DNA METHYLATION, CHROMATIN AND GLOBIN GENE SILENCING

DNA 甲基化、染色质和珠蛋白基因沉默

• 批准号: 7349825
• 负责人: JOSEPH DESIMONE
• 金额: $ 0.7万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7349825
• 关键词: DNA; METHYLATION; CHROMATIN; GLOBIN; GENE

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.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。镰状细胞性贫血患者胎儿血红蛋白（HbF）水平升高在临床上是有益的。在狒狒模型中进行的实验表明，使用药物如5-氮杂-2'-脱氧胞苷（地西他滨）、丁酸盐和羟基脲可以提高HbF水平。这些药物对镰状细胞病患者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次数、急性胸综合征的发生率和输血需求。HbF水平的微小变化证明了相当数量（10-40%）的治疗难治性。此外，由于增加的HbF在红细胞中分布不均，很大比例的红细胞仍然不受细胞内脱氧hbs分子聚合的保护。因此，必须开发新的和改进的药物和治疗方法，在更大比例的患者中提高HbF的水平，并最大限度地提高F细胞的产量。我们的目标是在使用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中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。目前，利用FACS、亚硫酸盐测序和甲醛固定染色质片段（CHIP）免疫沉淀结合PCR，可以分析少量高纯度造血祖细胞中基因的甲基化和组蛋白乙酰化状态。我们建议跟踪γ -珠蛋白基因表达、DNA甲基化和组蛋白乙酰化在胎儿发育和正常红细胞分化过程中的变化，以及地西他滨和组蛋白去乙酰化酶抑制剂诱导HbF产生的增加。我们将使用体外培养系统和体内狒狒模型系统来研究这些机制，我们已经使用了20年。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方法来增加镰状细胞病患者HbF的产生。