The Role of CCAAT Displacement Protein in Neutrophil-Specific Gene Expression.

Abstract

CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε)and its downstream targets the neutrophil secondary granule proteins (SGPs), including lactoferrin (LF). We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Moreover, LF is not expressed in NB4 cells, a human acute promyelocytic cell line which contains the t(15;17) PML-RARα translocation. CDP has been found to bind persistently to the LF promoter in NB4 cells after morphological differentiation following ATRA induction. Several attempts at generating a CDP knockout mouse have been performed, but all have produced incomplete knockouts. We have generated short hairpin RNA (shRNA) constructs to knock down mouse CDP in myeloid cells by RNA interference. Single cell clones stably expressing the mouse shRNA have been generated in 32Dwt18 cells, and have been shown to decrease CDP expression. Knockdown of CDP does not change the phenotype of the cells, which remain IL3 dependent and undergo phenotypic maturation upon G-CSF induction. However, increased LF gene expression can be seen in uninduced cells expressing CDP shRNA, and the level of LF expression in single cell clones expressing the shRNA for CDP appears to correlate with the level of CDP repression. Control clones do not express LF until induced with G-CSF for several days. The knockdown of CDP does not appear to affect the expression of C/EBPε, suggesting that LF expression reflects direct modulation of CDP binding to its promoter and is not an indirect effect of increased C/EBPε expression. This suggests that CDP can function as the sole negative regulatory element for LF gene expression, and that relief of CDP repression can increase LF expression independent of increased binding of positive regulatory factors.