The function and regulation of CCAAT/enhancer binding protein ε

Abstract

In recent years, studies on the structure, function, and regulation of the C/EBPε gene have become an essential topic in the field of many diseases. CCAAT/enhancer-binding protein ε (C/EBPε) is the fifth member of the transcription factor CCAAT/C/EBP family of transcription factors. It plays crucial roles in cell proliferation, differentiation, immunity, energy metabolism, and hematopoiesis. C/EBPε plays essential roles in regulating the hematopoietic system, including myeloid cell development and maturation, participation in the body’s immune responses, and prevention of infections. C/EBPε function is regulated by phosphorylation, acetylation, methylation, and other types of genes. This review related to C/EBPε structure, function and regulation provides a theoretical basis for subsequent research in this area. C/EBPε is an emerging therapeutic target and thus provides new strategies for disease prevention and control.

Introduction

The transcription process of eukaryotes is very complex and often needs the assistance of a variety of protein factors. Transcription, the first step, is regulated by transcription factors (TFs), which represent one of the largest families of human genes. Göös H et al. identified 6703 and 1536 protein–protein interactions for 109 different human transcription factors (TFs), they found that TFs preferred to form more transient or proximal interactions than stable protein complexes. TFs were found to interact with proteins involved in chromatin remodeling, transcription, mRNA splicing and RNA processing.That study provided a rich resource of human TF interactions and also acted as a starting point for future studies aimed at understanding TF-mediated transcription. ¹

The C/EBP family of basic leucine zipper (bZIP) transcription factors includes six members (C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, C/EBPε and C/EBPζ). It is upregulated in the hematopoietic system, highest in myeloid progenitor cells and granulocyte differentiation, regulates neutrophil proliferation and maturation, and is also involved in the transcriptional regulation of a series of myeloid-specific genes.^2,3

Structure of C/EBPε

C/EBPε is present on human and mouse chromosome 14 and is the most important factor in the final granulation of both animals.^4,5 C/EBPε contains an intron with two potential shear acceptor sites and five cysteines, three of which (CYS-34, Cys-148, Cys-280) are relatively conserved in the C/EBP family and do not play an essential role in transactivation of C/EBPε or disulfide bond formation in vivo. C/EBPε mRNA expression was only detected in human peripheral blood mononuclear cells, tissues involved in the immune system, and ovaries, and in mice hematopoietic tissues, including embryonic liver and adult bone marrow and spleen.^6,7 Dimer formation of C/EBPε was not detected using both reducing and non-reducing SDS-polyacrylamide gel electrophoresis with Western blot analysis from either bacterial or mammalian expressed C/EBPε. ²

C/EBPε has the following four isomers: P32, P30, P27 and P14, ⁸ which have different activation potentials. ⁹ P32 and P30 have transcriptional activation function, P27 has the transcriptional inhibition function, P14 has the dominant negative regulation function and lacks the transcriptional activity, which may inhibit the transcriptional activation of the full-length C/EBPε.^2,10–12 The C/EBPε gene has two type of specific promoter cells (Pα and Pβ) and three exons (Ex). The third exon contains bZIP, and because of the different ways of transcribing the product, several transcripts of different lengths (2.5 bp and 1.3–1.5 bp) can be produced, thus increasing the complexity of transcriptional regulation. ¹³ The N-terminus of C/EBPε 27 read is the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε 27 isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation. ¹⁴ The functional domains, modification sites and isoforms of C/EBPε, and the dimer structure of bZIP domain are displayed in Figures 1 and 2.

Figure 1.

Functional domains, modification sites and isoforms of C/EBPε. Green: bZIP domain; Blue: phosphorylation site; Red: the bZIP domain region in four C/EBPε isoforms.

Figure 2.

The dimer structure of bZIP domain (the BR domain and the LZ domain are labeled on each monomer).

