Combinatorial Profiling and Characterization of AML1 Function in Hematopoiesis

AML1 造血功能的组合分析和表征

• 批准号: 8710324
• 负责人: Kentson Lam
• 金额: $ 4.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710324
• 关键词: Acute Myelocytic Leukemia; Adult; Affect; Agonist; Antibodies; Biological Assay; Blood; Blood Cells; Blood Platelet Disorders; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Breeding; Cannabinoids; Cell Differentiation process; Cell Maintenance; Cell Maturation; Cell Proliferation; Cell physiology; Cells; ChIP-seq; Chromosomes, Human, Pair 8; Coupled; DNA Binding; DNA-Binding Proteins; Data Set; Development; Disease; Dysmyelopoietic Syndromes; Embryo; Gene Expression; Gene Targeting; Genes; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic System; Homeostasis; Human Cloning; In Vitro; Knock-out; Knockout Mice; Lead; Light; Link; Literature; Luciferases; Maintenance; Massive Parallel Sequencing; Measures; Methods; Microarray Analysis; Mus; Mutation; Myeloproliferative disease; Non-Malignant; Pathway Analysis; Pathway interactions; Patients; Phenotype; Play; Population; Predisposition; Process; Proliferating; RUNX1 gene; Research Project Grants; Reverse Transcriptase Polymerase Chain Reaction; Role; Small Interfering RNA; Sorting - Cell Movement; Stem cells; Testing; Time; Tissue-Specific Gene Expression; Transplantation; Up-Regulation; base; blood treatment; cannabinoid receptor; cell motility; chromatin immunoprecipitation; combinatorial; embryonic stem cell; gene function; genome-wide analysis; improved; in vivo; insight; interest; mouse model; mutant; promoter; research study; self-renewal; stem; transcription factor

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.

描述（由申请人提供）：本研究项目的长期目标是了解AML 1（也称为Runx 1）在造血中的功能。AML 1是一种DNA结合转录因子，首先在造血干/祖细胞（HSPC）中表达，并调节它们如何分化为成熟血细胞。AML 1通常被认为是造血的主要调节因子。对AML 1敲除（KO）小鼠模型的分析表明，AML 1在确定性造血中起着重要作用。对AML 1有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，并且它们的HSPC在竞争性造血细胞再增殖、维持和自我更新方面存在缺陷。AML 1最初是从人急性髓性白血病细胞中克隆的，其易位涉及染色体8和21。此外，AML 1突变或AML 1单倍不足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生相关，并有发生急性髓性白血病的倾向。这些结果表明，AML 1在血细胞分化和血液相关疾病中起着至关重要的作用。在这里，我建议继续表征AML 1功能的假设，即AML 1的直接目标调节HSPCs的关键功能。我们已经对野生型和AML 1缺陷型HSPC进行了差异基因表达研究，并对AML 1转录因子占用进行了全基因组分析。为了进一步确定AML 1在HSPCs中的作用，我将首先使用染色质免疫沉淀-定量PCR研究和启动子-荧光素酶测定来证实AML 1与其靶基因的位点相互作用以影响基因表达。其次，将在AML 1缺陷的背景下调节候选靶基因的表达和/或活性，并进行体外分析，包括集落形成、集落再铺板和长期培养起始细胞测定。这些试验将揭示AML 1与其靶基因之间的相互作用如何改变HSPC分化、自我更新和长期再增殖潜力的功能。最后，将使用移植研究和竞争性造血细胞再增殖测定来测试这些靶基因在AML 1缺陷型HSPC中的体内功能。这三个特定目标将导致AML 1直接靶点的鉴定和表征及其在AML 1相关血液发育中的功能。总的来说，这项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方法和更好的骨髓移植方法。

项目成果

Response: the role of RUNX1 isoforms in hematopoietic commitment of human pluripotent stem cells.

• DOI: 10.1182/blood-2013-04-494914
• 发表时间: 2013-06
• 期刊: Blood
• 影响因子: 20.3
• 作者: D. Ran;K. Lam;Wei-Jong Shia;Miao-Chia Lo;Jun-Bao Fan;D. Knorr;Patrick I. Ferrell;Zhaohui Ye;Ming Yan;Linzhao Cheng;D. Kaufman;Dong-er Zhang