Binding Protein Epsilon Research Articles

Analysis of telomerase activity and RNA expression in a patient with acute promyelocytic leukemia treated with all‐trans retinoic acid

In this study, we show that all-trans retinoic acid (ATRA) treatment leads to a rapid decrease in telomerase activity, which was associated with the reduction in myeloblasts and occurs before the appearance of myelocytes, in a patient with acute promyelocytic leukemia (APL). Microarray analysis by ATRA treatment for 48 hr in peripheral blood mononuclear cells (in vivo) and in cultured bone marrow mononuclear cells (in vitro) from a patient with APL revealed upregulation of CD11b, CD11c, CCAAT enhancer binding protein epsilon, Rb1, Mad, and tumor necrosis factor-related genes; and downregulation of hTERT, c-Myc, WT1, bcl-2, and eukaryotic translation elongation factor 1alpha2. The results might offer the potential to define the molecular mechanism underlying ATRA-induced granulocytic differentiation in patients with APL, and provide clues to identify novel molecular therapeutic targets.

Protein partners of C/EBPε

CCAAT-enhancer binding protein-epsilon (C/EBPepsilon) is a nuclear transcription factor implicated in the regulation of terminal myeloid differentiation. Using a yeast two-hybrid screen, potential interaction partners of C/EBPepsilon involved in myeloid development were identified. C/EBPepsilon was found to associate with other C/EBP family members, including C/EBPepsilon and CHOP as well as other proteins that are known to contain a leucine-zipper protein interaction motif including CREB2, LDOC1, E6TP1, and AF-17. In addition, C/EBPepsilon demonstrated the potential for interaction with proteins that do not possess a leucine-zipper motif, including proteins that may be involved in sumoylation (protein inhibitor of activated STAT1 [PIAS1] and ubiquitin-conjugating enzyme E2I). As expected, the association of C/EBPepsilon with other C/EBP family members depends on the presence of a functional leucine-zipper motif. Mapping studies of C/EBPepsilon with PIAS1 (as an example of a nonleucine-zipper-containing protein) showed that C/EBPepsilon interacts with the amino-terminal domain of PIAS1. The function of C/EBPepsilon interacting proteins was further investigated. Co-expression of C/EBPepsilon with C/EBPdelta resulted in potent transactivation in a lactoferrin reporter system. A gel mobility shift assay suggests that C/EBPepsilon, C/EBPalpha, and C/EBPdelta proteins can bind as heterodimers to a C/EBP consensus DNA-binding site. As CHOP is known to represent a transcriptional repressor, the functional interaction between C/EBPepsilon and CHOP was investigated. Co-expression of C/EBPepsilon and c-Myb with CHOP caused marked transcriptional repression of target reporter genes. Our results suggest heterodimeric partners of C/EBPepsilon modulate the function of C/EBPepsilon in mediating gene transcription during myelopoiesis.

CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation, and Does Not Require but Is Enhanced by DNA-Binding.

The CCAAT/enhancer binding protein epsilon (C/EBPε) is critical for the terminal differentiation and lineage-specific gene expression of granulocytes, and expression of C/EBPε32 and its shorter 27 kD and 14 kD isoforms is developmentally regulated during neutrophil granulocyte differentiation. We have defined a novel role for the unique 27 kD isoform (C/EBPε27) as a potent antagonist of GATA-1-mediated transactivation of the promoter of the gene encoding the eosinophil secondary granule protein, major basic protein (MBP) (Du et al, J. Biol. Chem. 2002; 277:43481–43394). We also showed that these two transcription factors physically interact in eosinophil cell lines in vivo. In the present studies, we performed the first structure-function analyses of the C/EBPε27 isoform to map its potent repressor domains, with comparisons to the C/EBPε32 and C/EBPε14 isoforms, using transactivation assays of the MBP P2 promoter in the presence of GATA-1. Our results show that the repression of GATA-1 is mediated in part by the unique N-terminus of C/EBPε27 (not shared with other C/EBPε isoforms) in combination with part of a previously identified RDI domain (shared with full length C/EBPε32). We show further that this repressor activity is independent of DNA binding (via deletion of the basic region of C/EBPε27) as well as of sumoylation of the RDI “VKEEP” sumoylation consensus site present in both the C/EBPε32 and C/EBPε27 isoforms, and conserved in the C/EBPε proteins of many other species. Thus, our findings identify the unique N-terminus of the C/EBPε27 isoform, a distinct 68 amino acid sequence not shared with any other C/EBPε isoforms or other C/EBP family members, as the minimum repressor domain required for potent antagonism of GATA-1 activity. Of interest, fusion of this novel 68 amino acid sequence to the N-terminus of full length C/EBPε32 converted it into a partial repressor of GATA-1, but did not alter the transactivation potential of the C/EBPε32 isoform itself. The mechanism for maximal C/EBPε27 attenuation of GATA-1 activity requires a combination of both GATA-1-C/EBPε27 protein-protein interaction and C/EBPε27 binding to the proximal C/EBP consensus site immediately upstream in the target promoter. Neither C/EBPε32 nor C/EBPε14 inhibited C/EBPε27 antagonism of GATA-1, supporting a protein-protein interaction mechanism for its repressor activity that is enhanced by, but does not require, DNA binding to a proximal C/EBP site. Expression of the C/EBPε27 isoform likely serves to titrate and/or turn off expression of secondary granule protein genes such as MBP during eosinophil terminal differentiation, when these genes are ultimately silenced in the mature cell. These studies illustrate the unique regulatory (activating versus repressor) activities for the various C/EBPε isoforms, activities consistent with their developmentally regulated expression and lineage-specific activities during granulocyte (both neutrophil and eosinophil) differentiation.

