MOLECULAR MECHANISMS BY WHICH IRF-8 REGULATES MYELOPOIESIS

IRF-8 调节骨髓细胞生成的分子机制

• 批准号: 8917207
• 负责人: Amy M Becker
• 金额: $ 15.26万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8917207
• 关键词: Binding; Candidate Disease Gene; ChIP-seq; Chronic Myeloid Leukemia; Common Lymphoid Progenitor; Complex; DNA; DNA Binding; Data; Dendritic Cells; Development; Disease; Dysmyelopoietic Syndromes; Factor Analysis; Gene Expression; Gene Targeting; Genes; Goals; Health; Hematopoietic stem cells; Human; IFN consensus sequence binding protein; In Vitro; Indium; Knowledge; Lead; Lymphoid; Modeling; Molecular; Molecular Profiling; Multipotent Stem Cells; Mus; Mutate; Mutation; Myelogenous; Myeloid Leukemia; Myelopoiesis; Neutropenia; Pathway interactions; Phenotype; Production; Proteins; Research; SPI1 gene; Staging; Stem cells; Testing; base; blocking factor; in vivo; insight; mutant; neutrophil; new therapeutic target; overexpression; progenitor; programs; promoter; proto-oncogene protein Spi-1; reconstitution; stem cell differentiation; transcription factor

DESCRIPTION (provided by applicant): The transcription factor, IRF-8, blocks neutrophil lineage commitment at several stages of hematopoietic stem cell differentiation. Previous findings revealed that Irf8-/- mice as well as humans and mice with select IRF-8 mutations overproduce neutrophils. We found that IRF-8 is necessary and sufficient to block neutrophil differentiation in common myeloid and common lymphoid progenitor cells, CMPs and CLPs, respectively. Gene expression profiles in Irf8-/- CMPs and CLPs compared to controls revealed that distinct target genes were dysregulated in each of these developmental pathways. The proposed research focuses on the molecular mechanisms by which IRF-8 regulates commitment to the neutrophil lineage. IRF-8 must heterodimerize with other transcription factors in order to bind DNA. In Aim 1, we will determine the co-factors necessary for IRF-8 to regulate neutrophil differentiation and determine the direct target genes of these complexes. IRF-8 forms complexes with the transcription factor, PU.1. Interestingly, reduced PU.1 levels also result in increased neutrophil differentiation. We hypothesize that IRF-8/PU.1 complexes regulate the expression of genes that are required to block neutrophil differentiation in CMPs and CLPs. To test this, we will use previously described IRF-8 mutants that lack the ability to bind co-factors and/or DNA. Importantly, some of these mutants recapitulate the neutrophil phenotype observed in Irf8-/- mice while others result in normal neutrophil differentiation. We will analyze the abiliy of these mutants to bind to PU.1 and transactivate known IRF-8/PU.1 target genes. To identify the IRF-8/PU.1 target genes that block neutrophil differentiation in CMPs and CLPs, IRF-8 and PU.1 ChIP-seq profiles will be compared and contrasted between progenitor cells that express wild type IRF-8 or mutant IRF-8. We will determine whether these candidate factors can block neutrophil differentiation in Irf8-/- progenitor cells in vitro. Some genes that are required to blck neutrophil lineage commitment downstream of IRF-8 might not be direct targets of IRF-8 or might not be exclusively regulated by IRF-8/PU.1 complexes. Thus, in Aim 2 we will employ a candidate-based approach to identify genes regulated by IRF-8 in CMPs and CLPs that limit neutrophil differentiation. We have selected candidate transcription factors that were shown to be downregulated in Irf8-/- CMPs or CLPs via microarray. These factors block neutrophil production or decrease the expression of myeloid genes in vitro. We hypothesize that these candidate genes are the functional downstream targets of IRF-8 that block neutrophil differentiation. We will determine whether these factors block the aberrant neutrophil development in Irf8-/- CMPs and CLPs in vitro and in vivo. Very little is known about the molecular mechanisms by which IRF-8 regulates neutrophil differentiation. This data will significantly increase our understanding of neutrophil lineage commitment and could uncover novel therapeutic targets for diseases in which neutrophils are dysregulated, including myelodysplastic syndromes, myeloid leukemias and various neutropenias.

描述（由申请人提供）：转录因子IRF-8在造血干细胞分化的几个阶段阻断中性粒细胞谱系定型。先前的研究结果显示，Irf 8-/-小鼠以及具有选择性IRF-8突变的人类和小鼠过度产生中性粒细胞。我们发现，IRF-8是必要的和足够的，以阻止中性粒细胞分化的共同骨髓和共同淋巴祖细胞，CMP和CLP，分别。与对照相比，Irf 8-/-CMP和CLP中的基因表达谱显示，不同的靶基因在这些发育途径中的每一个中失调。拟议的研究重点是IRF-8调节中性粒细胞谱系承诺的分子机制。IRF-8必须与其他转录因子异源二聚体化以结合DNA。在目的1中，我们将确定IRF-8调节中性粒细胞分化所必需的辅助因子，并确定这些复合物的直接靶基因。IRF-8与转录因子PU.1形成复合物。有趣的是，PU.1水平降低也导致中性粒细胞分化增加。我们假设IRF-8/PU.1复合物调节阻断CMP和CLP中中性粒细胞分化所需的基因的表达。为了测试这一点，我们将使用先前描述的缺乏结合辅因子和/或DNA的能力的IRF-8突变体。重要的是，这些突变体中的一些重现了在Irf 8-/-小鼠中观察到的中性粒细胞表型，而其他突变体导致正常的中性粒细胞分化。我们将分析这些突变体与PU.1结合并反式激活已知IRF-8/PU.1靶基因的能力。为了鉴定在CMP和CLP中阻断中性粒细胞分化的IRF-8/PU. 1靶基因，将在表达野生型IRF-8或突变型IRF-8的祖细胞之间比较IRF-8和PU. 1 ChIP-seq谱。我们将确定这些候选因子是否可以在体外阻断Irf 8-/-祖细胞中的中性粒细胞分化。阻断IRF-8下游中性粒细胞谱系定型所需的一些基因可能不是IRF-8的直接靶点，或者可能不完全受IRF-8/PU.1复合物的调节。因此，在目标2中，我们将采用基于候选人的方法来鉴定限制中性粒细胞分化的CMP和CLP中受IRF-8调控的基因。我们已经通过微阵列选择了在Irf 8-/-CMP或CLP中显示下调的候选转录因子。这些因子在体外阻断中性粒细胞的产生或降低髓样基因的表达。我们假设这些候选基因是阻断中性粒细胞分化的IRF-8的功能性下游靶点。我们将确定这些因素是否在体外和体内阻断Irf 8-/-CMP和CLP中异常的中性粒细胞发育。关于IRF-8调节中性粒细胞分化的分子机制知之甚少。这些数据将显着增加我们对中性粒细胞谱系定型的理解，并可能发现中性粒细胞失调疾病的新治疗靶点，包括骨髓增生异常综合征，髓性白血病和各种血小板减少症。