CCAAT/enhancer binding protein epsilon is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing

Abstract

CCAAT/enhancer binding protein (C/EBP) epsilon is a recently cloned member of the C/EBP family of transcription factors and is expressed exclusively in cells of hematopoietic origin. The human C/EBPepsilon gene is transcribed by two alternative promoters, Palpha and Pbeta. A combination of differential splicing and alternative use of promoters generates four mRNA isoforms, of 2.6 kb and 1.3-1.5 kb in size. These transcripts can encode three proteins of calculated molecular mass 32.2 kDa, 27.8 kDa, and 14.3 kDa. Accordingly, Western blots with antibodies specific for the DNA-binding domain, that is common to all forms, identify multiple proteins. C/EBPepsilon mRNA was greatly induced during in vitro granulocytic differentiation of human primary CD34(+) cells. Retinoic acid treatment of HL60 promyelocytic leukemia cells for 24 hr induced C/EBPepsilon mRNA levels by 4-fold, while prolonged treatment gradually reduced mRNA expression to pretreatment levels. Transient transfection experiments with expression vectors for two of the isoforms demonstrated that the 32.2-kDa protein is an activator of transcription of granulocyte colony-stimulating factor receptor promoter, while the 14.3-kDa protein is not. Thus, C/EBPepsilon is regulated in a complex fashion and may play a role in the regulation of genes involved in myeloid differentiation.

Figure 1

Modular structure of the C/EBPɛ gene and its distinct RNA transcripts. (A) The transcription start sites of each of the two promoters, Pα and Pβ, are indicated by arrows. Boxes represent coding regions and thick lines represent untranslated regions of the mature mRNA. Nos. 1–5 correspond to primers RY46, RY44, RY66, RY68, and RY77, respectively. The location of the relevant start codons for each transcript and the common TGA stop codon is also shown. Vertical lines in the bZIP region indicate the presence of the leucine repeat. (B) RT-PCR reaction products amplified by the combination of C/EBPɛ specific primers indicated for each panel were analyzed by Southern blotting, using an internal primer as a probe (primer 4, A). All products were verified by sequence. β-Actin specific primers were used as controls. Fragment sizes are shown on the right, and the name of each transcript is at the left. The combination of primers 1 and 5 in primary T cells and macrophages amplifies a crossreacting product that is not related to epsilon based on sequence analysis of this fragment (Middle). A Pα1 RT-PCR-generated fragment of 1,826 bp is detected with longer exposure times in neutrophils (not shown).

Figure 3

Three proteins with identical bZIP domains are encoded by the C/EBPɛ gene. (A) Western blot analysis of nuclear extracts from cell lines. Equivalent amounts of cell extracts from each of the cell lines shown were loaded. Molecular mass markers are shown on the left side. (B) The three proteins C/EBPɛ³², C/EBPɛ²⁷, and C/EBPɛ¹⁴ are shown. Boxed residues represent identical amino acids. • indicate the position of each leucine residue of the zipper.

Figure 2

Sequence of the two putative human C/EBPɛ promoter regions. The Pβ region is the major promoter used in HL60 cells, because the majority of the clones (>60%) identified by RACE–PCR were initiated at this site. The transcriptional start sites of the C/EBPɛ gene were determined by 5′-RACE–PCR and sequence analysis of several independent clones. Large arrows indicate sequence ends of the majority of cDNA clones for each promoter region. Small arrows show ends of less frequent RACE–PCR clones. The translation initiation codon is shown in uppercase and bold characters. Purine stretches are boxed.

Figure 4

(A) C/EBPɛ mRNA is preferentially expressed in granulocytic cells. Primary human CD34⁺ cells were enriched from peripheral blood of healthy donors and plated in medium supplemented with GM-CSF and M-CSF (GM + M; lanes 4–5), G-CSF (G; lanes 6–7), or erythropoietin (Epo, lanes 8–9). Northern blot was performed after 8 days (lanes 4, 6, 8) and 11 days (lanes 5, 7, 9) of culture. Lane 1, KG1a cells; lane 2, enriched CD34⁺ cells (83% CD34⁺); lane 3, primary CD34⁺ cells (CD34⁺) used for in vitro differentiation (lanes 4–9); lane 10, HL60 cells. (Upper) Hybridization to a C/EBPɛ-specific probe. (Lower) The blot was stripped and hybridized to a 28S RNA oligonucleotide control probe. (B) Mature human neutrophils express the long isoform of C/EBPɛ. Human monocytes and neutrophils were purified from peripheral blood of healthy donors. HeLa cells (lane 1), human monocytes (lane 2), human neutrophils (lane 3), and HL60 cells (lane 4) are shown. Migration of the 28S and 18S ribosomal RNA is shown to the left. (C) C/EBPɛ is up-regulated during granulocytic differentiation of human leukemic cell lines. K562 cells were cultured in medium supplemented with 1.5% DMSO (+DMSO) for 5 days to induce erythrocytic differentiation. U937 and HL60 cells were treated for 2 days with 1.3 × 10⁻⁷ M TPA (+TPA) for monocytic differentiation or with 10⁻⁶ M retinoic acid (+RA) for 4 days for granulocytic differentiation. Northern blot was performed at time points indicated above each lane, as described above with the exception that RNA was normalized to 18S ribosomal RNA (Lower).

Figure 5

Activation of G-CSF receptor promoter-luciferase reporter construct by C/EBPα and C/EBPɛ in cotransfected HeLa cells. The G-CSF receptor-luciferase wild type (WT) and mutant (Mut) have been described (24). Increasing amounts of expression plasmids for C/EBPɛ or 0.5 μg of C/EBPα were cotransfected with a constant amount (5 μg) of both G-CSF receptor-luciferase reporter plasmids and 1 μg of β-galactosidase (pCMVβ) reporter. The amount of transfected DNA was adjusted with vector DNA to 10 μg per dish. The values are presented as a percentage of fold transactivation over the reporter. The experiments were repeated at least four times.