Growth Factor Independence 1 (Gfi-1) Plays a Role in Mediating Specific Granule Deficiency (SGD) in a Patient Lacking an Abnormality in the C/EBPε Gene.

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype as SGD patients and prompted examination of the human C/EBPε gene for mutation in this disease. Mutations in the C/EBPε gene have been identified by others in two patients with SGD, resulting in loss of gene expression. However, other patients with a similar disease phenotype have a normal C/EBPε coding sequence, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a C/EBPε mutation demonstrated an elevated level of the C/EBPε protein in the patient's peripheral blood neutrophils as compared to the level in normal control neutrophils. Microarray analysis of this patient's bone marrow compared with that of a normal control revealed, among other genes, elevated levels of the transcription factors C/EBPε and PU.1. As a consequence, several PU.1 target genes showed increased expression in the SGD patient bone marrow. This observation was confirmed by both real time PCR and western blot analysis. PU.1 is a hematopoietic-specific transcription factor belonging to the Ets family of DNA binding proteins and plays a critical role in B-cell, macrophage and late stage neutrophil development. Sequence analysis of the PU.1 gene from our SGD patient however, did not reveal any mutation in the coding region of the gene. Western blot analysis of nuclear extracts prepared from peripheral blood neutrophils from this patient did however reveal significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1), although no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. Gfi-1 belongs to a family of zinc finger containing transcriptional repressor oncoproteins. Mice lacking Gfi-1 were found not only to be neutropenic, but also demonstrated defects in neutrophil differentiation, including loss of neutrophil secondary and tertiary granule proteins, reminiscent of SGD. More recently, heterozygous germline mutations of Gfi-1 were shown to cause severe congenital neutropenia (SCN) in humans. It has been suggested that Gfi1 represses neutrophil elastase (Ela2), germline mutations within which are a major contributor to hereditary neutropenias. Our data suggest that decreased levels of Gfi1 in our SGD patient result in increased levels of PU.1 and C/EBPε; an effect consistent with observations first made in the neutrophils of Gfi-1-null mice. The increased PU.1 levels might then act to sequester C/EBPε protein via direct protein-protein interaction. This in turn could explain the loss of secondary granule protein gene expression in the SGD patient by inducing functional C/EBPε deficiency.

The Role of CCAAT Displacement Protein in Neutrophil-Specific Gene Expression.

