GENOMIC APPROACHES TO MYELOID SPECIFICATION

骨髓规范的基因组学方法

• 批准号: 6946268
• 负责人: SHERMAN Morton WEISSMAN
• 金额: $ 26.19万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6946268
• 关键词: RNA interference; SDS polyacrylamide gel electrophoresis; acute myelogenous leukemia; affinity chromatography; biological signal transduction; cell differentiation; cell proliferation; cytokine; enzyme linked immunosorbent assay; gene induction /repression; genetically modified animals; hematopoietic stem cells; laboratory mouse; mass spectrometry; monocyte; myeloid stem cell; neutrophil; polymerase chain reaction; transcription factor; western blottings

Analysis of RNA expression patterns has indicated that forty or more transcription factors are consistently up or down regulated during differentiation of a promyelocytic cell line to more mature neutrophils. These include a number of factors already recognized to have a role in cell proliferation or in myeloid differentiation, but also at least fifteen factors that have not been studied in the context of granulocytic maturation. Several such factors may be involved in gene silencing during differentiation while others may have a more general role in cell lineage determination. We propose to take a genomic approach to study the role of the latter factors in neutrophil and monocyte differentiation of the human NB4 cell line and in normal neutrophils. The methods we propose to use include 1) studying the effects of RNAi knockdown on the morphology and function of differentiating NB4 cells, 2) using genomic and promoter tiling arrays to measure sites of DNA binding of these factors and 3) determining the effects of knock-down on chromatin modification at target promoters. We will also use protein affinity chromatography to measure changes in association of other proteins with these factors during differentiation. Results in NB4 cells will be correlated with established murine models of myeloid differentiation and in differentiating primary marrow progenitors.

RNA表达模式的分析表明，在早幼粒细胞系分化为更成熟的中性粒细胞期间，40种或更多种转录因子一致地上调或下调。这些因子包括许多已经被认为在细胞增殖或髓样分化中起作用的因子，但也包括至少15种尚未在粒细胞成熟背景下研究的因子。几个这样的因素可能涉及在分化过程中的基因沉默，而其他可能有一个更普遍的作用，在细胞谱系的决定。我们建议采取基因组的方法来研究后者的因素在中性粒细胞和单核细胞分化的人NB4细胞系和正常中性粒细胞的作用。我们建议使用的方法包括1）研究RNAi敲除对分化的NB 4细胞的形态和功能的影响，2）使用基因组和启动子平铺阵列来测量这些因子的DNA结合位点，以及3）确定敲除对靶启动子处的染色质修饰的影响。我们还将使用蛋白质亲和层析来测量分化过程中其他蛋白质与这些因子的相关变化。NB4细胞中的结果将与建立的髓系白血病小鼠模型相关。 分化和分化初级骨髓祖细胞。