REGULATION OF FETAL HEMOGLOBIN SYNTHESIS

镰状细胞病患者的临床病程较轻 当Hb-F水平升高时。目前正在进行试验，以评估 HU对刺激Hb-F合成和修饰临床表达的影响 疾病。从胎儿到胎儿转换的详细分子理解 成体球蛋白以及操纵这种开关的能力 许多严重的人类血红蛋白疾病和地中海贫血的希望 综合症。这项提案的总体目标是1)定义造血学 祖细胞对长期HU治疗的反应；以及2)定义 胎儿珠蛋白表达升高的分子基础 BPFH的非删除形式。具体目标包括：(1) 外周血中BFU-ES的数量、每个菌落的细胞数、 将监测S期的BFU-ES和伽马/β珠蛋白mRNA比率 镰状细胞病患者HU治疗前后的变化 表达不同的Hb F水平，以检验以下假设 外周血中BFU-ES在Hb-F水平为 从药理上讲是增加的。(2)LCR和A-伽马3‘的作用 增强剂在增强相对启动子强度差异中的作用 -175非缺失形式的hpfh和野生型伽马启动子将 在各种红系中使用瞬时表达分析进行评估 和非红系环境。与-175区域结合的蛋白质 BP变化和/或直接与GATA-1交互将被隔离并 特色化的。将尝试表达这些蛋白质和GATA- 1试图在非红系患者中反式激活-175 HPFH启动子 环境。(3)我们将尝试在#年激活胎儿珠蛋白计划 这位HPFH患者的淋巴细胞。暂态异核体将被制造出来 通过将表达成体珠蛋白程序的MEL细胞与转化的 正常人和HPFH患者的淋巴细胞。计时示意图位于 融合后将进行活体检查，以监测蛋白质的差异 与胎儿LCR、3‘增强子和紧邻5’侧翼的结合 珠蛋白基因。消减文库的表达与差异 将由只表达成体或胚胎的MEL细胞制成 成人程序，以努力识别对激活 人胎儿珠蛋白在瞬时异核体中的程序。

The clinical course in patients with sickle cell disease is more mild when Hb F levels are elevated. Trials are ongoing to evaluate effects of HU on stimulating Hb F synthesis and modifying clinical expression of disease. A detailed molecular understanding of the switch from fetal to adult globins together with the ability to manipulate this switch holds promise for many of the severe human hemoglobinopathies and thalassemia syndromes. General goals of this proposal are 1) to define hematopoietic progenitor response to prolonged HU treatment; and 2) to define the molecular basis for elevated expression of fetal globins, in a non-deletional form of BPFH. Specific aims include the following: (1) Numbers of BFU-Es in peripheral blood, cells per colony, percentage of BFU-Es in S phase and gamma/beta globin mRNA ratios will be monitored before and after HU treatment in patients with sickle cell disease expressing various Hb F levels in order to test the hypothesis that BFU-Es in peripheral blood decrease when Hb F levels are pharmacologically increased. (2) The role of the LCR and A-gamma 3' enhancer in potentiating relative promoter-strength differences between the -175 non-deletional form of HPFH and wild type gamma promoter will be evaluated using transient expression assays in a variety of erythroid and nonerythroid environments. Proteins binding to the area of the -175 bp change and/or directly interacting with GATA- 1 will be isolated and characterized. Attempts will be made to express these proteins and GATA- 1 in an effort to transactivate the -175 HPFH promoter in a non-erythroid environment. (3) We will attempt to activate the fetal globin program in lymphocytes from this HPFH patient. Transient heterokaryons will be made by fusing MEL cells expressing an adult globin program with transformed lymphocytes from normals and the patient with HPFH. Timed footprints in vivo will be done after fusion to monitor for differences in protein binding to the LCR, 3' enhancer and immediate 5' flank of the fetal globin genes. Expression and difference or subtractive cDNA libraries will be made from MEL cells either expressing only adult or embryonic and adult programs in an effort to identify cDNAs critical to activation of the human fetal globin program in transient heterokaryons.