CCAAT displacement protein (CDP) is a highly conserved, ubiquitously expressed homeodomain protein with extensive homology to the Drosophila cut protein. CDP contains three highly conserved DNA-binding repeats called cut repeats, as well as a conserved homeodomain sequence. CDP is a transcriptional repressor of several developmentally regulated genes, including the phagocyte-specific cytochrome heavy chain gene, gp91-phox, and CCAAT enhancer binding protein epsilon (C/EBPε)and its downstream targets the neutrophil secondary granule proteins (SGPs), including lactoferrin (LF). We have previously shown that CDP binds to and represses both the C/EBPε and lactoferrin (LF) gene promoters thereby preventing expression of secondary granule proteins (SGPs) both directly and indirectly. CDP represses expression of SGPs in 32Dcl3 cells, an IL-3 dependent murine myeloid cell line that undergoes differentiation in response to IL-3 withdrawal and G-CSF stimulation. Moreover, LF is not expressed in NB4 cells, a human acute promyelocytic cell line which contains the t(15;17) PML-RARα translocation. CDP has been found to bind persistently to the LF promoter in NB4 cells after morphological differentiation following ATRA induction. Several attempts at generating a CDP knockout mouse have been performed, but all have produced incomplete knockouts. We have generated short hairpin RNA (shRNA) constructs to knock down mouse CDP in myeloid cells by RNA interference. Single cell clones stably expressing the mouse shRNA have been generated in 32Dwt18 cells, and have been shown to decrease CDP expression. Knockdown of CDP does not change the phenotype of the cells, which remain IL3 dependent and undergo phenotypic maturation upon G-CSF induction. However, increased LF gene expression can be seen in uninduced cells expressing CDP shRNA, and the level of LF expression in single cell clones expressing the shRNA for CDP appears to correlate with the level of CDP repression. Control clones do not express LF until induced with G-CSF for several days. The knockdown of CDP does not appear to affect the expression of C/EBPε, suggesting that LF expression reflects direct modulation of CDP binding to its promoter and is not an indirect effect of increased C/EBPε expression. This suggests that CDP can function as the sole negative regulatory element for LF gene expression, and that relief of CDP repression can increase LF expression independent of increased binding of positive regulatory factors.

Identification of murine and human XCP1 genes as C/EBP-epsilon-dependent members of FIZZ/Resistin gene family.

The CCAAT enhancer binding protein epsilon (C/EBP-epsilon) transcription factor is expressed predominantly in granulocytes. Mice with a disruption of the C/EBP-epsilon gene fail to produce mature granulocytes and eosinophils. Cells derived from the peritoneal exudates of C/EBP-epsilon -/- mice lack the expression of a number of chemokines and chemokine receptor genes. We have found a novel C/EBP-epsilon-dependent promyelocyte-specific gene, mXCP1. mXCP1 belongs to a family of XCP/FIZZ/Resistin genes, which includes four murine genes and two human genes, hXCP1 and hXCP2. These genes have four exons and encode short secreted proteins sharing a ten-cysteine motif. Murine mXCP1, mXCP2 and mXCP3 genes map to murine chromosome 16 and mXCP4 is positioned on chromosome 8; the hXCP1 and hXCP2 genes are located at homologous regions of chromosomes 3 and 19. Introduction of an inducible C/EBP-epsilon gene into the NIH3T3 and myeloid cells from C/EBP-epsilon-null mice line revealed that the conditional expression of C/EBP-epsilon induced mXCP1. The HXCP1 gene was identified as a C/EBP-epsilon-dependent regulatory homologue of mXCP1. The expression data for other members of XCP/FIZZ gene family are presented. Further studies indicate that XCP1 is a secreted protein that is chemotactic to myeloid cells from C/EBP-epsilon-null mice and is able to interact directly with alpha-defensin.

C/EBPϵ interacts with retinoblastoma and E2F1 during granulopoiesis

AbstractCCAAT enhancer binding protein epsilon (C/EBPϵ) is a myeloid specific transcription factor that is essential for terminal granulocytic differentiation. Retinoblastoma (Rb) and E2F1 are critical cell cycle regulators that also have been implicated in several differentiation systems. Here, we demonstrate that C/EBPϵ interacts with Rb and E2F1 during granulocytic differentiation in NB4 and U937 human myeloid cells and in 32Dcl3 murine myeloid precursor cells. The interaction between C/EBPϵ and Rb enhances C/EBPϵ-mediated transcription of myeloid specific genes both in reporter assays and endogenously. The C/EBPϵ-E2F1 interaction results in repression of E2F1-mediated transcriptional activity. Finally, overexpression of C/EBPϵ in human myeloid cells leads to down-regulation of c-Myc. We propose that the interactions between C/EBPϵ, a tissue-specific transcription factor, and the broad-spectrum proteins, Rb and E2F1, are important in C/EBPϵ-induced terminal granulocytic differentiation.

Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein

GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein epsilon (C/EBPepsilon) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBPepsilon isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBPepsilon(27) isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBPepsilon(27) are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBPepsilon(32/30) and epsilon(27) physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU.1, and C/EBPepsilon isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBPepsilon(27).

C/EBP epsilon mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut).

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBP epsilon) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBP epsilon activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBP epsilon(-/-) mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBP epsilon and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBP epsilon in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBP epsilon mRNA. We therefore hypothesize that C/EBP epsilon positively regulates SGP gene expression, and that C/EBP epsilon is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBP epsilon promoter is regulated by CDP/cut during myeloid differentiation.

