Review ArticleThe CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors is a multifaceted highly-regulated system for gene regulation
Introduction
Recent developments in molecular biology have resulted in a greater understanding of gene regulation by transcription factors. Despite the ubiquitous nature of transcription factor binding sites, these elements mediate accurate transcriptional responses. Many genes are transcribed in specific cell types or at distinct developmental stages [1], [2], [3]. One of the key issues throughout the process is the mechanism by which transcription factors can precisely control the diversity and specificity of the complex patterns of eukaryotic gene regulation. One explanation involves fine-tuning of tissue-specific and signal-dependent transcription factors, and the arrangement of activator/repressor binding sites on genes. Combinations of interactions among activators, repressors, and their functional modifiers further generate considerable diversity. In addition, post-translational modification provides further opportunities to regulate gene products with an almost unlimited regulatory potential. One such transcription factor family is the CCAAT/enhancer-binding protein (C/EBP) family. The general subject of physiological and pathological roles of C/EBP proteins in gene regulation has been covered by a vast number of excellent original reports and reviews. These include overviews with a specific focus on cell-cycle and cancer [4], [5], [6], [7], extracellular signaling [8], [9], [10], tissue development [11], [12], [13], [14], [15], [16], and viral pathogenesis [17]. Although some of these reviews may appear chronologically dated, they remain extremely relevant. Consequently, this survey is not intended to replicate previous accounts, except for clarification, extension and recent advances on this topic. Although not all of the examples described herein involve classic cytokine pathways, both the activation of C/EBP by cytokines and their involvement in cytokine gene expression underscore the importance of a thorough understanding of biochemical and structural nuances in evaluating relevant mechanisms directly related to cytokine function.
Section snippets
The C/EBP family of bZIP transcription factors
Most transcription factors consist of a collection of structural and functional domains. Factors that interact directly with DNA possess a DNA binding domain (DBD) that usually provides cognate recognition of specific sequence information. Some carry transcription activation domains (TAD) important for protein–protein interactions that lead directly or indirectly to the initiation of transcription. Multimerization domains provide for the formation of homomers and heteromers. Heteromeric
DNA binding and dimerization of C/EBP via the bZIP domain
The bZIP domain interaction with DNA has been structurally characterized for several members of the superfamily. However the X-ray structure for C/EBPα bZIP–DNA complex [22] reveals the nature of target site specificity for this family in which there is a common basic region providing palindromic α-helical recognition of the DNA major groove. One important characteristic of the structure is that it provides at least one explanation for the asymmetric nature of a binding site interacting with a
Binding of C/EBP to gene regulatory elements
C/EBP was initially considered to be a protein involved in liver-specific gene expression and designated as C/EBPα [18], [26]. C/EBPβ was identified as a nuclear factor that binds to an IL-1-responsive element in the IL6 gene [34]. In early studies, C/EBPβ-binding sequences were found in regulatory regions of several genes associated with the acute-phase response, inflammation, and hematopoiesis. These include those that code for: C reactive protein; α1-acid glycoprotein; α2-macroglobuin;
Intra- and inter-family C/EBP homotypic heterodimerization
In addition to homodimers, C/EBP proteins can form homotypic heterodimers, especially with members of the CREB/ATF family, via the leucine zipper dimerization domain [47] greatly expanding the repertoire of DNA-binding specificities and altering functional activities and trans-activation potential (Fig. 3).
Such unique transcriptional specificities have been reported in C/EBPζ and C/EBPγ (Fig. 4). C/EBPζ (induced in the acute-phase response, adipocytic differentiation and cellular stress) is
Translational regulation of C/EBP isoforms
Eukaryotic protein translation requires the formation of the eukaryotic initiation factor (eIF4F) translation initiation complex that consists of the cap-binding protein eIF4E, the ATP-dependent helicase eIF4A, and a large scaffolding protein eIF4G. Alternative C/EBP translation initiation from a single mRNA generates modified isoforms with distinct transcription activation potential. The mTOR signaling pathway controls the ratio of C/EBP isoform expression through eIF4E [74]. This mechanism
Phosphorylation of C/EBPβ
The signal transduction pathway mediated by cytokines is initiated by activation of multiple protein kinases. C/EBP proteins contain various phosphorylation sites (Fig. 1 and Table 4). There have been numerous reports, dealing with phosphorylation of C/EBP family members. Phosphorylation and dephosphorylation can cause conformational changes and affect DNA binding to cognate sequences or cooperative activity with cofactors, resulting in modulation of protein–protein interactions and
Other post-translational modifications of C/EBP
Members of the C/EBP family often reveal post-translational modifications that can affect activity and intracellular localization. In addition to phosphorylation, acetylation, and sumoylation have also been implicated in such regulation and are nicely described in two reviews [8], [9]. In general, phosphorylation plays mostly a positive role in activating C/EBPα and C/EBPβ [8], [9], although there may be circumstances where it is inhibitory for C/EBPα [88], [105]. In contrast, sumoylation
Formation of multi-protein complexes on DNA
Activation of eukaryotic genes in response to extracellular signals requires assembly of stereospecific multi-protein complexes on DNA. Eukaryotic transcription factors bind to enhancers and promoters as multi-protein complexes. The assembly of architectural proteins mediated by DNA looping allows multi-protein–DNA interactions normally retarded by the high energetic cost of DNA bending and twisting. The resulting bend and twist of DNA provides additional specificity for the interaction. In
C/EBPβ/α and Spi-1 play crucial roles in hematopoietic cell differentiation
C/EBPα as well as Spi-1 are up-regulated during myelomonocytic development, while down-regulated in megakaryo-erythroid differentiation. Suh et al. [119] showed increased erythropoiesis in C/EBPα-/- mice and that over-expression of C/EBPα in hematopoietic progenitors from 5-FU-treated mice inhibits erythroid development, while promoting myeloid development. Murine erythroleukemia (MEL) cells expressing C/EBPα exhibit inhibited erythroid differentiation and induction of myeloid-specific genes
Unique compensatory roles of C/EBPβ and C/EBPδ
MyD88 is an important signaling pathway adaptor protein downstream of TLR. MyD88-deficient macrophages exhibit severely impaired cytokine production such as IL-12 p40, IL-6 and TNFα, but can activate both p65 and p50 NF-κB subunits in response to stimuli. However, in MyD88-deficient macrophages, C/EBPδ expression was undetectable, and LPS-induction of C/EBPβ was also markedly suppressed. In addition, the fact that C/EBPβδ double knockout in mouse embryonic fibroblasts (MEF) showed impaired
Summary and perspectives
The C/EBP family, which includes C/EBPα, the founding member of the bZIP superfamily [129], has been a consistently studied collection of transcription factors for more than two decades. During that time, investigations have led to the demonstration of the numerous regulatory mechanisms presented in this review. Recent reports have provided detailed insight into the crucial functional and physiological roles of family members in molecular processes during cellular responses to external signals.
Acknowledgments
Dr. Deborah L. Galson at The University of Pittsburgh is acknowledged for discussions and assistance in the preparation of this manuscript. P.E.A. was supported by funds from the Hunkele Dreaded Disease Fund and the Bayer School of Natural and Environmental Sciences at Duquesne University.
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