Analysis of GATA transcription factors functions on development and differentiation of hematopoietic cells.

1)体内加塔转录因子的功能分析小鼠中的加塔-1生殖系突变由于缺陷性红系细胞成熟而导致胚胎致死，因此其它造血加塔因子不能补偿加塔-1的损失。为了确定加塔-1在红系细胞中的专性存在是否是由于其独特的生化特性或时空模式，我们试图用置于加塔-1基因转录控制下的造血加塔因子cDNA来拯救加塔-1突变小鼠。我们发现，转基因表达的加塔-1 cDNA完全废除了加塔-1缺陷型。令人惊讶的是，从相同的调控盒表达的加塔-2和加塔-3因子也挽救了加塔-1突变的胚胎致死表型。这些结果表明，在红细胞生成过程中，决定加塔-1适当积累的转录控制是加塔-1活性的最关键决定因素。2）体内加塔-1的结构域分析。Linea ...更多信息 生殖系加塔-1突变体的基因特异性转基因拯救揭示了细胞培养模型未揭示的加塔-1特异性结构域的新特性。N-末端和N-指结构域赋予加塔-1体内功能互补但明显可区分的性质。3）加塔因子的转录调节。为了确定加塔-1基因造血增强子（G1 HE）的边界，制备了一系列缺失构建体，并在转染和转基因小鼠分析中进行了测试。结果表明，原始红细胞和定形红细胞中加塔-1基因的调控活性均存在于G1 HE的149 bp核心区，而巨核细胞中G1 HE的调控活性则存在于更宽的区域，该149 bp核心区包含一个加塔、一个GAT和一个E-box。突变分析表明，只有加塔框是基因调控活性的关键。因此，这些结果证明存在用于加塔-1基因表达的加塔因子网络，并且G1 HE由决定红细胞或巨核细胞谱系特异性的两个元件组成。少

1) Functonal analyses of GATA transcription factoers in vivo. GATA-1 germline mutation in mice results in embryoniclethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of GATA-1. To determine whether GATA-1's obligate presence in erythroid cellsis due to its distinct biochemical properties or spatiotemporal patterning, we attempted to rescue GATA-1 mutant mice with hematopoietic GATA factor cDNAs placed under the transcriptional control of the GATA-1 gene. We found that transgenic expression of a GATA-1 cDNA fully abrogated the GATA-1-deficient phenotype. Surprisingly, GATA-2 and GATA-3 factors expressed from the same regulatory cassette also rescued the embryoniclethal phenotype of the GATA-1 mutation. These results demonstrate that the transcriptional control dictating proper GATA-1 accumulation is the most critical determinant of GATA-1 activity during erythropoiesis.2) Domain analysis of GATA-1 in vivo. Linea … More ge-specific transgenic rescue of the germ line GATA-1 mutant revealed novel properties od specific domains of GATA-1 that are not revealed by cell culture models. The N-terminal and N-finger domains confer complementing but clearly distingulsihable properties to GATA-1 function in vivo.3) Transcriptional regulation of GATA factors. To define the boundaries of the GATA-1 gene hematopoieticenhancer(G1HE), a series of deletion constructs was prepared and tested in transfection and transgenic mice analyses. The results clearly indicated that a strong GATA-1 gene regulatory activity in both primitive and definitive erythroid cell exists in a 149 bp core region with in G1HE, while that in megakaryocyte exists in more wide region of G1HE.This 149 bp core region contains one GATA, one GAT and one E-box. Mutational analyses revealed that only the GATA box is critical for gene regulatory activity. Thus these results demonstrate the presence of a network of GATA factors for GATA-1 gene expression and G1HE consist of two elements which determine erythroid or megakaryocyte lineage specificity. Less