GLOBIN GENE REGULATION BY RC TRANSCRIPTION FACTOR GATA-1

这项建议的广泛的长期目标是阐明 红系转录因子对珠蛋白基因调控的机制 加塔-1。 具体目标是鉴定编码DNA的氨基酸残基 结合特异性和转录激活，阐明了 特异性加塔-1位点功能中的结合亲和力，并表征 加塔-1与其他转录因子的相互作用对 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 酸残基指导序列特异性和DNA结合的稳定性。 加塔-1结构域与GAL的DNA结合结构域的融合将在 红系细胞系激活转录和相互作用的能力 与其他转录因子。 加塔-1的突变体将用于 重建来自缺乏造血干细胞的ES细胞系的红细胞生成。 功能性加塔-1基因。 选自人β-珠蛋白的加塔位点 将测定基因座的结合亲和力，并测定具有不同结合亲和力的位点。 将比较亲和力的活性和发育特异性。 我们 将从加塔-1、CACCC和AP-1构建最小红系启动子。 1/NFE-2结合位点，并测定其活性和发育 在红系细胞系中的特异性。 顺式元件结合其他 转录因子将与最小启动子中的加塔-1位点连接 并在瞬时测定中测试相互作用。 红细胞“小基因”将 由编码加塔-1位点的寡核苷酸构建， 侧接基质附着区（MARs）的顺式元件， LacZ的表达。 这些将在转基因小鼠中进行测试， 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.