Defining the role of Drip27, a novel long noncoding RNA, in erythropoiesis

 描述（由申请人提供）：本申请描述了候选人对红细胞发育（红细胞生成）遗传学的研究，该研究将在5年指导职业发展计划的背景下进行。他的最终目标是成为一名独立的医生，科学家在该地区的实验室为基础的，学术血液学-肿瘤学。在费城儿童医院（CHOP）的主要研究导师Gerd Blobel博士和St. Jude儿童研究医院的共同导师Mitchell韦斯博士的指导下，Paralkar博士制定了一个结构化的培训计划，包括密集的实验室研究，RNA生物学和生物信息学课程，定期参加科学会议，并由经验丰富的教师咨询委员会监督。Blobel和韦斯博士在培训科学家和医学科学家方面拥有丰富的经验，CHOP和宾夕法尼亚大学的教育/培训环境非常出色。Paralkar博士的实验室工作集中在长非编码RNA（LncRNA）上，这是一种最近受到重视的遗传物质，可调节基因表达并调节正常和病理性哺乳动物发育的各个方面。据信存在数千种LncRNA，但只有少数被详细研究。Paralkar博士发现了一种名为Drip27的新型LncRNA，它可以调节关键的细胞周期抑制剂p27。该提案研究了Drip27 LncRNA直接激活p27转录，从而调节红细胞生成过程中的细胞分裂和复制的假设。这项研究的长期目标是确定Drip27在正常造血发育和相关疾病（如血癌）中的作用。在初步研究中，Paralkar博士使用全面的RNA测序和生物信息学工具来鉴定小鼠和人类红细胞前体中的数百种新型LncRNA。他选择了一种名为“Drip27”的新型保守LncRNA，并使用CRISPR技术在红细胞系中删除了它。缺失导致关键的细胞周期抑制因子p27的表达显著降低。该提案旨在通过产生具有截短的Drip27转录物的小鼠ES细胞并将其分化成成红细胞来研究Drip27转录物缺失对红细胞生成的影响（目的1）。将通过对Drip27 RNA分子的亚细胞定位进行成像，并通过评估Drip27和p27基因座之间是否存在染色体环来检查Drip27发挥功能的机制（目的2）。已发表的工作表明，许多LncRNA通过与转录因子/辅因子/染色质修饰复合物的物理相互作用来调节基因表达，所述转录因子/辅因子/染色质修饰复合物协调全局基因表达。为了进一步确定Drip27作用于p27基因座的机制，将鉴定与Drip27结合的蛋白质（目的3）。这三个目标放在一起，将建立Drip27在红细胞生成中的作用及其调节靶基因的机制。如果成功，这项工作将阐明红细胞生成和非编码RNA功能范例的新调控机制。反过来，这些发现可能会产生对各种血液疾病的见解，包括贫血，骨髓增生性疾病和骨髓增生异常综合征。在一个严格的正式培训计划的背景下，在一个高度支持的学术环境中进行这项研究将为候选人提供必要的数据和专业工具，以促进成为一个独立的位置，作为一个以研究为导向的学术物理学家，科学家。

 DESCRIPTION (provided by applicant): This application describes the candidate's research on the genetics of red blood cell development (erythropoiesis) to be performed within the context of a 5-year mentored career development plan. His ultimate goal is to become an independent physician-scientist in the area of laboratory-based, academic hematology- oncology. Under the guidance of his primary research mentor Dr. Gerd Blobel at the Children's Hospital of Philadelphia (CHOP), and his co-mentor Dr. Mitchell Weiss at St. Jude Children's Research Hospital, Dr. Paralkar has developed a structured training plan consisting of intensive laboratory research, coursework in RNA biology and bioinformatics, regular participation at scientific conferences, and oversight by an experienced faculty advisory committee. Drs. Blobel and Weiss have extensive experience in training scientists and physician-scientists, and the educational/training environments at CHOP and the University of Pennsylvania are outstanding. Dr. Paralkar's laboratory work centers on long non-coding RNAs (LncRNAs), a recently appreciated class of genetic material that modulates gene expression and regulates diverse aspects of normal and pathological mammalian development. Thousands of LncRNAs are believed to exist, but only few have been studied in detail. Dr. Paralkar has identified a novel LncRNA named Drip27 that regulates the critical cell cycle inhibitor p27. This proposal investigates the hypothesis that Drip27 LncRNA directly activates p27 transcription, and thereby regulates cell division and replication during erythropoiesis. The long term goal of this research is to define the role of Drip27 in normal hematopoietic development and associated diseases such as blood cancers. In preliminary studies, Dr. Paralkar used comprehensive RNA-Sequencing and bioinformatic tools to identify hundreds of novel LncRNAs in mouse and human erythroid precursors. He chose a novel conserved LncRNA named `Drip27,' and he used CRISPR technology to delete it in an erythroid cell line. Deletion led to a significant reduction i the expression of the crucial cell cycle inhibitor p27. This proposal aims to study the effect of Drip27 transcript loss on erythropoiesis by generating mouse ES cells with truncated Drip27 transcript and differentiating them into erythroblasts (Aim 1). The mechanisms by which Drip27 functions will be examined by imaging the subcellular localization of Drip27 RNA molecules and by assessing for the presence of chromosomal looping between the Drip27 and the p27 loci (Aim 2). Published work demonstrates that many LncRNAs regulate gene expression through physical interactions with transcription factors/co-factors/chromatin modifying complexes that orchestrate global gene expression. To further define the mechanism by which Drip27 acts on the p27 locus, proteins binding to Drip27 will be identified (Aim 3). These three aims, put together, will establish the role played by Drip27 in erythropoiesis and the mechanism by which it regulates its target gene. If successful, this work will illuminate novel regulatory mechanisms in erythropoiesis and paradigms for noncoding RNA function. In turn, these findings are likely to produce insights into various blood diseases including anemias, myeloproliferative disorders and myelodysplastic syndromes. Performing this research in the context of a rigorous formal training plan within a highly supportive academic enviroment will provide the candidate essential data and professional tools for advancement into an independent position as a research oriented academic physician-scientist.