Regulation of hemoglobin production in normal and disease states

项目摘要/摘要 逆转胎儿(HBF)向成人血红蛋白的发育转换是一种重要的治疗方法 镰状细胞病(SCD)的治疗方法HBF在健康成人、SCD患者或治疗后的表达 药理学的HBF诱导剂不均匀地分布在称为“F细胞”的细胞子集中；增加 F细胞的数量和每个F细胞中HBF的含量对SCD的治疗都是重要的。我有过 人类原代F细胞的纯化和鉴定技术的发展及其与 等同的A细胞，不表达HBF。我的初步数据显示，这两种细胞的主要不同之处在于 它们的珠蛋白含量，但不在任何已知的HBF调节器的表达中，并且F细胞已经增加了很长时间。 基因座控制区(LCR)增强子与g-珠蛋白启动子之间的范围染色质接触 基因。我的初步研究和这项提议中提出的实验是第一次尝试 通过与A细胞的直接比较来确定F细胞的特征。我建议进一步鉴定来自健康成年人的F细胞， SCD患者，并使用尖端技术使用三种药物HBF诱导剂治疗 转录破裂动力学、染色质可及性和远距离染色质研究技术 互动。此外，我还将探讨三种染色质重塑酶的功能。 HbF表达调控因子的功能遗传筛选。我提出的研究将进一步加深我们对 HbF调节机制及HbF在临床重要背景和意愿中表达的异质性 指导开发更有效的治疗镰状细胞病的方法。 这份提案描述了我独立研究生涯发展的五年培训计划： 研究红细胞生物学和血红蛋白调节的儿科血液学家、内科医生和科学家。我是 宾夕法尼亚大学儿科学讲师和 费城儿童医院(CHOP)血液学，曾接受过细胞和分子方面的博士培训 生物学和红细胞生物学。该奖项的目标是发展和完善基本技能， 作为一名成功的独立调查人员的职业生涯所必需的，包括基因监管方面的专业知识， 表观遗传学和生物信息学。我的这个奖项的导师是格德·布洛贝尔博士，一位国际公认的领导者 在表观遗传学中，红系基因调控和血红蛋白转换的发育控制。我也有过 招募了一个由科学家和具有免费专业知识的内科科学家组成的咨询委员会 和指导经验，并拥有CHOP和宾夕法尼亚大学的全部资源 为了完成我的研究和职业发展目标。在指导下完成本建议书 环境将给我作为一名独立的内科科学家的职业生涯留下最好的位置。

PROJECT SUMMARY / ABSTRACT Reversing the developmental switch from fetal (HbF) to adult hemoglobin is an important therapeutic approach in sickle cell disease (SCD). HbF expression in healthy adults, patients with SCD, or those treated with pharmacologic HbF inducers is distributed heterogeneously in a subset of cells called “F-cells”; increasing the number of F-cells as well as the HbF content in each F-cell are both important for treatment of SCD. I have developed techniques for purification and characterization of primary human F-cells and their comparison to equivalent A-cells, which do not express HbF. My preliminary data show that the two cell types differ primarily in their globin content but not in expression of any known HbF regulators, and that F-cells have increased long- range chromatin contacts between the locus control region (LCR) enhancer and the promoters of the g-globin genes. My preliminary studies and the experiments proposed in this proposal represent the first attempt to characterize F-cells by direct comparison to A-cells. I propose to further characterize F-cells from healthy adults, patients with SCD, and following treatment with three pharmacologic HbF inducers using cutting-edge techniques for studies of transcriptional bursting kinetics, chromatin accessibility and long-range chromatin interactions. In addition, I will explore the function of three chromatin remodeling enzymes identified in a loss of function genetic screen for modulators of HbF expression. My proposed studies will further our understanding of the mechanisms of HbF regulation and heterogeneity of HbF expression in clinically important contexts and will guide the development of more effective therapeutics for sickle cell disease. This proposal describes a five-year training plan for the development of my independent research career as an academic pediatric hematologist physician-scientist studying red cell biology and hemoglobin regulation. I am an Instructor in Pediatrics at the University of Pennsylvania and an attending physician in the Division of Hematology at the Children’s Hospital of Philadelphia (CHOP) with previous PhD training in cell and molecular biology and red blood cell biology. The goals for this award are to develop and refine the essential skills that will be required for a successful career as an independent investigator, including expertise in gene regulation, epigenetics, and bioinformatics. My mentor for this award is Dr. Gerd Blobel, an internationally recognized leader in epigenetics, erythroid gene regulation, and developmental control of hemoglobin switching. I have also enlisted an advisory committee composed of scientists and physician-scientists with complimentary expertise and mentoring experience, and have the full resources of CHOP and the University of Pennsylvania available for the completion of my research and career development goals. Completion of this proposal in a mentored environment will leave me optimally positioned for a career as an independent physician-scientist.