GENOMIC APPROACHES TO MYELOID SPECIFICATION

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的过程中，四十种或更多种转录因子持续上调或下调。其中包括许多已被认为在细胞增殖或骨髓分化中起作用的因素，但也包括至少十五个尚未在粒细胞成熟背景下研究的因素。一些这样的因素可能与分化过程中的基因沉默有关，而其他因素可能在细胞谱系决定中发挥更普遍的作用。我们建议采用基因组方法来研究后者因素在人类 NB4 细胞系的中性粒细胞和单核细胞分化以及正常中性粒细胞中的作用。我们建议使用的方法包括 1) 研究 RNAi 敲低对分化 NB4 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，3) 确定敲低对目标启动子处染色质修饰的影响。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关联变化。 NB4 细胞的结果将与已建立的小鼠骨髓模型相关 分化和分化原代骨髓祖细胞。

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.