title: Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor.
abstract: Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-specific genes in heterologous cell types, such as embryonic fibroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, 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 stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of primers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel 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 full-length human C/EBP-epsilon was cloned from a promyelocyte-late myeloblast-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 extension analysis of C/EBP-epsilon mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-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 Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression 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-epsilon (amino acids 1 to 115) showed that C/EBP-epsilon is a 32-kDa nuclear phosphoprotein. The human C/EBP-epsilon protein exhibited strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of the C/EBP-epsilon sense and antisense expression constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-specific c-mim and human myeloperoxidase promoters suggested a role for C/EBP-epsilon transcription factor in the regulation of a subset of myeloid-cell-specific genes. Transient tranfection of a promyelocyte cell line (NB4) with a C/EBP-epsilon expression plasmid increased cell growth by sevenfold, while antisense C/EBP-epsilon caused a fivefold decrease in clonal growth of these cells.
authors.affiliation: Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Chumakov
authors.forename: I
authors.initials: I
authors.lastname: Grillier
authors.forename: E
authors.initials: E
authors.lastname: Chumakova
authors.forename: D
authors.initials: D
authors.lastname: Chih
authors.forename: J
authors.initials: J
authors.lastname: Slater
authors.forename: H P
authors.initials: HP
authors.lastname: Koeffler
mesh_terms: D000123:Acetyltransferases; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes / Q000235:genetics*; D006098:Granulocytes; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009195:Peroxidase / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; C489468:Cebpe protein, mouse; D010750:Phosphoproteins; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C070790:CEBPE protein, human; D009195:Peroxidase; D000123:Acetyltransferases; C077509:mim-1 protein, Gallus gallus