DNase I hypersensitivity analysis of the human CCAAT enhancer binding protein epsilon (C/EBPepsilon) gene.

Human C/EBPepsilon is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue-specific manner; it is expressed almost exclusively in myeloid cells. To understand the mechanism by which the expression of C/EBPepsilon gene is controlled, we cloned a large genomic region surrounding the C/EBPepsilon gene and performed a DNase I hypersensitivity analysis of this locus. These sites probably represent areas of binding of proteins modulating gene transcription. Hypersensitive (HS) regions in 30 kb of DNA surrounding the C/EBPepsilon gene were examined in C/EBPepsilon high-expressing (NB4, HL-60), low-expressing (Jurkat), very-low-expressing (KG-1), and non-expressing (K562) hematopoietic cells as well as in non-hematopoietic-non-expressing cells (MCF-7, DU 145, PC-3). Three HS sites were detected near the first exon of C/EBPepsilon gene. They were found only in hematopoietic cells and were especially prominent in C/EBPepsilon expressing cells, suggesting that these sites play an important role in transcribing the gene. These hypersensitive bands did not change when the cells were cultured with retinoids. Gel-shift assays using 200 bp of nucleotide sequences that encompassed the hypersensitive sites and nuclear extracts from NB4 and Jurkat cells (C/EBPepsilon expressing) as well as K562 and MCF-7 cells (non-expressing) showed different retarded bands on gel electrophoresis. A fourth HS site, located about 11 kb upstream of exon 1, was found only in cells highly expressing C/EBPepsilon. Two sites, one about 4.5 kb upstream of exon 1 and another about 8.5 kb downstream of exon 2, were positive only in non-expressing cell lines, suggesting that repressors may bind in these areas. Taken together, we have found six specific DNase I hypersensitive sites in the region of C/EBPepsilon that may be involved in regulating transcription of this gene.

Representational difference analysis using myeloid cells from C/EBP epsilon deletional mice.

C/EBP epsilon is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue specific manner; it is expressed exclusively in myeloid cells. C/EBP epsilon-deficient mice developed normally but failed to generate functional neutrophils and eosinophils, and these mice died of opportunistic infections suggesting that C/EBP epsilon may play a central role in myeloid differentiation. To identify myelomonocytic genes regulated by the C/EBP epsilon gene, we performed representational difference analysis (RDA), a polymerase chain reaction (PCR)-based subtractive hybridization using neutrophils and macrophages from wild-type and C/EBP epsilon knockout mice. We identified a set of differentially expressed genes, including chemokines specific to myelomonocytic cells. Several novel genes were identified that were differentially expressed in normal myelomonocytic cells. Taken together, we have found several genes whose expression might be enhanced by C/EBP epsilon. (Blood. 2000;96:3953-3957)

CCAAT/enhancer binding protein epsilon is a potential retinoid target gene in acute promyelocytic leukemia treatment.

The CCAAT/enhancer binding protein epsilon (C/EBPepsilon) is a nuclear transcription factor expressed predominantly in myeloid cells and implicated as a potential regulator of myeloid differentiation. We show that it was rapidly induced in the acute promyelocytic leukemia (APL) cell line NB4 during granulocytic differentiation after exposure to retinoic acid (RA). Our data suggest that induction of C/EBPepsilon expression was through the retinoic acid receptor alpha (RARalpha) pathway. Reporter gene studies showed that C/EBPepsilon promoter/enhancer activity increased in a retinoid-dependent fashion via the retinoic acid response element (RARE) present in the promoter region of C/EBPepsilon. The RA-induced expression of C/EBPepsilon markedly increased in U937 myelomonoblasts that were induced to express promyelocytic leukemia/RARalpha (PML/RARalpha), but not in those induced to express promyelocytic leukemia zinc finger/RARalpha (PLZF/RARalpha). In retinoid-resistant APL cell lines, C/EBPepsilon either is not induced or is induced only at very high concentrations of RA (>/=10(-6) M). In addition, forced expression of C/EBPepsilon in the U937 myelomonoblastic leukemia cells mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b/CD66b expression, and induction of secondary granule protein expression. Our data strongly suggest that C/EBPepsilon is a downstream target gene responsible for RA-induced granulocytic differentiation of APL cells.

Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP epsilon).

Human C/EBP epsilon is a newly cloned gene coding for a CCAAT/enhancer binding protein that may be involved in the regulation of myeloid differentiation. Our studies showed that levels of C/EBP epsilon mRNA were markedly increased in NB4 cells (promyelocytic leukemia line), because they were induced by 9-cis retinoic acid (9-cis RA) to differentiate towards granulocytes. Accumulation of C/EBP epsilon mRNA occurred as early as 1 hour after exposure of NB4 cells to 9-cis RA (5 x 10(-7) mol/L); and at 48 hours, levels were increased by 5.1-fold. Dose-response studies showed that 10(-7) to 10(-6) mol/L 9-cis RA (12 hours) resulted in peak levels of C/EBP epsilon mRNA; but even 10(-10) mol/L 9-cis RA increased levels of these transcripts. NB4 cells pulse-exposed (30 minutes) to all-trans retinoic acid (ATRA), washed, and cultured (3 days) with either dimethylsulfoxide (DMSO) or hexamethylene bisacetamide (HMBA) had a prominent increase in levels of C/EBP epsilon mRNA and an increase in granulocytic differentiation, but exposure to either DMSO or HMBA alone had no effect on base levels of C/EBP epsilon and did not induce differentiation. Macrophage-differentiation of NB4 reduced levels of C/EBP epsilon mRNA. Nuclear run-off assays and half-life studies showed that accumulation of C/EBP epsilon mRNA by 9-cis RA was due to enhanced transcription. Furthermore, this C/EBP epsilon mRNA accumulation did not require synthesis of new protein factors because 9-cis RA induced C/EBP epsilon mRNA accumulation in the absence of new protein synthesis. ATRA also induced expression of C/EBP epsilon protein in NB4 cells, as shown by Western blotting. In contrast to the increase of C/EBP epsilon in 9-cis RA-mediated granulocytic differentiation, the DMSO-induced differentiation of HL-60 cells down the granulocytic pathway was associated with an initial reduction of C/EBP epsilon mRNA levels. In summary, we have discovered that expression of C/EBP epsilon mRNA is markedly enhanced as the NB4 promyelocytes are induced by retinoids to differentiate towards granulocytes. This induction of C/EBP epsilon mRNA expression is transcriptionally mediated and occurs in the absence of synthesis of additional protein factors. We suspect that the C/EBP epsilon promoter/enhancer contains a retinoic acid-response element that is directly stimulated by retinoids.