GLOBIN GENE REGULATION BY RC TRANSCRIPTION FACTOR GATA-1

这项提议的广泛的长期目标是澄清 红系转录因子调控珠蛋白基因的机制 GATA-1。其具体目的是识别编码dna的氨基酸残基。 结合特异性和激活转录，阐明其作用 结合亲和力在特定GATA-1位点的作用，并表征 GATA-1与其他转录因子的相互作用 红系细胞的启动子活性和发育特异性。 我们将继续进行定点突变研究，以确定氨基 酸残基决定了DNA结合的序列特异性和稳定性。 GATA-1结构域与GAL DNA结合域的融合将在 激活转录和相互作用能力的红系细胞系 与其他转录因子结合。GATA-1的突变体将用于 由缺乏A基因的ES细胞系产生的重建红细胞生成 功能性GATA-1基因。从人类β-珠蛋白中选择的GATA位点 将分析位点的结合亲和力，以及具有不同 亲和力将在活动性和发育专一性方面进行比较。我们 将从GATA-1、CACCC和AP-1构建最小的红系启动子- 1/NFE-2结合位点，并测定其活性和发育 红系细胞的特异性。绑定其他的顺式元素 转录因子将在最小启动子中连接到GATA-1位点 并在瞬变测试中测试相互作用。红系“迷你型”将 由编码GATA-1位点并相互作用的寡核苷酸构建而成 以矩阵附着区(MARS)为侧翼的顺式元件，并驱动 LacZ的表达。这些将在转基因小鼠身上进行测试 发育阶段特异性。

The broad long-term objective of this proposal is to elucidate the mechanism of globin gene regulation by the erythroid transcription factor GATA-1. The specific aims are to identify amino acid residues encoding DNA binding specificity and activation of transcription, clarify the role of binding affinity in the function of specific GATA-1 sites, and characterize the effect of interactions of GATA-1 with other transcription factors on promoter activity and developmental specificity in erythroid cells. We will continue site-directed mutagenesis studies to identify the amino acid residues directing sequence specificity and stability of DNA binding. Fusion of GATA-1 domains to the DNA binding domain of GAL will be tested in erythroid cell lines for the ability to activate transcription and interact with other transcription factors. Mutants of GATA-1 will be used to reconstitute erythropoiesis derived from an ES cell line lacking a functional GATA-1 gene. GATA sites selected from the human beta-globin locus will be assayed for binding affinity, and sites with differing affinities will be compared for activity and developmental specificity. We will construct minimal erythroid promoters from GATA-1, CACCC, and AP- 1/NFE-2 binding sites, and assay their activity and developmental specificity in erythroid cell lines. Cis-elements binding other transcription factors will be linked to GATA-1 sites in minimal promoters and tested for interaction in transient assays. Erythroid "minigenes" will be constructed from oligonucleotides encoding GATA-1 sites and interacting cis-elements flanked by matrix attachment regions (MARs), and driving expression of LacZ. These will be tested in transgenic mice for developmental stage specificity.