Combinatorial Profiling and Characterization of AML1 Function in Hematopoiesis

描述（由申请人提供）：本研究项目的长期目标是了解AML1（也称为Runx1）在造血中的功能。AML1是一种DNA结合转录因子，首先在造血干细胞/祖细胞（HSPCs）中表达，并调节它们如何分化为成熟血细胞。AML1通常被认为是造血的主要调节因子。对AML1敲除（KO）小鼠模型的分析表明，AML1在最终造血中起着重要作用。对AML1条件敲除的成年小鼠的研究表明，这些小鼠在正常血细胞成熟方面存在异常，其HSPCs在竞争性造血细胞再生、维持和自我更新方面存在缺陷。AML1最初是从8号和21号染色体易位的人类急性髓性白血病细胞中克隆出来的。此外，AML1突变或AML1单倍功能不全的患者与骨髓增生异常综合征、骨髓增生性疾病和家族性血小板疾病的发展有关，并有发展为急性髓系白血病的倾向。这些结果表明，AML1在血细胞分化和血液相关疾病中都起着至关重要的作用。在这里，我建议通过验证AML1的直接靶点调节HSPCs的关键功能的假设来继续表征AML1的功能。我们已经对野生型和AML1缺失型HSPCs进行了差异基因表达研究，并对AML1转录因子占用率进行了全基因组分析。为了进一步确定AML1在HSPCs中的作用，我将首先通过染色质免疫沉淀-定量PCR研究和启动子荧光素酶测定来证实AML1与其靶基因的位点相互作用，从而影响基因表达。其次，候选靶基因的表达和/或活性将在AML1缺乏的背景下进行调节，并进行体外分析，包括集落形成、集落复制和长期培养启动细胞试验。这些试验将揭示AML1与其靶基因之间的相互作用如何改变HSPC分化、自我更新和长期再生潜力的功能。最后，这些靶基因在aml1缺陷造血干细胞中的体内功能将通过移植研究和竞争性造血细胞再生实验进行测试。这三个特异性目标将导致AML1直接靶点的鉴定和表征及其在AML1相关血液发育中的功能。总的来说，本研究揭示的见解将提供对造血的更好理解，从而改善血液相关疾病的治疗和更好的骨髓移植方法。

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the function of AML1, also known as Runx1, in hematopoiesis. AML1 is a DNA binding transcription factor that is first expressed in hematopoietic stem/progenitor cells (HSPCs) and regulates how they differentiate into mature blood cells. AML1 is often considered a master regulator of hematopoiesis. Analyses of AML1 knockout (KO) mouse models demonstrate that AML1 plays a fundamental role in definitive hematopoiesis. Studies of adult mice with AML1 conditionally knocked out showed these mice have abnormalities in normal blood cell maturation and that their HSPCs were defective in competitive hematopoietic cell repopulation, maintenance and self-renewal. AML1 was originally cloned from human acute myeloid leukemia cells with a translocation involving chromosomes 8 and 21. In addition, patients with mutations in AML1 or with haploinsufficiency of AML1 were linked to the development of myelodysplastic syndrome, myeloproliferative disease, and familial platelet disorder with propensity to develop acute myeloid leukemia. These results have revealed that AML1 serves a vital role in both blood cell differentiation and blood-associated diseases. Here, I propose to continue the characterization of AML1 function by testing the hypothesis that direct targets of AML1 regulate critical functions in HSPCs. We have already performed differential gene expression studies on wild type and AML1-deficient HSPCs, and have conducted genome-wide analysis of AML1 transcription factor occupancy. To further establish the role of AML1 in HSPCs, I will first confirm that AML1 interacts with the loci of its target genes to affect gene expression by using chromatin immunoprecipitation-quantitative PCR studies and promoter-luciferase assays. Second, the expression and/or activity of candidate target genes will be modulated in the background of AML1 deficiency and in vitro analyses including colony formation, colony replating, and long-term culture initiating cell assays will be performed. These assays will reveal how interactions between AML1 and its target genes may alter the functions of HSPC differentiation, self-renewal, and long-term repopulating potential. Finally, the in vivo functions of these target genes in AML1-deficient HSPCs will be tested using transplantation studies and competitive hematopoietic cell repopulation assays. These three Specific Aims will result in the identification and characterization of AML1 direct targets and their functions in AML1 related blood development. Overall, the insights revealed in this study will provide a better understanding of hematopoiesis, leading to improved treatments for blood-related disorders and better methods for bone marrow transplantation.