NOTCH SIGNALING IN MYELOID DEVELOPMENT

描述(由申请者提供)：这个项目描述了一个为期5年的职业发展计划，旨在促进血液学研究和血液病理学的学术生涯。拟议的研究计划将建立在现有的知识基金的基础上，利用华盛顿大学现有的独特资源，该大学在培养内科科学家方面有着良好的记录。蒂莫西·莱伊博士是髓系发育和癌症基因组学方面的专家，也是美国血液学会导师奖的前获得者，他将担任研究导师。这项建议的目标是获得科学方法论、技术技能、拨款撰写和科学出版方面的实用和/或教学培训，目的是建立一个专注于造血发展的独立研究计划。建议的研究将集中在Notch通路上，以确定Notch信号改变髓系发育的机制(S)。我们最近的研究表明，表达PML-RARA癌基因的髓系祖细胞(使用Ctsg-PML-RARA小鼠模型)可以增强自我更新，这种自我更新可以通过Notch抑制在体外被取消。这些观察结果将利用已建立的Notch失活的小鼠模型(条件性DN-MAML-GFP)在体内扩展：特定目标1：我们将研究Notch信号在体内自我更新和髓系发育中的作用。Notch阻断对正常和Ctsg-PML-RARA来源的造血干细胞/祖细胞(HSPC)的影响将通过体内方法来评估祖细胞的适合性和信号，以及髓系肿瘤的长期发展；具体目标2：我们将确定Notch靶基因和早期髓系祖细胞的表观遗传学变化。DN-MAML1-GFP融合上的GFP部分将用于染色质免疫沉淀，然后进行Illumina配对末端测序(ChIP-Seq)，以确定c-Kit阳性细胞(富含髓系干/祖细胞)中Notch激活的基因组靶点。这些研究将与转录图谱和额外的CHIP-SEQ实验配对，以监控组蛋白的修饰。这些研究将确定Notch通路影响髓系发育的机制，并将确定髓系特有的Notch功能，最终可能指导髓系疾病的新治疗方法。

DESCRIPTION (provided by applicant): This project describes a 5-year career development program designed to foster an academic career in hematologic research and hematopathology. The proposed research program will build on an existing fund of knowledge using the unique resources available at Washington University, which has a proven track record of developing physician scientists. Dr. Timothy Ley, an expert in myeloid development and cancer genomics and a previous recipient of the American Society of Hematology Mentor Award, will serve as the research mentor. The goals of this proposal are to obtain practical and/or didactic training in scientific methodology, technical skills, grant writing, and scientific publication for the purposeof establishing an independent research program focused on hematopoietic development. The proposed research will focus on the Notch pathway to determine the mechanism(s) by which Notch signaling alters myeloid development. Our recent studies have shown that myeloid progenitors expressing the PML-RARA oncogene (using the Ctsg-PML-RARA mouse model) have enhanced self-renewal, which can be abrogated in vitro by Notch inhibition. These observations will be extended in vivo using an established mouse model of Notch inactivation (conditional DN-MAML-GFP) in the following Aims: Specific Aim 1: We will investigate the role of Notch signaling in self-renewal and myeloid development in vivo. The impact of Notch blockade in both normal and Ctsg-PML-RARA-derived hematopoietic stem/progenitor cells (HSPCs) will be assessed by in vivo methods to assess progenitor fitness and signaling, as well as the long-term development of myeloid neoplasms; and Specific Aim 2: We will identify Notch target genes and epigenetic changes in early myeloid progenitors. The GFP moiety on the DN-MAML1-GFP fusion will be targeted for chromatin immunoprecipitation followed by Illumina paired-end sequencing (ChIP-Seq) to determine the genomic targets of Notch activation in c-Kit positive cells (enriched in myeloid stem/progenitors). The studies will be paired with transcriptional profiling and additional ChIP-Seq experiments to monitor histone modifications. These studies will define the mechanisms by which the Notch pathway influences myeloid development and will identify myeloid-specific Notch functions, which may ultimately guide novel therapeutic approaches for myeloid diseases.