Function of C/EBPε

C/EBPε and myeloid cells

C/EBPε is an important regulator of myeloid cell development and also controls cell proliferation, cell cycle, and maturation. Williams et al. ⁷ have confirmed the presence of C/EBPε mRNA in human Jurkat T cell line, but not in any other human lymphoid cell lines, and it is not expressed at significant levels in the thymus. However, the C/EBPε was strongly expressed in mouse spleen, a major source of B cells, and the reported existence of a common precursor for both macrophages and B cells, ¹⁵ which indicated a role for C/EBPε in B cell development or function. The expression of C/EBPε was evidently restricted in both mouse and human, which indicated that its primary function is within the myeloid lineage of hematopoietic cells. ⁷ Furthermore, the gene encoding macrophage colony-stimulating factor receptor (M-CSFR) is a potential C/EBPε target gene that may support a role for M-CSFR in the regulation of myeloid differentiation.

Halene et al. ³ found that C/EBPε deficiency not only results in incomplete differentiation of granulocytes, but also impairs their hematopoietic function in the bone marrow, resulting in an intermediate type of cell with characteristics of monocytes and granulocytes. Neutrophils showed abnormal chemotactic activity, including less migration of ATRA-induced ePRO KO cells to high concentrations of IL-8, but the cause of their potential abnormal chemotaxis was unknown. Hlx1 plays a crucial role in fetal liver development. In the bone marrow, Hlx1 expression is upregulated during myeloid differentiation and is able to increase expression levels in granulocytes and monocytes, and this suggests an important role in myeloid differentiation. In addition, Hlx-1 mRNA levels in primary myeloid cells and mature neutrophils from WT and C/EBPε^−/− mice were examined and found that the deletion of C/EBPε expanded the immature cell population. Co-expression of monocyte and neutrophil markers and an inclination toward monocytic differentiation indicated that C/EBPε KO cells have reduced expression of Hlx transcription factors and that Hlx transcription factors are involved in myeloid maturation and migration. They have some protective effect against the chemotaxis defect of KO cells.^3,16

C/EBPε and infection

Targeted disruption of C/EBPε has been shown to result in abnormal macrophage maturation and phagocytosis and reduced production of macrophage-associated cytokines and costimulation molecules associated with phagocytosis. in C/EBPε^−/− mouse macrophages, IL-10 mRNA and protein expression was markedly decreased, and the expression of IL-12 mRNA, CD14, and IL-6 was found to be lower in the macrophages and lymphocytes. This indicates the importance of C/EBPε in innate immunity.^17,18 The immune function of mice lacking C/EBPε was severely impaired, with a survival of fewer than 5 months and 60% dying from Pseudomonas aeruginosa infection. ¹² Neutrophils deficient in C/EBPε have internal and external defects such as reduced regulatory bacterial uptake, reduced phagocytosis due to reduced or no expression of secondary granule proteins, poor migration due to abnormal expression of CD11b integrin and L-selectin, and increased expression of TNF-α, IL-1Ra. Neutrophil responses to inflammation, such as decreased expression of TNF-α and IL-1Ra, are believed to contribute to the abnormal microenvironment. Kyme P, et al. ¹⁹ utilized WT and C/EBPε^−/− mice infected with different doses of Staphylococcus aureus. Study results showed that C/EBPε-/mice had significantly decreased body weight compared to WT mice, following S. aureus infection. The lesion area was significantly increased, a large number of neutrophils and macrophages were observed, and CFU was also significantly increased, suggesting higher chemokine levels in CXCL-1 and CXCL-2 and higher chemokine levels at the site of infection in C/EBPε^−/− mice. Phagocyte accumulation failed to inhibit S. aureus infection, suggesting that the antimicrobial mechanism of C/EBPε^−/− phagocytes is severely lacking. WT and C/EBPε^−/− blood were protected with gentamicin; S. aureus was easily removed from WT blood, but not from C/EBPε^−/− blood, it was found to continue to grow in the cells of C/EBPε^−/− blood. Staphylococcus aureus showed that neutrophils from C/EBPε^−/− mice had no antimicrobial activity against the host. Furthermore, nicotinamide (NAM) was found to increase the activity of C/EBPε. This leads to immune-mediated effective clearance of S. aureus.

