REPRESSOR ACTIVITY OF CCAAT DISPLACEMENT PROTEIN IN HL-60 MYELOID-LEUKEMIA CELLS

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 pro moter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approa ches to ascertain whether CDP/cut serves as a repressor of gp91-phox g ene expression, First, we used transient transfection assays in 3T3 ce lls to demonstrate that the CDP/cut binding site from the gp91-phox pr omoter acts as a negative regulatory element in artificial promoter co nstructs, Second, we isolated a stable transformant of HL-60 myeloid c ells constitutively expressing transfected CDP/cut cDNA, Stable transf ormants carrying expression vector alone or expressing CDP/cut mRNA we re induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retin oic acid/dimethylformamide, Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosol ic components, p47 and p67, In the stable transformant expressing tran sfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whe reas morphologic differentiation and induction of mRNA for myeloid oxi dase components p47 and p67 were unaffected, These data provide persua sive evidence that CDP/cut acts to repress the gp91-phox gene.