CLONING OF THE NOVEL HUMAN MYELOID-CELL-SPECIFIC C EBP-EPSILON TRANSCRIPTION FACTOR/

Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-spec ific genes in heterologous cell types, such as embryonic fibroblasts. In humans, similar effects were observed with homologous members of th e CCAAT/enhancer-binding protein (C/EBP) family of transcriptional reg ulators, especially the human homolog of chicken NF-M, C/EBP-beta (NF- IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inflammatory stim uli, making it an unlikely candidate to have an exclusive role as a co mbinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of prim ers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a no vel human gene encoding a member of the C/EBP gene family, identified as the human homolog of CRP1, C/EBP-epsilon. A 1.2-kb cDNA encoding fu ll-length human C/EBP-epsilon was cloned from a promyelocyte-late myel oblast-derived lambda gt11 library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer ext ension analysis of C/EBP-epsilon mRNA detected a single major transcri ption start site approximately 200 bp upstream of the start codon, The putative promoter area is similar to those of several other myeloid-c ell-specific genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the factors of the Et s family of transcriptional regulators. Northern blot analyses indicat ed a highly restricted mRNA expression pattern,,vith the strongest exp ression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipitation studies using rabbit anti-C/EBP- epsilon antibodies raised against the N-terminal portion of C/EBP-epsi lon (amino acids 1 to 115) showed that C/EBP-epsilon is a 32-kDa nucle ar phosphoprotein. The human C/EBP-epsilon protein exhibited strong an d specific binding to double-stranded DNA containing consensus C/EBP s ites. Cotransfection of the C/EBP-epsilon sense and antisense expressi on constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-specific c-mim and human myeloperoxid ase promoters suggested a role for C/EBP-epsilon transcription factor in the regulation of a subset of myeloid-cell-specific genes. Transien t tranfection of a promyelocyte cell line (NB4) with a C/EBP-epsilon e xpression plasmid increased cell growth by sevenfold, while antisense C/EBP-epsilon caused a fivefold decrease in clonal growth of these cel ls.