Granulocyte-colony stimulating factor (G-CSF) is an essential cytokine, which contributes to proliferation and differentiation of granulocyte precursor cells in the bone marrow. Despite recent progress in understanding G-CSF signaling events, the mechanisms that underlie the distinct spectrum of biological functions attributed to G-CSF-mediated gene expression remain unclear. Previous studies have identified a number of genes, which are up-regulated in G-CSF-stimulated myeloid precursor cells. In this study, we sought to identify additional target genes of G-CSF-mediated proliferation and/or differentiation. cDNA representational difference analysis was used with the 32Dcl3 cell line as a model system to isolate genes, which are up-regulated in an immediate-early manner upon G-CSF stimualtion. We isolated p120 nucleolar-proliferation antigen (NOL1), a highly conserved, nucleolar-specific, RNA-binding protein of unknown function, and confirmed its expression by Northern blot analysis in 4-h, G-CSF-induced 32Dcl3 cells. Isolation of a mouse p120 genomic clone revealed the presence of a signal tranducer and activator of transcription (STAT)-binding site in the first intron of the gene. We demonstrate the importance of STAT3 and STAT5 in mediating the G-CSF response with respect to p120 expression by transient transfection analysis, oligonucleotide pull-down assays, and the loss of p120 expression in the bone marrow of mice lacking normal STAT3 signaling. In addition, overexpression of p120 in G-CSF-induced 32D cells revealed normal, morphologic maturation and growth characteristics but loss of lactoferrin expression, a marker of normal neutrophil maturation, suggesting that inappropriate expression of the p120 gene can result in aberrant neutrophil maturation.
- Representation difference analysis (RDA)