Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor

Mol Cell Biol. 1997 Mar;17(3):1375-86. doi: 10.1128/MCB.17.3.1375.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetyltransferases*
  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Base Sequence
  • CCAAT-Enhancer-Binding Proteins*
  • Cell Line
  • Cloning, Molecular
  • Exons / genetics
  • Gene Expression Regulation*
  • Genes / genetics*
  • Granulocytes*
  • HL-60 Cells
  • Humans
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Peroxidase / genetics
  • Phosphoproteins / genetics
  • Promoter Regions, Genetic / genetics
  • Proteins / genetics
  • RNA, Messenger / analysis
  • RNA, Messenger / genetics
  • Recombinant Fusion Proteins
  • Sequence Analysis, DNA
  • Transcription Factors / chemistry
  • Transcription Factors / genetics*
  • Transcription Factors / physiology
  • Transcription, Genetic / genetics
  • Transcriptional Activation

Substances

  • CCAAT-Enhancer-Binding Proteins
  • Cebpe protein, mouse
  • Phosphoproteins
  • Proteins
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Transcription Factors
  • CEBPE protein, human
  • Peroxidase
  • Acetyltransferases
  • mim-1 protein, Gallus gallus

Associated data

  • GENBANK/U80982