DNA-BINDING PROTEINS AND FETAL HEMOGLOBIN REGULATION

建议的研究针对的是相关的分子机制 用于调节胎儿血红蛋白(HBF)和成人血红蛋白(HBA) 在狒狒体内合成。我们之前的研究结果表明， 从生产HBA到HbF的反向转换发生在狒狒身上 遭受急性红系应激或接受DNA治疗 去甲基化试剂5-氮杂胞苷。这种反向切换也会发生在 镰状细胞性贫血和β-地中海贫血患者的治疗 5-氮胞苷。升高HbF对这些患者的临床效果是 前景看好，但可能的致癌性阻碍了进一步的研究。一个 更好地理解HbF对HBA的分子机制 转换可能表明更好的治疗方法，而不会产生 无法忍受的副作用。我们的研究集中在非组蛋白的作用上。 核蛋白(DNA序列特异性结合蛋白)在调控中的作用 HBF在妊娠过程中的表达。核蛋白将从 促性腺激素基因转换前后的有核红系细胞 确定胎儿中是否存在DNA结合蛋白的差异 合成HbF与动物合成HBA的比较。DNA绑定将是 使用含有已知珠蛋白调节基因的DNA片段进行检测 序列。我们计划测试这些DNA的功能意义- 结合蛋白的体外转录实验，并对其进行纯化 参与珠蛋白基因切换的蛋白质。调节蛋白 基因将被克隆用于结构和功能研究。这些cdna 克隆还将用于转基因实验，以评估其 治疗潜力。

The proposed studies are directed at the molecular mechanisms responsible for the regulation of fetal hemoglobin (HbF) and adult hemoglobin (HbA) synthesis in the baboon. Our previous results have demonstrated that a reverse switch from the production of HbA to HbF occurs in baboons subjected to acute erythropoietic stress or treatment with the DNA hypomethylating agent, 5-azacytidine. This reverse switch also occurs in patients with sickle cell anemia and Beta thalassemia who are treated with 5-azacytidine. The clinical effect of increased HbF in these patients was promising, but possible carcinogenicity has hindered further studies. A better understanding of the molecular mechanisms responsible for HbF to HbA switching may indicate better approaches to therapy without producing intolerable side effects. Our studies focus on the role of non-histone nuclear proteins (DNA sequence-specific binding proteins) in controlling HbF expression during gestation. Nuclear proteins will be extracted from nucleated erythroid cells of pre- and post-switch baboon fetuses to determine whether differences in DNA-binding proteins exist in fetuses synthesizing HbF compared to animals synthesizing HbA. DNA binding will be examined using DNA fragments containing known globin gene regulatory sequences. We plan to test for the functional significance of these DNA- binding proteins by in vitro transcription assays, and to purify those proteins that participate in globin gene switching. The regulatory protein genes will be cloned for use in structure and function studies. These cDNA clones will also be used in transfection experiments to evaluate their therapeutic potential.