DNA-BINDING PROTEINS AND FETAL HEMOGLOBIN REGULATION

拟议的研究是针对分子机制负责 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。 我们以前的结果表明， 在狒狒中发生从HbA到HbF的反向转换 经受急性红细胞生成应激或用DNA治疗 低甲基化剂，5-氮杂胞苷。 这种反向转换也发生在 镰状细胞性贫血和β地中海贫血患者， 5-氮杂胞苷。 在这些患者中，HbF升高的临床影响是 但可能的致癌性阻碍了进一步的研究。 一 更好地理解HbF到HbA的分子机制 转换可能意味着更好的治疗方法， 无法忍受的副作用 我们的研究集中在非组蛋白的作用 核蛋白（DNA序列特异性结合蛋白） 妊娠期HbF表达。 核蛋白质将从 转换前和转换后狒狒胎儿的有核红细胞， 确定胎儿中是否存在DNA结合蛋白的差异 与合成HbA的动物相比， DNA结合将是 使用含有已知珠蛋白基因调控的DNA片段进行检测 序列的 我们计划测试这些DNA的功能意义- 通过体外转录测定结合蛋白，并纯化那些 参与珠蛋白基因转换的蛋白质。 调节蛋白 基因将被克隆用于结构和功能研究。 这些cDNA 克隆也将用于转染实验以评估它们的 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.