A Genomics Approach to Gamma-Globin Regulation

应聘者：我非常有动力，非常有资格从事学术工作 血液学领域的医学。在进入医学院之前，我获得了生物化学博士学位。 在三年半的时间内完成一个为期六年的项目。作为一名三年级的医科学生，我决定攻读 镰状细胞病(SCD)的联合研究和临床生涯，包括成人和儿科专科 培训，并在12年的培训中执行了这一计划。我的团契研究让我了解了 基因组学的强大潜力，我已经应用这项技术来识别与胎儿相关的罕见变异 血红蛋白水平(HbF，α2，2)，以了解-珠蛋白的调节，并开发新的，智能的 为患有血红蛋白疾病的个人设计的治疗方法。我已经利用了遗传学和基因组学 贝勒医学院(BCM)、德克萨斯儿童医院的大量SCD人口和 贝勒大学和全国各地都提供血液学方面的特殊指导，以制定我的研究计划。 我有一个有希望的结果，表明FOXO_3在金的支持下，作为-珠蛋白的正向调节因子的作用 标准的体外功能研究。我打算将我在分子生物学、基因组学和SCD方面的专业知识应用于 揭示FOXO_3及其途径中的其他基因调控-珠蛋白的机制。这项研究将 增加了我们对珠蛋白转换和红细胞生成的理解，并可能引导我们产生新的HBF诱导 治疗。 研究职业发展计划：我将利用我的导师团队和广泛的教育 以及在休斯顿的研究机会，成为珠蛋白转换和红细胞生成方面的专家。我会有所收获的 在尖端分子技术方面的专业知识，以完成我的目标，研究 FOXO_3在-珠蛋白调节中的作用贝勒大学生物医学科学研究生项目提供的课程包括 需要培训分子生物学方法、生物统计学、生物信息学和基因组学，以及伦理学和 科学写作。我将经常与我的联合初选导师古德尔博士会面，审查CHIP-SEQ的数据 (Aim 1c)和RNA-Seq(Aim 2b)实验，并将讨论对结果的解释和见解 他们和我的共同小学导师米奇·韦斯一起提供红细胞生成和-珠蛋白调节。Dr。 伯文克尔将继续为我对整个外显子组测序(WES)数据的基因组分析提供指导 (目标1)。我将继续享受BCM和我的儿科部门的特殊支持 血液学/肿瘤学，85%的保护时间保证作为终身教职的助理教授， 实验室空间，一名研究助理，以及用于可能超过K08的研究材料的部门资金 在整个赠款期间的预算。我的指导团队将帮助我实现我的目标 对我们理解-珠蛋白调节和红细胞生成有重大贡献，并提交 竞争激烈的R01应用程序将在第3年末完成。 研究项目：几种血红蛋白疾病，最明显的是SCD和β地中海贫血，可能是 通过增加-珠蛋白的表达而有效治疗。对-珠蛋白调控有更全面的了解 可以促进HBF诱导剂的靶向设计。在这项提案的初步数据中，我描述了 171例SCD患者WES确定的罕见变异的创新基因基础分析。这一分析 确定FOXO_3为-珠蛋白的正向调节因子。然后我验证了与功能研究的联系 研究-珠蛋白调节的最佳体外系统，人原代红系培养。我现在建议使用 来自更大范围的镰状细胞病患者(n=1000)的WES数据证实了这种关系 FOXO_3和-珠蛋白水平之间的关系，并确定在-珠蛋白中起作用的其他FOXO_3途径基因 监管。我将测量不同程度的FOXO_3敲除对-珠蛋白表达的影响，以 复制体内FOXO_3杂合变异状态，测定FOX_3对-珠蛋白的剂量效应 级别。FOXO_3途径基因在-珠蛋白中的功能将通过shRNA敲除来研究 人原代红系培养。我还将开始分析七个独特的FOX03的功能 在我们的初步研究中发现了变种。 FOXO_3和FOX_3途径基因调控-珠蛋白的机制将被阐明 通过几种方式。我将确定FOXO_3对其他红系细胞表达水平的影响 通过对红系前体细胞的RNA进行RNA-Seq，对红系前体细胞的RNA进行RNA-Seq，从而获得整个红系成熟过程中的基因 在没有FOXO_3的情况下，红细胞生成的所有五个阶段都被击倒。红系特异性FOXO_3结合位点将是 通过在人类原代红系细胞中进行的全球芯片序列鉴定。表达数据，DNA结合位点 通过分析WES数据确定的与HBF水平相关的基因的数据和路径分析将是 结合在一起，得出了FOXO_3调节-珠蛋白水平所涉及的因素的完整图景。我的 工作和未来的职业生涯将集中在-珠蛋白调节上，并将这些信息应用于开发新的 针对患有血红蛋白疾病的个人的治疗。

The Candidate: I am highly motivated and exceptionally qualified to pursue a career in academic medicine in the field of Hematology. I earned a PhD in Biochemistry prior to attending medical school, completing a 6 year program in three and a half years. As a third year medical student, I decided to pursue a combined research and clinical career in sickle cell disease (SCD), including adult and pediatric subspecialty training, and carried out this plan over twelve years of training. My fellowship research introduced me to the powerful potential of genomics, and I have applied this technology to identify rare variants associated with fetal hemoglobin levels (HbF, α22), in order to understand –globin regulation and develop novel, intelligently designed therapies for individuals with hemoglobinopathies. I have capitalized on the genetics and genomics strengths of Baylor College of Medicine (BCM), the large SCD population of Texas Children's Hospital, and the exceptional mentorship in hematology available at Baylor and across the country to develop my research plan. I have promising results indicating a role for FOXO3 as a positive regulator of –globin, supported by gold standard in vitro functional studies. I intend to apply my expertise in molecular biology, genomics and SCD to unravel the mechanism by which FOXO3 and other gene sin its pathway regulate –globin. This research will add to our understanding of globin switching and erythropoiesis, and is likely to lead us to novel HbF induction therapies. Research Career Development Plan: I will utilize my mentorship team and the wide array of educational and research opportunities in Houston to become an expert in globin switching and erythropoiesis. I will