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.
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