C/EBP-ε: Chromosomal mapping and mutational analysis of the gene in leukemia and preleukemia

doi:10.1016/S0145-2126(97)00072-6

Leukemia Research

Volume 21, Issue 9, September 1997, Pages 833-839

https://doi.org/10.1016/S0145-2126(97)00072-6 Get rights and content

Abstract

We and others have cloned a novel human gene CCAAT/enhancer-binding protein ε (C/EBP-ε) encoding a member of the C/EBP gene family. It is exclusively expressed in myeloid and T-lymphoid cells and appears to have an important role in inducing expression of several myeloid-specific genes. We used a polymerase chain reaction (PCR)-based technique to examine DMA from 93 hamster/human radiation hybrid clones in order chromosomally to map C/EBP-ε to 14q11.2 (between D14S264 and D14S275) which is telomeric to the T-cell receptor α and δ genes and centromeric to several other myeloid gene products including Cathepsin G (CTSG) and Chymase-1 (CMA1). To determine whether C/EBP-ε behaves as an altered tumor-suppressor gene, samples from patients with acute myelogenous leukemia (AML and myelodysplastic syndrome (MDS) evolving to AML were studied for loss of heterozygosity (LOH) using microsatellite sequences that we identified within 0.2 kb of the amino-terminus of the human C/EBP-ε, gene. Allelic loss of the C/EBP-ε gene was detected in four out of 20 (20%) evolving MDS cases and in none of the 17 AML and 17 T-cell leukemia cases. Mutational analysis of the gene was performed using PCR-SSCP on 37 AML and 40 MDS cases including those with LOH at the gene. No abnormalities were found suggesting that the altered gene in this region is not C/EBP-ε. Also, C/EBP-ε was examined by Southern blot analysis on DNA samples from 20 AML patients and 10 AML cell lines. No rearrangements or amplifications of the gene were detected. Taken together, we have mapped C/EBP-ε to 14q11.2, a region containing other myeloid and T-lymphoid specific genes. Furthermore, no structural alterations were detected in the C/EBP-ε gene.

