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 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.
Lievens
P M
PM
Division of Pediatric Hematology/Oncology, Children's Hospital, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
Donady
J J
JJ
Tufarelli
C
C
Neufeld
E J
EJ
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D004274:DNA, Recombinant; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004126:Dimethylformamide / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D005914:Globins / Q000235:genetics; D018398:Homeodomain Proteins; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics*; D008264:Macrophages / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009504:Neutrophils / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors; D014170:Transformation, Genetic; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C080632:CUX1 protein, human; C000624101:Cux1 protein, mouse; D004274:DNA, Recombinant; D018398:Homeodomain Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D014212:Tretinoin; D004126:Dimethylformamide; D005914:Globins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide