DNA METHYLATION, CHROMATIN AND GLOBIN GENE SILENCING

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。胎儿血红蛋白（HbF）水平升高对镰状细胞贫血患者的临床有益.在狒狒模型中进行的实验表明，使用药理学试剂如5-氮杂-2 '-脱氧胞苷（地西他滨）、丁酸盐和羟基脲可升高HbF水平。这些药物在镰状细胞病患者中的有效性在许多临床试验中得到证实。MSH研究表明，羟基脲治疗减少了患者疼痛危象的数量、急性胸部综合征的发生率和输血需求。相当多的患者（10-40%）是治疗难治性的，HbF水平的变化极小。此外，由于增加的HbF在红细胞中分布不均匀，因此大百分比的红细胞保持不受脱氧HbS分子的细胞内聚合的保护。因此，必须开发新的和改进的药剂和疗法，其在更大比例的患者中将HbF增加到更高水平，并使产生的F细胞的数量最大化。我们的目标是在去甲基化药物地西他滨、组蛋白去乙酰化酶抑制剂和生长因子的基础上，为镰状细胞病患者开发一种更好的治疗方案。我们打算通过确定DNA甲基化和组蛋白乙酰化在成人珠蛋白基因表达的发育调节和HbF表达的再激活中的作用来研究这些药物的作用机制。在少量的高度纯化的造血祖细胞中的基因的甲基化和组蛋白乙酰化状态的分析，现在可以使用流式细胞仪，亚硫酸氢盐测序和免疫沉淀的甲醛固定的染色质片段（CHIP）结合PCR。我们建议在胎儿发育和正常红细胞分化过程中，以及在地西他滨和组蛋白去乙酰化酶抑制剂诱导的HbF产生增加后，跟踪γ-珠蛋白基因表达、DNA甲基化和组蛋白乙酰化的变化。我们将使用体外培养系统和体内狒狒模型系统，我们已经使用了20年来研究这些机制。这些研究将确定γ-珠蛋白基因沉默的机制，并将有助于开发新的程序来增加镰状细胞病患者的HbF产生。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Increased level of fetal hemoglobin (HbF) is clinically beneficial ill patients with sickle cell anemia. Experiments performed in the baboon model demonstrated that HbF levels could be elevated using pharmacologic agents such as 5-aza-2'-deoxycytidine (decitabine), butyrates, and hydroxyurea. The usefulness of these drugs in patients with sickle cell disease was confirmed in a number of clinical trials. The MSH study demonstrated that hydroxyurea therapy reduced the number of pain crises, incidence of acute chest syndrome, and transfusion requirements in patients. A significant number (10-40%) are refractory to treatment as evidenced by minimal changes in HbF levels. Furthermore, because the increased HbF is distributed heterogeneously among red cells, a large percentage of erythrocytes remain unprotected from intracellular polymerization of deoxy-HbS molecules. New and improved agents and therapies must therefore be developed which increase HbF to higher levels in a greater proportion of patients and maximize the number of F cells produced. It is our goal to develop a better therapeutic regimen for patients with sickle cell disease based upon the use of the demethylating drug decitabine, histone deacetylase inhibitors, and growth factors. We intend to investigate the mechanism of action of these agents by determining the role of DNA methylation and histone acetylation in both the development regulation of globin gene expression and the reactivation of HbF expression in the adult. Analysis of the methylation and histone acetylation status of genes in small numbers of highly purified hematopoietic progenitor cells is now possible using FACS, bisulfite sequencing and immunoprecipitation of formaldehyde-fixed chromatin fragments (CHIP) in combination with PCR. We propose to follow changes in gamma-globin gene expression, DNA methylation, and histone acetylation during fetal development and normal erythroid differentiation, and following augmentation of HbF production induced by administration of decitabine and histone deactylase inhibitors. We will use an in vitro culture system and an in vivo baboon model system that we have used for the past 20 years to study these mechanisms. These studies will define the mechanisms of gamma-globin gene silencing, and will aid in the development of new procedures to augment HbF production in patients with sickle cell disease.