Regulation of AP‐3 enhancer activity during hematopoietic differentiation

Abstract

Phorbol ester treatment of the human leukemic cell line U937 induces macrophage differentiation over 24-48 hr. This differentiation is mediated by the activation and/or repression of specific gene transcription by proteins, enhancer binding factors, that bind to the DNA upstream of the start site of transcription. We find that differentiation of U937 cells induced by phorbol esters and bryostain 1, activators of protein kinase C, and the phosphatase inhibitor, okadaic acid, stimulates transcription from an enhancer sequence which contains multimerized AP-3 binding sequences but not from one that contains multimerized AP-2 binding motifs. Electrophoretic mobility shift assays (EMSA) demonstrate that AP-3 DNA binding activity peaks at 24 hr, remains elevated for 24 hr, and then decreases thereafter. Southwestern blotting demonstrates that the AP-3 enhancer sequence binds to a 48 kDa protein present in these leukemic cells. Because the AP-3-oligomer also contains an overlapping NF-kappa B-like site, the role of NK-kappa B proteins in regulating transcription from this multimerized oligonucleotide was investigated. Transfection of U937 cells with NF-kappa B family members demonstrated activation of AP-3-mediated transcription by rel A but little effect induced by NFKB1 and c-rel. It is unlikely, however, that phorbol ester-induced transcription from this AP-3 sequence is solely mediated by this NF-kappa B family member since treatment of U937 cells with antisense rel A oligodeoxynucleotides did not block phorbol ester-mediated transcription from the AP-3 site. These data demonstrate that AP-3, but not AP-2 sequences, functions to activate mRNA transcription during phorbol ester-induced hematopoietic differentiation and suggests a complex interaction between NF-kappa B and AP-3 proteins in the regulation of this enhancer element.

A Novel Transcription Factor Regulates Expression of the Vacuolar H+-ATPase B2 Subunit through AP-2 Sites during Monocytic Differentiation

“…AP-2 binding sites were found to be required for tumor necrosis factor ␣ expression induced by granulocyte-macrophage colony-stimulating factor but not by phorbol ester (45). AP-2 binding sequences were also found not to have any enhancer activity for transcription of downstream reporters during phorbol ester-induced differentiation of U-937 cells (46). Although these findings appear to conflict with our results, we have shown that THP-1 cells closely mimic primary blood monocytes in their ability to express V-ATPase during differentiation, whereas U-937 cells do not (18).…”

Aging-related Deficiency of CD28 Expression in CD4+ T Cells Is Associated with the Loss of Gene-specific Nuclear Factor Binding Activity

“…Elk1 and Ets1 are structurally related proteins (34,35) that have been shown to play an important role in the expression of T cell-specific genes (36 -40). In contrast, AP3 and AP3-like factors are not known to mediate T cell-specific gene transcription, although they have been implicated in the differentiation of myeloid cells (41) and in the synthesis of an IgE-induced mast cell-derived cytokine (42).…”

“…However, it is also unlikely that the observed differential binding activities of site ␤ for nuclear extract from CD28ϩ and CD28 null CD4ϩ T cells represent an AP3 or an AP3-like factor. AP3 has not been documented to play any role in T cell-specific gene transcription, although it has been shown to be important in the differentiation of myeloid cells (41). An AP3-like transcription factor has also been implicated in the inducible expression of a novel mast cell-derived cytokine (42).…”