z-logo
open-access-imgOpen Access
Cloning of the Novel Human Myeloid-Cell-Specific C/EBP-ε Transcription Factor
Author(s) -
Alexey M. Chumakov,
I Grillier,
E. A. Chumakova,
Doris Y. Chih,
Jacquelyn S. Slater,
H. Phillip Koeffler
Publication year - 1997
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.17.3.1375
Subject(s) - biology , microbiology and biotechnology , transcription factor , enhancer , gene , genetics , promoter , complementary dna , gene expression
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.

The content you want is available to Zendy users.

Already have an account? Click here to sign in.
Having issues? You can contact us here