Regulation of hemoglobin production in normal and disease states

项目总结/摘要 逆转从胎儿（HbF）到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。在健康成人、SCD患者或用抗-HBF治疗的患者中的HbF表达 药理学HbF诱导剂不均匀地分布在称为“F细胞”的细胞亚群中; F细胞的数量以及每个F细胞中的HbF含量对于SCD的治疗都是重要的。我有 开发了用于纯化和表征原代人F细胞的技术，并将其与 等同的A细胞，其不表达HbF。我的初步数据显示，这两种细胞类型的区别主要在于 他们的珠蛋白含量，但没有任何已知的HbF调节剂的表达，和F细胞增加了长期， 基因座控制区（LCR）增强子和g-珠蛋白启动子之间的染色质接触范围 基因.我的初步研究和本提案中提出的实验代表了第一次尝试， 通过与A细胞直接比较来表征F细胞。我建议进一步表征健康成人的F细胞， SCD患者，并在使用三种药物HbF诱导剂治疗后， 研究转录爆发动力学、染色质可及性和长程染色质的技术 交互.此外，我将探讨三种染色质重塑酶的功能，这些酶在一个细胞的丢失中被鉴定出来。 HbF表达调节剂的功能遗传筛选。我提出的研究将进一步加深我们对 HbF调节机制和HbF表达的异质性在临床上重要的背景下，将 指导开发更有效的镰状细胞病疗法。 这份建议书描述了一个五年的培训计划，以发展我的独立研究事业， 一位研究红细胞生物学和血红蛋白调节的儿科血液学家。我是 宾夕法尼亚大学儿科讲师， 费城儿童医院（CHOP）血液学，曾接受细胞和分子学博士培训 生物学和红细胞生物学。该奖项的目标是发展和完善的基本技能， 作为一名独立的研究者，需要一个成功的职业生涯，包括基因调控的专业知识， 表观遗传学和生物信息学。我获得这个奖项的导师是Gerd Blobel博士，他是一位国际公认的领导者 表观遗传学、红细胞基因调控和血红蛋白转换的发育控制。我也 招募了一个由科学家和医学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 完成我的研究和职业发展目标。在一个有指导的 环境将使我最适合作为一名独立的医生科学家的职业生涯。

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.