DNA Methylation, Chromatin, and Globin Gene Silencing

DNA 甲基化、染色质和球蛋白基因沉默

• 批准号: 6739076
• 负责人: JOSEPH DESIMONE
• 金额: $ 31.17万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-18 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739076
• 关键词: DNA methylation; acetylation; amidohydrolases; azacitidine; baboons; cell differentiation; chromatin; deoxycytidine; enzyme inhibitors; flow cytometry; gene expression; gene induction /repression; globin; hematopoietic stem cells; hemoglobin F; histones; immunoprecipitation; nucleic acid sequence; tissue /cell culture

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：胎儿血红蛋白（HbF）水平的升高对于患有镰状细胞性贫血的患者具有临床益处。在狒狒模型中进行的实验表明，使用 5-aza-2'-脱氧胞苷（地西他滨）、丁酸盐和羟基脲等药物可以提高 HbF 水平。这些药物对镰状细胞病患者的有效性已在多项临床试验中得到证实。 MSH 研究表明，羟基脲治疗减少了疼痛危象的数量、急性胸部综合征的发生率以及患者的输血需求。 HbF 水平的微小变化证明了相当多的人 (10-40%) 难以治疗。此外，由于增加的 HbF 在红细胞中分布不均匀，因此很大一部分红细胞仍未受到脱氧 HbS 分子细胞内聚合的保护。因此，必须开发新的和改进的药物和疗法，将更多患者的 HbF 提高到更高水平，并最大限度地增加 F 细胞的产生数量。我们的目标是基于使用去甲基化药物地西他滨、组蛋白脱乙酰酶抑制剂和生长因子，为镰状细胞病患者开发更好的治疗方案。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 表达重新激活中的作用来研究这些药物的作用机制。现在可以使用 FACS、亚硫酸氢盐测序和甲醛固定染色质片段 (CHIP) 免疫沉淀与 PCR 相结合，分析少量高度纯化的造血祖细胞中基因的甲基化和组蛋白乙酰化状态。我们建议追踪胎儿发育和正常红细胞分化过程中γ-珠蛋白基因表达、DNA 甲基化和组蛋白乙酰化的变化，以及地西他滨和组蛋白脱乙酰酶抑制剂诱导的 HbF 产生的增加。我们将使用我们过去 20 年使用的体外培养系统和体内狒狒模型系统来研究这些机制。这些研究将明确 γ-珠蛋白基因沉默的机制，并将有助于开发新的程序以增加镰状细胞病患者的 HbF 产生。