C/EBPε and SGD

Neutrophil-specific granule deficiency (SGD) is a rare hematologic disorder characterized by an autosomal recessive inherited immunodeficiency with neutrophil and monocyte dysfunction. It lacks expression of lactoferrin and gelatinase B, shows delayed chemotaxis, repeated infections, and is life-threatening.^12,20–23 One patient with SGD had a 5 bp deletion in exon 2 of the C/EBPε gene with a reduced transcript count. Lactoferrin was not detected and only one fragment was shown as a normal PCR product, which had homozygous alleles. In SGD patients with C/EBPε 27 kDa and 14 kDa subtypes, there was the complete loss of secondary particles and selective loss of primary defensins. ¹³ Wada et al. ²⁴ reported another 2-amino acides (2-aa) deletion in the bZIP domain of C/EBPε in SGD. In addition to the typical morphological abnormalities of SGD, this patient also showed deletions of CD 15, CD16b, and CD66 B, the characteristic surface phenotype of SGD C/EBPε. ΔRS (p.Arg247_Ser248del) is located in the bZIP domain and results from a 6 bp homozygous deletion in exon 2 of the C/EBPε gene, forming a 2-aa deletion. ΔRS mutant protein still forms a heterodimer with the nuclear WT C/EBPε protein, resulting in a truncated frameshift mutation that impairs protein interaction with transcription factors GATA 1 and PU.1, and loss of transcriptional co-activation. bZIP domain and activation by disrupting dimerization and binding to DNA, these results in the C/EBPε leucine zipper domain further confirm its essential function and importance in its molecular pathogenesis.

In the study of Khanna-Gupta A et al., ²⁵ DNA sequence analysis in SGD patients revealed a heterozygous substitution at amino acid 218 (NM_001805) that lies within the highly conserved DNA-binding basic region of the C/EBPε gene. The mutant C/EBP (V218A) may promiscuously activate inappropriate downstream targets with C/EBP-like elements in their promoters.Göös H et al. ²⁶ reported a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency.

Regulatory modes involved in C/EBPε

Phosphorylation of C/EBPε

C/EBPε is a phosphoprotein and phosphorylation is one of the most widely studied mechanisms regulating the activity of transcription factors. Phosphorylation is one of the most widely studied mechanisms regulating transcription factor activity, affecting DNA binding and functions. Williamson et al. ²⁶ found that C/EBPε is phosphorylated in vivo at three serine residues (Ser 109181 and 188) and one threonine residue (Ser 75), but it has been shown that C/EBPε can also be phosphorylated in vitro by various kinases, including ERK 2, p38 MAP kinase, and PKA (protein kinase A). In the N-terminal reverse activation domain of C/EBPε, Ser75 was found to be phosphorylated only by p38 MAP kinase, producing a protein that can more effectively bind the consensus DNA sequences, resulting in increased transactivation on myeloid-specific promoters. The DNA-binding capacity of C/EBPε was significantly enhanced, and phosphorylation of this residue also upregulated the expression of myeloid-specific secondary granule genes in vivo. Phosphorylation of Ser 181 gave a C/EBPε protein that could bind DNA as well as wild-type C/EBPε but was inactive on a myeloid-specifific promoter.C/EBPε S181 A and C/EBPε S188D were able to activate the mim promoter-luciferase construct in these assays but were not as effective as the wild-type C/EBPε. However, the other mutations C/EBPε S181D and C/EBPε S188 A did not significantly activate the mim promoter. On the other hand, phosphorylation of Ser 181 was found to allow a C/EBPε protein to bind to DNA, similar to non-phosphrylated C/EBPε. However, phosphrylated C/EBPε does not activate the myeloid-specific promoter. We further examined the expression and biological effects of C/EBPε phosphorylation mutants in granulocyte differentiated cell lines and found that both C/EBPε S181D and C/EBPε S188D could up-regulate the expression of the secondary granule genes, but the secondary granule genes NGAL and B9 were upregulated in cells expressing wild-type C/EBPε and C/EBPε T75D. This result indicates that threonine is phosphorylated by p38 MAP kinase at consistent MAP kinase sites in C/EBPε.