gain expertise in the cutting edge molecular techniques needed to accomplish my goal of investigating the role of FOXO3 in –globin regulation. The graduate programs at Baylor in biomedical science provide the courses I need to train in molecular biology methods, biostatistics, bioinformatics and genomics, as well as ethics and scientific writing. I will meet frequently with my co-primary mentor, Dr. Goodell, to review data from ChIP-Seq (Aim 1c) and RNA-Seq (Aim 2b) experiments, and will discuss the interpretation of the results and the insights they provide into erythropoiesis and –globin regulation with my co-primary mentor, Mitch Weiss. Dr. Boerwinkle will continue to provide guidance for my genomic analysis of whole exome sequencing (WES) data (Aim 1). I will continue to enjoy exceptional support from BCM, and my division of Pediatric Hematology/Oncology, with 85% protected time guaranteed as a tenure-track assistant professor, with laboratory space, a research assistant, and department funds for research materials that may exceed the K08 budget throughout the duration of the grant. My mentorship team will help me accomplish my goals of making a significant contribution to our understanding of –globin regulation and erythropoiesis, and submit a competitive R01 application by the end of year 3. Research Project: Several hemoglobinopathies, most notably SCD and beta thalassemia, could be effectively treated by increasing –globin expression. A more complete understanding of –globin regulation could facilitate targeted design of a HbF inducer. In the preliminary data of this proposal, I describe the innovative gene-bases analysis of rare variants identified by WES of 171 patients with SCD. This analysis identified FOXO3 as a positive regulator of –globin. I then verified the association with functional studies in the best in vitro system for studying –globin regulation, human primary erythroid culture. I now propose to use the WES data from a much larger cohort of patients (n=1000) with sickle cell disease to confirm the relationship between FOXO3 and –globin levels, and identify additional FOXO3 pathway genes that play a role in –globin regulation. I will measure the effect of various degrees of FOXO3 knockdown on –globin expression, to replicate the in vivo heterozygous FOXO3 variant state, and determine the dose effect of FOXO3 on –globin levels. The function of FOXO3 pathway genes in –globin will be investigated through shRNA knockdown in human primary erythroid culture. I will also begin to analyze the functionality of the seven unique FOXO3 variants identified in our pilot study. The mechanisms by which FOXO3 and FOXO3 pathway genes regulate –globin, will be elucidated through several modalities. I will determine the effect of FOXO3 on the expression levels of other erythroid genes throughout erythroid maturation by performing RNA-Seq on RNA from erythroid precursors with and without FOXO3 knockdown at all five stages of erythropoiesis. Erythroid specific FOXO3 binding sites will be identified by global ChIP-Seq performed in human primary erythroid cells. Expression data, DNA binding site data and pathway analysis of genes associated with HbF levels identified through analysis of WES data will be combined to produce a complete picture of the factors involved in FOXO3 regulation of –globin levels. My work and future career will focus on –globin regulation, and applying this information to developing new therapies for individuals with hemoglobinopathies.