥功能的DNA结合结构域。 与 携带条件活性GATA-因子（GATA-雌激素）的G1 E细胞 受体融合），系统搜索RNA诱导（或抑制） 在这些因素的直接控制下， 将进行成熟。 这些“差异化”成绩单， 最终提供的试剂，以了解为什么缺乏 加塔-1的基因突变导致发育停滞。 最后，功能 红系早期加塔-1和加塔-2的冗余 发育，这是由加塔-1-红系的表型所暗示的 前体，将通过生成有针对性的ES进行正式测试 缺乏加塔-1和加塔-2功能的细胞。 最终，这些研究 将揭示加塔-1和其他GATA因子在红细胞内的功能 细胞来编程它们的分化。