Novel molecular targets of RUNX in hematopoietic stem/progenitor cells

描述（由申请人提供）：本研究的长期目标是阐明造血干细胞增殖和分化为成熟血细胞的遗传机制。更具体地说，该项目将重点关注Runxl转录因子在造血干细胞功能中的作用。Runxl是DNA结合转录因子。然而，其在造血中的关键靶基因尚不清楚。Runxl的染色体易位和突变参与白血病和骨髓增生异常综合征的发病机制。小鼠中Runxl基因破坏由于完全缺乏确定的造血而导致胚胎死亡。在成年生活中有条件地敲除该基因会引起各种造血谱系中正常增殖和分化的扰动。该提议将检验Runxl直接调节参与造血干细胞的维持和分化的一组关键基因的表达的假设。该项目将分三个步骤进行。在特定目的1中，小鼠多能造血干细胞系EML（正常造血的良好体外模型）将用于ChIP芯片分析。ChIP芯片分析结合了染色质免疫沉淀和DNA平铺微阵列分析，是一种用于全基因组搜索转录因子靶基因的强大技术。在特定目标2中，将对来自Runxl条件性敲除小鼠的造血干细胞进行表达微阵列分析。这些细胞的转录谱将揭示其表达受RUNX1控制的基因。然后将通过在MDS/AML患者中发现的Runxl（Ala224fsTer228）突变的转录谱来探讨Runxl在疾病中的作用。在具体目标3中，具体目标1和2中获得的数据将被组合以描绘在Runxl转录因子直接控制下的基因网络。主要的靶基因和受该基因影响的下游通路将集中在造血干细胞功能上。将选择最重要的基因，并使用体外和体内方法测试其在造血系统中的意义。正常造血是造血干细胞中各种基因的受控表达的结果。Runxl是造血干细胞中基因表达的主要调节因子。尽管在该领域进行了大量的研究，但对于正常造血最关键的Runxl靶基因仍然是未知的。该项目的目标是发现正常造血不可缺少的基因。了解控制造血的精确分子机制对于开发更好的造血疾病治疗药物至关重要。

DESCRIPTION (provided by applicant): The long-term objective of this research is to clarify the genetic mechanisms by which hematopoietic stem cells proliferate and differentiate into mature blood cells. More specifically, this project will focus on the Runxl transcription factor in hematopoietic stem cell function. Runxl is a DNA binding transcription factor. However, its critical target genes in hematopoiesis are not known. Chromosomal translocations and mutations of Runxl are involved in the pathogenesis of leukemia and myelodysplastic syndrome. Runxl gene dysruption in mice causes embryonic lethality due to complete lack of definitive hematopoiesis. Conditional knockout of this gene in adult life causes perturbation of normal proliferation and differentiation in various hematopoietic lineages. This proposal will test a hypothesis that Runxl directly regulates the expression of a group of critical genes involved in the maintenance and differentiation of hematopoietic stem cells. This project will proceed in three steps. In Specific Aim 1, the mouse multipotent hematopoietic stem cell line EML, which is a good in vitro model of normal hematopoiesis, will be used for ChIP on chip analysis. ChIP on chip analysis combines chromatin immunoprecipitation and DNA tiling microarray analysis, and is a powerful technique for genome-wide search for transcription factor target genes. In Specific Aim 2, expression microarray analysis will be performed on hematopoietic stem cells from the Runxl conditional knockout mouse. The transcriptional profile of these cells will reveal the genes whose expression is controlled by RUNX1. The role of Runxl in illness will then be approached throuh the transcriptional profile of the Runxl (Ala224fsTer228) mutation, found in a patient with MDS/AML. In Specific Aim 3, the data obtained in Specific Aims 1 and 2 will be combined to picture the network of genes that are under direct control of the Runxl transcription factor. The major target genes and the downstream pathways affected by this gene will be characterized focusing on hematopoietic stem cell function. The most significant genes will be selected and tested for their significance in the hematopoietic system, using both in vitro and in vivo approaches. Normal hematopoiesis is a result of controlled expression of various genes in the hematopoietic stem cell. Runxl is a master regulator of gene expression in hematopoietic stem cells. Despite intense research in the field, the most critical Runxl target genes for normal hematopoiesis is still unknown. The goal of this project is to discover the genes indispensable for normal hematopoiesis. Understanding of the precise molecular mechanisms that control hematopoiesis is essential for the development of better therapeutic drugs for hematopoietic disorders.