Knock-Down of CCAAT Displacement Protein (CDP) Restores Specific Granule Protein (SGP) Gene Expression in NB4 Acute Promyelocytic Leukemia Cells.

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 involved in neutrophil maturation, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε ). It also represses expression of neutrophil secondary granule proteins (SGPs), including LF, which are downstream targets of C/EBPε . 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. Additionally, we have demonstrated that overexpression of 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. Conversely, knocking down CDP expression by RNA interference in 32D cells upregulates levels of LF mRNA in uninduced cells. NB4 cells are an acute promyelocytic cell line that contains the t(15;17) PML-RARα translocation and can be induced toward neutrophil maturation with all-trans-retinoic Acid (ATRA). We have previously demonstrated that NB4 cells uniformly fail to express LF and all the SGP genes upon ATRA induction. In further studies, we showed that this failure of expression occurs despite appropriate binding of C/EBP factors known to upregulate SGP expression. We further demonstrated persistent binding of CDP to the LF promoter in NB4 cells upon ATRA induction, suggesting that failure of LF expression was due to persistent binding of CDP rather than a failure of C/EBP transactivation. In order to test this hypothesis, we knocked down CDP expression in NB4 cells using short hairpin RNA (shRNA) constructs and examined the effect of reduced CDP levels on SGP expression. ShRNA CDP constructs were introduced into NB4 cells by nucleofection using an Amaxa® nucleofector. Half the transfected cells were induced with ATRA, while the other half remained uninduced. After 24-hours of induction, total RNA was extracted from the cells and RTqPCR performed to assess the levels of SGP mRNA. We demonstrate that decreased CDP expression in ATRA- induced NB4 cells is sufficient to restore LF, collagenase, and gelatinase expression in the leukemic cell line. SGP expression remains absent in uninduced CDP-shRNA NB4 cells, as well as in both uninduced and induced cells transfected with an empty vector. Our observations reaffirm the critical role played by CDP in regulating the expression of LF and other SGP proteins during neutrophil development. How the PML-RARα gene product functions to alter CDP activity in NB4 cells at the molecular level, thereby restricting SGP expression, is currently being addressed.