DNA binding of the transcription factor PO-B is regulated during differentiation of HL-60 cells.
We have previously purified a transcription factor, PO-B, whose DNA binding capacity is increased by dephosphorylation and which contributes significantly to the basal transcription of genes such as pro-opiomelanocortin (Wellstein A., et al., J. Biol. Chem., 266: 12234-12241, 1991). In the present study, we describe several new properties of PO-B which suggest that the function of this transcription factor is not confined to regulation of gene expression in the pituitary. Furthermore, we present the first evidence for a signal transduction pathway that modulates the interactions of PO-B with DNA. We detected PO-B DNA binding activity in a number of mammalian cell lines (HeLa, C127, and AtT-20). However, PO-B was undetectable in extracts from undifferentiated HL-60 (U-HL-60) and CV-1 cells. Further characterization of these PO-B-negative extracts, by mixing experiments with PO-B-positive extracts, revealed that the U-HL-60 extracts, but not CV-1, contained enzymatic activity capable of increasing the mobility of the PO-B-DNA complex on nondenaturing gels. Concomitantly, there was also a reduction in the overall amount of PO-B bound to its cognate element. Immunoprecipitation with an antiserum to the protein kinase ERK 1 removed the modulatory activity from the U-HL-60 extracts, as did incubation with an ERK substrate peptide. Whole cell extracts from HL-60 cells which had been treated for 96 h with the macrophage-differentiating phorbol ester 12-O-tetradecanoylphorbol-13-acetate contained no modulatory activity. Furthermore, PO-B could be detected in these extracts. We conclude that an ERK or ERK-regulated protein in U-HL-60 cellular extracts regulates PO-B DNA binding and that some portion of the increase in PO-B DNA binding during HL-60 differentiation may arise from alterations in this regulatory activity.
Department of Pharmacology, Georgetown University School of Medicine, Washington, DC 20007.
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Dobrenski
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Zeft
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Wellstein
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Riegel
D001483:Base Sequence; D002454:Cell Differentiation / Q000502:physiology; D002457:Cell Extracts / Q000032:analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism*; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D011494:Protein Kinases