Repressor Activity of CCAAT Displacement Protein in HL-60 Myeloid Leukemia Cells

Abstract

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…Identifying the precise mechanism by which CKLiK interferes with neutrophil gene expression will require more detailed analyses, but CKLiK may target a repressor of neutrophil differentiation, such as the CCAAT displacement protein (CDP/cut) or the transcription factor PU.1. Both factors can repress neutrophil gene expression, and their activities have been suggested to be modulated by serine phosphorylation [24,[37][38][39][40][41]. Interestingly, CDP represses the expression of C/EBPε, lactoferrin and gp91 phox , all three of which were inhibited in cells expressing the constitutively active CKLiK.…”

CUX1, a haploinsufficient tumour suppressor gene overexpressed in advanced cancers

“…On the basis of reporter assays and in vitro DNA binding assays, early studies described p200 CUX1 as a transcriptional repressor that functions in myeloid precursor cells to downregulate the expression of genes that become expressed only in terminally differentiated cells [29][30][31][32][33] . However, it has not been possible to confirm the recruitment of p200 CUX1 to specific genomic sites in vivo using chromatin immunoprecipitation, because it is very difficult to immunoprecipitate the full-length CUX1 protein following cross-linking (R. Harada and A.N., unpublished observation).…”

CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland

“…The binding conditions used for DNase I footprinting experiments were the same as those described for EMSAs, except that samples contained purified, bacterially expressed CDP representing the C-terminal two-thirds of the protein (CR2-Cterm) (39). Full-length CDP is not soluble.…”

“…Single cells were obtained with trypsin, growth medium was added, and the cell count was determined. For transient transfections, cells were pelleted and resuspended to 10 7 /200 l in medium (lacking FBS) containing 40 g of pLC-LUC DNA (13) and 40 g of DNA with various ratios of a CDP expression vector (pRc/CMV CDP) (39) or an expression vector lacking the CDP insert (pcDNA3) (Invitrogen, Carlsbad, Calif.). To normalize for DNA uptake in transient-transfection assays, 5 g of pRSV/lacZ or up to 0.7 g of pRL-TK (Promega, Madison, Wis.) was added to all samples in the same transfection.…”