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.

Purification of PO-B, a protein that has increased affinity for the pro-opiomelanocortin gene promoter after dephosphorylation.

The region -15 to -3 of the pro-opiomelanocortin (POMC) gene promoter specifically binds a transcription factor previously designated PO-B. This region of the POMC gene is involved in the control of constitutive POMC gene expression since mutation of the PO-B DNA-binding site severely reduces transcription from the POMC promoter both in vivo and in vitro (Riegel, A. T., Remenick, J., Wolford, R., Berard, D., and Hager, G. (1990) Nucleic Acids Res. 18, 4513-4521). We have now purified PO-B from HeLa cells approximately 25,000-fold to greater than 90% homogeneity by a combination of ion exchange and reversed phase chromatography. In addition we have studied post-translational modifications that alter the affinity of purified PO-B for its cognate DNA binding site. In Southwestern analysis of column fractions, two bands of apparent molecular masses of 54 and 56 kDa bound specifically to the PO-B recognition sequence. The two copurified components have indistinguishable amino acid composition, are highly hydrophobic, and are heat and acid stable. DNA-binding specificity studies suggest that PO-B does not represent any previously described transcription factor. In addition, dephosphorylation of both species with acid phosphatase induced an about 30-fold increase in DNA binding but failed to produce any significant change in electrophoretic mobility. We conclude that the purified PO-B species represent products of the same gene and suggest that the in vivo function of PO-B may be regulated by its phosphorylation status.