The Myeloid-Cell-Specific c-fes Promoter Is Regulated by Sp1, PU.1, and a Novel Transcription Factor

Abstract

The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5′-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA elements within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3′-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (–408 to –386, –293 to –254, –76 to –65, and –34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5′-flanking sequences confer myeloid-cell-specific gene expression. Electro-phoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3′ of the PU.1/Elf-1 site and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3′ site (FP4-3′) within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assays demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.