Acetylation of C/EBPε

Bartels et al. ²⁷ analyzed the myeloid cell lines HL-60 and NB4 and found that acetylation reactions occur in C/EBPε (32/30 KD). Four acetyl lysines were isolated (K100 and K121 in inhibitory region I, K198 between Rd II and the base region, and K202 including the base and DNA binding regions). The acetylation level of CEBP was regulated by P300 acetylation and SIRT1 deacetylation, and the acetylation of K121 and K198 was found to be used to regulate regulatory domain motif (RDM) and regulatory domain 1 (RD1) functions and to regulate neutrophil transcriptional activity. Acetylation of C/EBPε was demonstrated to be required for neutrophil development, although not specifically involved in dimerization or nuclear localization. Furthermore, acetylation of K121 proved to be important for C/EBPε binding to DNA. Class III HDACs inhibitors such as NAM play an essential role in regulating the transcription factor C/EBPε by modifying histone and non-histone acetylation and affecting transcriptional expression by regulating chromatin condensation.^19,28–32 Exposure of human neutrophils to NAM for 6–12 h resulted in a 5-fold increase in the acetylation level of core histone H3 and a 4-fold increase in the acetylation level of C/EBPε in the promoter region of C/EBPε. The increased transcriptional activity of C/EBPε in macrophages was accompanied by increased histone H3 acetylation and protein acetylation levels in the C/EBPε promoter. ¹⁹

Methylation of C/EBPε

Musialik E et al. ³³ compared the methylation degree and expression levels of the CBP gene promoter in 78 AML patients, normal bone marrow and hematopoietic progenitor cells. They found that methylation levels of C/EBPα (37%), C/EBPδ (35.5%), and C/EBPε (56.7%) were higher than those of normal bone marrow and hematopoietic progenitor cells. Compared to normal bone marrow and CD-15 cells, promoter methylation of C/EBPε in AML and hematopoietic progenitor cells was relatively high. At the same time, the expression level of C/EBPε in AML was higher than in normal bone marrow, suggesting the presence of abnormal promoter methylation in C/EBPε. The high methylation in patients may reflect the undifferentiated nature of leukemia cells. We further classified the C/EBPε promoter into three groups according to the Fab system in terms of methylation level and expression. Immature group (M0 and M1), Granulocyte mature group (M2) and Monocyte mature group (M4 and M5). The methylation level was highest in the immature group, consistent with that of normal progenitor CD15 and BM cells C/EBPε methylation is dependent on FLT3 mutation status. NPM 1 without FLT3-ITD and with CEBPA mutation C/EBPε methylation levels in patients are generally low^33–35 and may be associated with normal bone marrow development. In vitro stimulation of CD34 progenitor cells reduced promoter methylation of this gene in normal hematopoietic differentiation. ³⁶

Regulation of C/EBPε protein interactions

C/EBPε can bind to a variety of proteins and activate transcriptional regulation, including other members of the C/EBP family (C/EBP δ, CHOP), and leucine zipper proteins (CREB2, LDOC1, E6TP1, AF-17), and proteins involved in signal transduction (STAT6, STAT16), Sgn3 (phosphorylated c-Jun, I-B α, p105), ubiquitin-like proteins (inhibitors of STAT1 [PIAS1]), and the ubiquitin-binding enzyme E2I. ³⁷ Many C/EBPε-interacting proteins have a basic DNA binding and C-terminal “leucine zipper” (BZIP) motif that binds to C/EBPε in vitro; C/EBPε does not mediate the leucine zipper motif, but the leucine zipper is required for the interaction. The BZIP domains of mouse C/EBP α, β, ε and rat C/EBP δ can form many different heterodimers. ⁵ Heterodimer-protein interactions may play an essential role in the regulation of C/EBPε activity during myelopoiesis. For example, in cells transfected with C/EBPε and C/EBP δ expression plasmids, the activity of the human lactoferrin promoter can be co-induced. As a homodimer, CHOP has a negative regulatory effect on C/EBPε-dependent transcriptional activation of the myeloid promoter and can markedly suppress the activation of the MIM-1 promoter by C/EBPε and the co-activation of c-MYB and C/EBPε. pIAS 1 (amino acid 1) interacts with C/EBPε in vivo via its N-terminal domain, and lysine mutants such as K121R, K15xR, and K198R regulate protein-protein interactions and may inhibit C/EBPε target genes.^37,38