The role of C/EBP(epsilon) in the terminal stages of granulocyte differentiation.

As a consequence of its characterization using both in vitro and knockout mouse models, the myeloid-specific transcription factor, CCAAT/enhancer binding protein (C/EBP)epsilon, has been identified as a critical regulator of terminal granulopoiesis and one of the causative mutations in the human disease, neutrophil-specific granule deficiency. C/EBPs are a family of transcription factors sharing numerous structural and functional features and to date include C/EBPalpha, -beta, -gamma, -delta, -epsilon, and -zeta. C/EBPalpha was the first family member isolated and characterized, its essential role in hepatocyte and adipocyte differentiation demonstrated in knockout mouse models. Subsequent analysis of the hematopoietic elements in fetal mouse liver revealed its critical role in myelopoiesis. Understanding the role of C/EBPepsilon in terminal granulopoiesis in the context of other known transcription factors is ongoing with analysis of deficient and conditionally expressing cell lines and knockout models. Mouse models with targeted gene disruptions have contributed greatly to our understanding of the transcriptional regulation of granulopoiesis. Further manipulation of these models and other conditional expression systems have bypassed some of the limitations of knockout models and helped delineate the interactions of different transcription factors in affecting granulocyte development. Phenotypic expression of the loss of C/EBPepsilon in mice is extreme, resembling absolute neutropenia with systemic infection with P. aeruginosa. Future work will need to explore the regulation of C/EBPepsilon expression, its functional interactions with other transcriptional regulators such as PU.1, and its role in monocyte differentiation and function in the mouse.

C/EBPepsilon -/- mice: increased rate of myeloid proliferation and apoptosis.

CCAAT/enhancer binding protein epsilon (C/EBPepsilon) is essential for terminal granulocytic differentiation. Its expression begins at the transition between the proliferative and non-proliferative compartments of myelopoiesis. We studied the effect of targeted disruption of the C/EBPepsilon gene on murine myeloid proliferation and apoptosis. Bone marrow cellularity of C/EBPepsilon -/- and wild-type mice was 95% and 65%, respectively. The C/EBPepsilon -/- mice had an expansion in the number of their CFU-GM/femur. The number of myeloid committed progenitor cells in the peripheral blood and the spleen of these mice was also increased. Bromodeoxyuridine (BrDU) pulse labeling studies demonstrated that the fraction of actively proliferating cells was two-fold higher in the bone marrow of C/EBPepsilon -/- mice. However, the number of myeloid colonies arising from purified Sca-1+/lin- early hematopoietic progenitor cells and from bone marrow mononuclear cells grown in different cytokine combinations was not significantly different between wild-type and knock-out mice. Also, long-term marrow growth, and CFU were not different between the wild-type and C/EBPepsilon -/- mice. The sensitivity to induction of apoptosis in the committed progenitor cell compartment after either withdrawal of growth factor or brief exposure to etoposide was normal. However, Gr-1 antigen-positive C/EBPepsilon -/- granulocytic cells showed an increased rate of apoptosis in comparison to their wild-type counterparts. In summary, the myeloid compartment appears to be expanded in mice lacking C/EBPepsilon. However, this is not the consequence of an intrinsic myeloproliferation but due to an indirect, possibly cytokine-mediated stimulation of myelopoiesis in vivo. C/EBPepsilon may have a role in the inhibition of apoptosis in maturing granulocytic cells.

AML1-MTG8 leukemic protein induces the expression of granulocyte colony-stimulating factor (G-CSF) receptor through the up-regulation of CCAAT/enhancer binding protein epsilon

The t(8;21) translocation is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). In this translocation, the AML1 (CBFA2/PEBP2aB) gene is disrupted and fused to the MTG8 (ETO) gene. The ectopic expression of the resulting AML1-MTG8 fusion gene product in L-G and 32Dcl3 murine myeloid precursor cells stimulates cell proliferation without inducing morphologic terminal differentiation into mature granulocytes in response to granulocyte-colony stimulating factor (G-CSF). This study found that the ectopic expression of AML1-MTG8 elevates the expression of the G-CSF receptor (G-CSFR). Analysis of the promoter region of the G-CSFR gene revealed that up-regulation of G-CSFR expression by AML1-MTG8 does not depend on the AML1-binding sequence, but on the C/EBP (CCAAT/enhancer binding protein) binding site. The results suggest that the overproduction of G-CSFR is at least partly mediated by C/EBPɛ, whose expression is activated by AML1-MTG8. The ectopic expression of G-CSFR in L-G cells induced cell proliferation in response to G-CSF, but did not inhibit cell differentiation into mature neutrophils. Overexpression of C/EBPɛ in L-G cells also stimulated G-CSF–dependent cell proliferation. High expression levels of G-CSFR were also found in the leukemic cells of AML patients with t(8;21). Therefore, G-CSF–dependent cell proliferation of myeloid precursor cells may be implicated in leukemogenesis.

