MOLECULAR MECHANISMS BY WHICH IRF-8 REGULATES MYELOPOIESIS

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承诺。先前的研究结果表明，Irf8-/-小鼠以及人类和选择IRF-8突变的小鼠过量产生中性粒细胞。我们发现IRF-8是阻断中性粒细胞分化的必要和充分的共同髓系和共同淋巴系祖细胞，cmp和CLPs。与对照组相比，Irf8-/- CMPs和CLPs中的基因表达谱显示，在这些发育途径中，不同的靶基因都出现了失调。提出的研究重点是IRF-8调节中性粒细胞谱系的分子机制。IRF-8必须与其他转录因子异二聚才能结合DNA。在Aim 1中，我们将确定IRF-8调节中性粒细胞分化所需的辅助因子，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，降低PU.1水平也会导致中性粒细胞分化增加。我们假设IRF-8/PU。1复合物调节cmp和CLPs中阻断中性粒细胞分化所需基因的表达。为了验证这一点，我们将使用先前描述的缺乏结合辅助因子和/或DNA能力的IRF-8突变体。重要的是，其中一些突变再现了在Irf8-/-小鼠中观察到的中性粒细胞表型，而其他突变则导致正常的中性粒细胞分化。我们将分析这些突变体结合PU.1和反激活已知IRF-8/PU的能力。1 .靶基因。识别IRF-8/PU。将在表达野生型IRF-8或突变型IRF-8的祖细胞中比较和对比阻断cmp和CLPs中中性粒细胞分化的1个靶基因，IRF-8和PU.1 ChIP-seq谱。我们将确定这些候选因子是否能在体外阻断Irf8-/-祖细胞的中性粒细胞分化。一些阻断IRF-8下游中性粒细胞谱系承诺所需的基因可能不是IRF-8的直接靶点，也可能不是由IRF-8/PU独家调节的。1复合物。因此，在Aim 2中，我们将采用一种基于候选物的方法来鉴定cmp和CLPs中受IRF-8调控的限制中性粒细胞分化的基因。我们通过微阵列选择了在Irf8-/- cmp或CLPs中被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系基因的表达。我们假设这些候选基因是IRF-8阻断中性粒细胞分化的功能性下游靶点。我们将在体外和体内确定这些因素是否阻断Irf8-/- cmp和CLPs中异常中性粒细胞的发育。关于IRF-8调节中性粒细胞分化的分子机制知之甚少。这一数据将显著增加我们对中性粒细胞谱系承诺的理解，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、髓性白血病和各种中性粒细胞减少症。

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.