Constitutive monocyte-restricted activity of NF-M, a nuclear factor that binds to a C/EBP motif

In a search for monocyte-specific nuclear factors, we analyzed in human cells the promoter of the chicken myelomonocytic growth factor, a gene that, in the chicken, is expressed in myeloid and myelomonocytic cells. Reporter gene constructs were active in monocytic Mono Mac 6 cells and in monoblastic THP-1 cells but not in the hematopoietic stem cell line K562. When a region with homology to the sequence recognized by CAAT enhancer-binding proteins (C/EBP) was inactivated by site-directed mutagenesis, the reporter activity was reduced by a factor of 10. Multimers of this region, termed F, in front of a heterologous promoter were active in Mono Mac 6 and THP-1 cells but not in K562 cells, WIL2 B cells, BT20 mammary carcinoma cells, MelJuso melanoma cells, or SK-Hep-1 hepatoma cells. Gel shift analysis with the F oligonucleotide identified DNA-binding activity in monocytic Mono Mac 6, monoblastic THP-1, and myelomonocytic HL60 cells. No binding was detected in myelomonocytic RC2A cells, in myeloid KG-1 cells, or in the hematopoietic stem cell line K562. Furthermore, a panel of solid tumor cell lines, representing various tissues, were also negative. Stimulation by PMA could not induce this binding factor in any of the negative cell lines. Analysis of primary cells (granulocytes, T cells, monocytes, and alveolar macrophages) revealed binding activity only in monocytes and macrophages. This DNA-binding factor, termed NF-M, was found to consist of two molecules, of 50 and 72 kDa, as determined by affinity cross-linking. Binding of NF-M was competed by the region F oligonucleotide and by the C/EBP motif from the albumin enhancer but not by an AP-2 motif. These data suggest that NF-M is a member of the C/EBP family of nuclear factors. The monocyte-restricted activity of NF-M suggests that this nuclear factor may be involved in regulation of monocyte-specific genes.