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CMTM5 is located at 14q11.2, a locus containing multiple genes associated with various carcinomas. Studies have shown that the abnormalities in the genes on chromosome 14q11.2 cause generation or growth of various cancers, such as nasopharyngeal carcinoma [14], leukemia [15], glioblastoma [16] and meningioma [17]. Herein, we provided the first examination of the tumor suppressor effects of CMTM5 in oral squamous cell carcinoma.
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CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that have been implicated in diverse cellular functions such as cellular differentiation and proliferation, and inflammatory processes. C/EBPζ, also known as GADD153, CHOP10, and DDIT3 has been found associated with the development of myxoid liposarcoma and the progression of melanoma. To investigate the correlation of C/EBPζ transcript levels with the development of leukemia, samples from 187 patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) were examined for C/EBPζ mRNA using real-time quantitative PCR (RQ-PCR). RQ-PCR analysis demonstrated the median levels of C/EBPζ were significantly decreased in MDS, AML, and CML patients compared with normal controls (1.40, 0.96, 2.60 versus 14.69, P < 0.0001). Significant differences were also observed between patients with CML and with AML or MDS. These results suggest that the insufficient dosage of C/EBPζ might be involved in the development of leukemia.
CCAAT/enhancer binding proteins repress the leukemic phenotype of acute myeloid leukemia
2003, Blood
Citation Excerpt :
Point mutations in C/EBPα have been identified in acute myeloid leukemias (AMLs)6,7; the AML with maturation-associated translocation fusion protein AML1-ETO inhibits C/EBPα expression8 and activity9; and the chronic myelogenous leukemia (CML) fusion protein BCR-ABL inhibits C/EBPα expression.10 Although C/EBPε mutations were not identified in patients with AML,11 such mutations have been observed in human patients with neutrophil-specific granule deficiency.12,13 We have suggested that altered function of C/EBPα and C/EBPε might also be important in the pathogenesis of acute promyelocytic leukemia (APL).14,15
CCAAT/enhancer binding proteins (C/EBPs) are a family of factors that regulate cell growth and differentiation. These factors, particularly C/EBPα and C/EBPε, have important roles in normal myelopoiesis. In addition, loss of C/EBP activity appears to have a role in the pathogenesis of myeloid disorders including acute myeloid leukemia (AML). Acute promyelocytic leukemia (APL) is a subtype of AML in which a role for C/EBPs has been postulated. In almost all cases of APL, a promyelocytic leukemia–retinoic acid receptor α (PML-RARα) fusion protein is expressed as a result of a t(15;17)(q22;q12) chromosomal translocation. PML-RARα inhibits expression of C/EBPε, whereas all-trans retinoic acid (tRA), a differentiating agent to which APL is particularly susceptible, induces C/EBPε expression. PML-RARα may also inhibit C/EBPα activity. Thus, the effects of PML-RARα on C/EBPs may contribute to both the development of leukemia and the unique sensitivity of APL to tRA. We tested the hypothesis that increasing the activity of C/EBPs would revert the leukemic phenotype. C/EBPα and C/EBPε were introduced into the FDC-P1 myeloid cell line and into leukemic cells from PML-RARA transgenic mice. C/EBP factors suppressed growth and induced partial differentiation in vitro. In vivo, enhanced expression of C/EBPs prolonged survival. By using a tamoxifen-responsive version of C/EBPε, we observed that C/EBPε could mimic the effect of tRA, driving neutrophilic differentiation in leukemic animals. Our results support the hypothesis that induction of C/EBP activity is a critical effect of tRA in APL. Furthermore, our findings suggest that targeted modulation of C/EBP activities could provide a new approach to therapy of AML.
Supplemental figure. C/EBPs suppress growth of murine myeloid leukemia in vivo. Bone marrow and spleen cells of leukemic mice were harvested and transduced with retroviruses expressing MIG (control), hC/EBPα, or hC/EBPε. At 24 ours after the second round of infection, GFP⁺ cells were sorted and intravenously injected into sublethally irradiated (4.5 Gy) healthy FVB/N females. (A) PML-RARα leukemia #1111, 50,000 cells per animal. Control mice n = 5, C/EBPα mice n = 5, C/EBPε mice n = 4. (B) PML-RARα leukemia #1111, 25,000 cells per animal. Control mice n = 4, C/EBPα mice n = 5, C/EBPε mice n = 5. (C) PML-RARα leukemia #935, 50,000 cells per animal. Control mice n = 6, C/EBPa mice n = 6, C/EBPε mice n = 6. (A, B) For PML-RARα leukemia #1111 recipients of C/EBP transduced cells survived longer. This prolongation of survival approached statistical significance for C/EBPε when 50,000 cells were used (A, p=.06) and was marked in recipients of 25,000 C/EBPε transduced cells (B, mean increase in survival 26 days, median 23 days, p=.02). (C) Both C/EBPα and C/EBPε modestly prolonged the survival of mice that received PML-RARα leukemia #935 cells (C/EBPα: mean increase in survival 3 days, median 3 days, P =.003; C/EBPε: mean increase in survival 6 days, median 6.5 days, P =.00001).
Neutrophil-specific granule deficiency: Homozygous recessive inheritance of a frameshift mutation in the gene encoding transcription factor CCAAT/enhancer binding protein-ε
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Citation Excerpt :
The polymorphic CA-repeat microsatellite sequence is located 35-bp 3′ of the polyadenylation site of the C/EBPεgene (Figure 1B). For analysis, the microsatellite was amplified by PCR (as described above) with the use of the primers KO691 (5′-ggcaaagagggcaggacccagc-3′) and KO692 (5′-ggtgcagacctagccacatgc-3′) and an annealing temperature of 55°C.30 The reactions were denatured by heating at 95°C for 5 minutes and electrophoresed through a denaturing 8M-urea, 5% polyacrylamide gel.
Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder. The neutrophils of individuals with SGD display atypical bi-lobed nuclei, lack expression of all secondary and tertiary granule proteins, and possess defects in chemotaxis, disaggregation, receptor up-regulation, and bactericidal activity, resulting in frequent and severe bacterial infections. Previously, a homozygous mutation in theCCAAT/enhancer binding protein–ε (C/EBPε) gene was reported for one case of SGD. To substantiate the role of C/EBPε in the development of SGD and elucidate its mechanism of inheritance, the mutational status of the gene was determined in a second individual. An A-nucleotide insertion in the coding region of the C/EBPε gene was detected. This mutation completely abolished the predicted translation of all C/EBPε isoforms. Microsatellite and nucleotide sequence analyses of the C/EBPε locus in the parents of the proband indicated that the disorder may have resulted from homozygous recessive inheritance of the mutant allele from an ancestor shared by both parents. The mutant C/EBPε₃₂ protein localized in the cytoplasm rather than the nucleus and was unable to activate transcription. Consistent with this, a significant decrease in the levels of the messenger RNAs (mRNAs) encoding the secondary granule protein human 18-kd cationic antimicrobial protein (hCAP-18)/LL-37 and the primary granule protein bactericidal/permeability-increasing protein were observed in the patient. The hCAP-18 mRNA was induced by overexpression of C/EBPε₃₂ in the human myeloid leukemia cell line, U937, supporting the hypothesis that C/EBPε is a key regulator of granule gene synthesis. This study strongly implicates mutation of theC/EBPε gene as the primary genetic defect involved in the development of neutrophil SGD and defines its mechanism of inheritance.
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Original articleC/EBP-ε: Chromosomal mapping and mutational analysis of the gene in leukemia and preleukemia

Abstract

A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family

EMBO Journal

Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells

Genes Development

Molecular cloning of a transcription factor, AGP/EBP, that belongs to member of the C/EBP family

Molecular and Cellular Biology

LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein

Genes Development

Characterization of cDNA clones for human myeloperoxidase: predicted amino acid sequence and evidence for multiple mRNA species

Nucleic Acids Research

Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses

Genes Development

Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP

Genes Development

CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription

Genes Development

A family of C/EBP related proteins capable of forming covalently leucine zipper dimers in vitro

Genes Development

Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3

IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP

Cell

Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types

Genes Development

A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells

Blood

CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter

Molecular and Cellular Biology

Cloning of the novel human myeloid-specific C/EBP transcription factor (MEF)

Molecular and Cellular Biology

A novel human CCAAT/enhancer binding protein gene, C/EBP-ε, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor αδ locus

Genomics

A method for constructing radiation hybrid maps of whole genomes

Nature Genetics

Allelotype analysis of childhood acute lymphoblastic leukemia

Cancer Research

Absence of WAF1 mutations in a variety of human malignancies

Blood

Constitutive monocyte-restricted activity of NF-M, a nuclear factor that binds to a C/EBP motif

Journal of Immunology

Original article
C/EBP-ε: Chromosomal mapping and mutational analysis of the gene in leukemia and preleukemia

A novel human CCAAT/enhancer binding protein gene, C/EBP-ε, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor $α δ$ locus