Others

miR-130a

miR-130a is an essential regulator of C/EBPε expression and has been found to be a regulator of normal granulation. However, aberrant regulation of miR-130a alters mRNA expression profiles by regulating C/EBPε. These changes in cell cycle regulation avoid premature arrest, altered maturation, and potential targeting of other cell cycle regulatory proteins during granulation. The immature phenotype was caused by decreased expression of miR-130a C/EBPε protein and lactoferrin (LTF, CAMP and Lcn-2 (Lcn-2) mRNA in mouse cell lines. Larsen et al. ³⁹ found that the target site of miR-130 is the human C/EBPε mRNA 3′-UTR and constructed a plasmid using the WT 3′-UTR of the C/EBPε gene. The results showed that miR-130a regulates C/EBPε expression by targeting the 3′-UTR of the C/EBPε transcript. In addition, mature neutrophil precursors had high endogenous miR-130a levels and substantial amounts of C/EBPε mRNA; C/EBPε protein was virtually absent. The C/EBPε transcript was not present in the mature neutrophil precursors. Transient transfection of these cells with LNA to inhibit the upregulation of C/EBPε protein induced by miR-130 resulted in increased expression of the C/EBPε target proteins cAMP and LTF. Transfection of MC/MM cells with LNA-miR-130a resulted in increased expression of C/EBPε protein, accompanied by an increase in LTF and cAMP.

NAM (vitamin B3)

Kyme et al. ¹⁹ injected NAM into uninfected WT mice and detected the expression of C/EBPε and downstream antimicrobial factors in mouse bone marrow mononuclear cells. The results showed that the levels of C/EBPε, cAMP, LTF mRNA and protein increased 72 h after NAM administration. It was also speculated that NAM, as an epigenetic regulator, may increase protein expression of several downstream targets via cAMP and LTF. We also found that cAMP gene expression via C/EBPε differed between humans and mice. WT mouse neutrophils did not show an increase in cAMP-induced by NAM, and other antimicrobial targets downstream of C/EBPε were also explored. Our results indicate that the expression of human and mouse neutrophils is not affected by NAM.

Bactericidal/permeability-increasing protein (BPI)

The work of Miyuki Tanaka et al. has shown that BPI is a direct target of C/EBPε. C/EBPε binds directly to the BPI promoter in vivo and mediates the expression of the BPI gene in vitro and in vivo. It has also been demonstrated that BPI expression in granulocytes requires C/EBPε and can be further mediated by C/EBPε in vitro and in vivo expression of the BPI gene in C/EBP-deficient BM cells require C/EBPε, ⁴⁰ which is a direct target of the BPI promoter in vitro and in vivo. ⁴⁰

MEF2D

MEF2D is a member of MEF2 family transcriptional factors and plays cellular functions in skeletal, cardiac, and neuronal development. CEBPE signifificantly increased after MEF2D knockout in AML cells, MEF2D negatively regulated CEBPE expression.CEBPE depletion largely abolished the increased leukemia cell differentiation caused by MEF2D knockout. MEF2D played a critical role in human MLL-r AML and uncover the MEF2D-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block.These results provided a possibility that disrupting the MEF2D-CEBPE regulatory axis might serve as a therapeutic strategy for AML patients. ⁴¹

C/EBPε regulates many downstream genes or proteins

C/EBPε is also directly involved in the regulation of many downstream genes or proteins, including IL-6, monocyte chemotaxis protein (MCP-1) gene, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and M-CSF-R. ⁴²

As noted above, C/EBPε plays an important role in the occurrence and development of various diseases,especially those related to immunity or infection. However, there are some scientific directions remain to be explored, and future study can focus on the following aspects: to reveal the mechanism of C/EBPε on the differentiation and immune responses between neutrophils, eosinophils and basophils; to develop new C/EBPε activators to effectively control inflammation or infection and improve survival in neutropenic patients; lack of C/EBPε affects eosinophil-specific gene expression as well as maturation, ⁴³ providing new interventions for diseases such as infection, sepsis and haematological disorders.

Conclusion

C/EBPε, a transcription factor, is regulated by various protein modifications (phosphorylation, acetylation and methylation) and some other genes or proteins, which affects transcription function. With the continuous in-depth study of C/EBPε on structure, function and regulation mechanism, C/EBPε would be a promising target for the prevention and intervention of some diseases.