Novel molecular targets of RUNX in hematopoietic stem/progenitor cells

描述(申请人提供)：这项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，该项目将重点研究Runx1转录因子在造血干细胞中的功能。Runx1是一种DNA结合转录因子。然而，其在造血中的关键靶基因尚不清楚。Runx1的染色体易位和突变参与了白血病和骨髓增生异常综合征的发病机制。由于完全缺乏明确的造血，小鼠的Runx1基因紊乱会导致胚胎死亡。在成年生活中，该基因的条件性敲除会导致不同造血系的正常增殖和分化受到干扰。这一提议将检验一种假设，即Runxl直接调控一组涉及造血干细胞维持和分化的关键基因的表达。这个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是一种良好的正常造血体外模型，将用于芯片上分析。芯片上分析结合了染色质免疫沉淀和DNA平铺微阵列分析，是一种强大的全基因组搜索转录因子靶基因的技术。在特定目的2中，将对Runx1条件基因敲除小鼠的造血干细胞进行表达芯片分析。这些细胞的转录图谱将揭示其表达受RUNX1控制的基因。然后将通过在MDS/AML患者中发现的Runx1(Ala224fsTer228)突变的转录图谱来探讨Runx1在疾病中的作用。在特定的目标3中，在特定的目标1和2中获得的数据将被组合，以描绘在Runx1转录因子的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以造血干细胞功能为重点进行描述。将选择最重要的基因，并使用体外和体内方法测试它们在造血系统中的重要性。正常的造血是造血干细胞中各种基因表达受控的结果。Runx1是造血干细胞基因表达的主要调节者。尽管在这一领域进行了密集的研究，但对正常造血最关键的Runx1靶基因仍然未知。该项目的目标是发现正常造血所必需的基因。了解控制造血的精确分子机制对于开发更好的治疗造血疾病的药物是至关重要的。

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.