Expression of Two Myeloid Cell-specific Genes Requires the Novel Transcription Factor, c-fes Expression Factor

The protein product of the c-fes proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and granulocytes). We have previously shown that 151 base pairs of c-fes5′-flanking sequences are sufficient for myeloid cell-specific expression and include functional binding sites for Sp1, PU.1, and a novel nuclear factor (Heydemann, A., Juang, G., Hennessy, K., Parmacek, M. S., and Simon, M. C. (1996) Mol. Cell. Biol.16, 1676–1686). This novel hematopoietic transcription factor, termed FEF (c-fes expression factor), binds to acis-acting element that is located at nucleotides −9 to −4 of the c-fes promoter between two Ets binding sites (at −19 to −15 and −4 to +1) which bind PU.1. We now show that a FEF binding site exists in the myeloid cell-specific regulatory region of a second gene, the −2.7-kilobase pair enhancer of chicken lysozyme. The lysozyme FEF site is immediately 5′ to a PU.1 site, analogous to their arrangement in the c-fes promoter, and allows the formation of a preliminary FEF consensus site, 5′-GAAT(C/G)A-3′. This consensus site does not match any sites for known transcription factors. Importantly, although PU.1 binds immediately 3′ of the FEF site in both the c-fes promoter and the chicken lysozyme enhancer (CLE), we show that they bind independently. The FEF sites are required for high levels of transcription by both the CLE and the c-fespromoter in transient transfection experiments. Importantly, elimination of the CLE FEF site abolishes all transcriptional activity of this enhancer element. Mutation of the adjacent PU.1 site in either the c-fes promoter or the CLE, reduces activity by approximately 50%. Therefore, transcription of both lysozyme andfes in myeloid cells requires FEF and PU.1. UV cross-linking experiments show that the FEF binding activity consists of a single 70-kDa protein in both human and murine cell lines. FEF binding activity is not affected by antibodies that specifically recognize a number of cloned transcription factors. Collectively, these data indicate that we have identified a novel transcription factor that is functionally important for the expression of at least two myeloid cell-specific genes.