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Repressor activity of CCAAT displacement protein in HL-60 myeloid leukemia cells
P M Lievens1, J J Donady, C Tufarelli
1Division of Pediatric Hematology/Oncology, Children's Hospital, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
Insights
CCAAT displacement protein (CDP)/cut represses the gp91-phox gene in myeloid leukemia cells. This transcriptional repressor selectively reduces gp91-phox mRNA induction during cell differentiation.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cell Differentiation
Background:
- CCAAT displacement protein (CDP)/cut functions as a transcriptional repressor.
- CDP/cut binding activity inversely correlates with gp91-phox mRNA expression in myeloid leukemia cells.
Purpose of the Study:
- To investigate if CDP/cut acts as a repressor of the gp91-phox gene.
- To elucidate the role of CDP/cut in myeloid cell differentiation and gene expression.
Main Methods:
- Transient transfection assays in 3T3 cells to assess promoter activity.
- Generation of HL-60 myeloid cell lines stably expressing CDP/cut cDNA.
- Northern blot analysis to quantify mRNA levels of gp91-phox, p47, and p67.
Main Results:
- The CDP/cut binding site from the gp91-phox promoter exhibited negative regulatory activity.
- Stable expression of CDP/cut in HL-60 cells selectively reduced gp91-phox mRNA induction.
- Morphological differentiation and induction of p47 and p67 mRNA were unaffected by CDP/cut expression.
Conclusions:
- CDP/cut acts as a specific repressor of the gp91-phox gene.
- CDP/cut's repressive function is selective and does not impact overall myeloid differentiation or other myeloid oxidase components.
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.