AML1-MTG8 leukemic protein induces the expression of granulocyte colony-stimulating factor (G-CSF) receptor through the up-regulation of CCAAT/enhancer binding protein epsilon

AbstractThe t(8;21) translocation is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). In this translocation, the AML1 (CBFA2/PEBP2aB) gene is disrupted and fused to the MTG8 (ETO) gene. The ectopic expression of the resulting AML1-MTG8 fusion gene product in L-G and 32Dcl3 murine myeloid precursor cells stimulates cell proliferation without inducing morphologic terminal differentiation into mature granulocytes in response to granulocyte-colony stimulating factor (G-CSF). This study found that the ectopic expression of AML1-MTG8 elevates the expression of the G-CSF receptor (G-CSFR). Analysis of the promoter region of the G-CSFR gene revealed that up-regulation of G-CSFR expression by AML1-MTG8 does not depend on the AML1-binding sequence, but on the C/EBP (CCAAT/enhancer binding protein) binding site. The results suggest that the overproduction of G-CSFR is at least partly mediated by C/EBPɛ, whose expression is activated by AML1-MTG8. The ectopic expression of G-CSFR in L-G cells induced cell proliferation in response to G-CSF, but did not inhibit cell differentiation into mature neutrophils. Overexpression of C/EBPɛ in L-G cells also stimulated G-CSF–dependent cell proliferation. High expression levels of G-CSFR were also found in the leukemic cells of AML patients with t(8;21). Therefore, G-CSF–dependent cell proliferation of myeloid precursor cells may be implicated in leukemogenesis.

Inherited Neutrophil Disorders: Molecular Basis and New Therapies.

Recent advances in our understanding of the molecular basis of inherited neutrophil disorders and complementary studies in transgenic mouse models have provided new insights into the normal mechanisms regulating myelopoiesis and the functional responses of mature neutrophils. Neutrophil specific granule deficiency is a rare disorder of neutrophil function characterized by a lack of neutrophil secondary granule proteins and associated with recurrent bacterial infections. The CCAAT/enhancer binding protein (C/EBP) epsilon, a leucine zipper transcription factor expressed primarily in myeloid cells, and C/EBPepsilon-deficient mice generated by gene targeting lack specific granules and have impaired host defense are discussed by Dr. Lekstrom-Himes in Section I. The similarity between these phenotypes led to the identification of a loss of function mutation in the C/EBPepsilon gene in a subset of patients with specific granule deficiency. Dr. Dale reviews the clinical features and management of congenital neutropenia and cyclic hematopoiesis in Section II. Inherited mutations in the neutrophil elastase gene have recently been identified in both disorders. Specific mutations identified in cyclic and congenital neutropenia are described along with possible mechanisms for regulation of hematopoiesis by neutrophil elastase. In Section III, Dr. Dinauer reviews the molecular genetics of chronic granulomatous disease and studies in knockout mouse models. This work has revealed important features of the regulation of the respiratory burst oxidase and its role in host defense and inflammation. Results from preclinical studies and phase 1 clinical trials for gene therapy for CGD are summarized, in addition to alternative approaches using allogeneic bone marrow transplantation with nonmyeloablative conditioning.

Neutrophil-specific granule deficiency results from a novel mutation with loss of function of the transcription factor CCAAT/enhancer binding protein epsilon.

Neutrophil-specific granule deficiency (SGD) is a rare disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins. CCAAT/enhancer binding protein (C/EBP)epsilon, a member of the leucine zipper family of transcription factors, is expressed primarily in myeloid cells, and its knockout mouse model possesses distinctive defects, including a lack of neutrophil secondary granule proteins. Sequence analysis of the genomic DNA of a patient with SGD revealed a five-basepair deletion in the second exon of the C/EBPepsilon locus. The predicted frame shift results in a truncation of the 32-kD major C/EBPepsilon isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The multiple functional defects observed in these early neutrophil progenitor cells, a consequence of C/EBPepsilon deficiency, define SGD as a defect in myelopoiesis and establish the requirement for C/EBPepsilon for the promyelocyte-myelocyte transition in myeloid differentiation.

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.