Interactive Fly, Drosophila

slow border cells


The DLK-1 kinase promotes CEBP-1 mRNA stability and local translation in C. elegans synapses and axon regeneration

Growth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. This study shows that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. These findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration (Yan, 2009).

Neurons respond to environmental stimuli and insults in a compartmentalized manner. Local protein synthesis in dendrites and axonal growth cones has emerged as a major mechanism allowing compartmentalized responses in growth cone guidance and neuronal plasticity. Several mRNAs are known to be transported and localized to growth cones or axons; transport of such mRNAs often is mediated by their 3' untranslated regions (3'UTRs) (Yan, 2009).

However, it remains controversial whether mature neurons employ axonal regulation of mRNA and local protein synthesis. mRNA-binding proteins, such as Zip-code binding proteins (ZBPs) and cytoplasmic polyadenylation element-binding proteins (CPEBs), are abundant in growing neurites but are mostly undetectable in axons and synapses of mature neurons. Polyribosomes are rarely seen in axons. Nonetheless, several reports suggest that axonal mRNA regulation must occur in mature neurons. In Aplysia neurons, mRNA for the peptide neurotransmitter Sensorin is concentrated at synapses upon contact with target motor neurons, and local translation and secretion of Sensorin promote synapse maturation and plasticity. A number of mRNAs are upregulated in axons of injured adult dorsal root ganglion (DRG) neurons. Regulation of Ran GTPase via local translation of RanBP1 is implicated in retrograde signaling of axon injury. Local translation thus can transmit injury signals and initiate local repair processes (Yan, 2009 and references therein).

CCAAT/enhancer-binding proteins (C/EBP) are widely expressed basic-leucine-zipper (bZip) domain transcription factors with long-studied roles in cell proliferation, differentiation, and stress. In neurons the transcriptional roles of C/EBP proteins have been linked to learning and memory. Learning and memory tasks trigger activation of Erk or p38 kinases, leading to phosphorylation of specific C/EBP isoforms. mRNAs of murine and leech C/EBP are also upregulated following axonal injury, and murine C/EBPβ can activate the transcription of an α-tubulin gene associated with injury responses. The pathways that induce C/EBP after injury are largely unknown (Yan, 2009 and references therein).

The function of the conserved ubiquitin E3 ligase RPM-1 in synaptogenesis and axon formation has been studied in C. elegans. C. elegans neurons have simple unipolar or bipolar axon trajectories and form synapses en passant. For example, the ALM and PLM mechanosensory neurons have a long axon that bifurcates into a branch exclusively forming synapses and another branch transducing mechanoreception. RPM-1 regulates the organization and stabilization of presynaptic terminals and axon termination in both mechanosensory and motor neurons. A major target of RPM-1 ubiquitination is the Dual-Leucine zipper Kinase DLK-1 MAPKKK, which acts in a MAPK cascade consisting of the MAPKK MKK-4 and the p38 kinase PMK-3. By controlling the level of DLK-1, RPM-1 keeps the activity of the DLK-1 cascade at optimal levels. This negative regulation of the DLK pathway by ubiquitin-mediated protein degradation is conserved in Drosophila and mammalian neurons (Yan, 2009 and references therein).

The targets of the DLK-1/p38 cascade have not previously been identified. This study reports the identification of MAK-2, a member of the MAPKAP kinase family, and CEBP-1, a member of the C/EBP class of bZip factors, as effectors of the C. elegans DLK-1 cascade. MAPKAPKs are conserved Ser/Thr kinases that are direct targets of p38 and Erk kinases. cebp-1 mRNA is destabilized by RPM-1 via the DLK-1/MAK-2 cascade, acting on the cebp-1 3.UTR. The DLK-1/MAK-2/CEBP-1 pathway is essential for regenerative regrowth of mature axons following laser axotomy, in part by regulating axonal cebp-1 mRNA stability and translation (Yan, 2009).

C/EBP protein interactions

Chronic exposure of pancreatic beta-cells to supraphysiologic glucose concentrations results in decreased insulin gene transcription. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta) is identified as a repressor of insulin gene transcription in conditions of supraphysiological glucose levels. C/EBPbeta is expressed in primary rat islets. After exposure to high glucose concentrations, the beta-cell lines HIT-T15 and INS-1 express increased levels of C/EBPbeta. The rat insulin I gene promoter contains a consensus binding motif for C/EBPbeta (CEB box) that binds C/EBPbeta. In non-beta-cells C/EBPbeta stimulates the activity of the rat insulin I gene promoter through the CEB box. Paradoxically, in beta-cells C/EBPbeta inhibits transcription, directed by the promoter of the rat insulin I gene by direct protein-protein interaction with a heptad leucine repeat sequence within activation domain 2 of the basic helix-loop-helix transcription factor E47 (Drosophila homolog: Daughterless). This interaction leads to the inhibition of both dimerization and DNA binding of E47 to the E-elements of the insulin promoter, thereby reducing functionally the transactivation potential of E47 on insulin gene transcription. It is suggested that the induction of C/EBPbeta in pancreatic beta-cells by chronically elevated glucose levels may contribute to the impaired insulin secretion in severe type II diabetes mellitus (Lu, 1997).

To define a mechanism by which retinoblastoma protein (See Drosophila Retinoblastoma-family protein) functions in cellular differentiation, primary fibroblasts from the lung buds of wild-type (RB+/+) and null-mutant (RB-/-) mouse embryos were examined. In culture, the RB+/+ fibroblasts differentiate into fat-storing cells, either spontaneously or in response to hormonal induction; otherwise syngenic RB-/- fibroblasts cultured in identical conditions do not. Ectopic expression of normal Rb, but not Rb with a single point mutation, enables RB-/- fibroblasts to differentiate into adipocytes. Rb appears in murine fibroblasts to activate CCAAT/enhancer-binding proteins (C/EBPs), a family of transcription factors crucial for adipocyte differentiation. Physical interaction between Rb and C/EBPs, demonstrated by reciprocal coimmunoprecipitation, occurs only in differentiating cells. Wild-type Rb also enhances the binding of C/EBP to cognate DNA sequences in vitro and the transactivation of a C/EBPß-responsive promoter in cells. Taken together, these observations establish a direct and positive role for Rb in terminal differentiation. Such a role contrasts with the function of Rb in arresting cell cycle progression in G1 by negative regulation of other transcription factors, like E2F-1 (Chen, 1996).

Transcriptional coactivators such as p300 and CREB-binding protein (CBP) function as important elements in the transcription factor network, linking individual transactivators via protein-protein interactions to the basal transcriptional machinery. An investigation was carried out as to whether p300 plays a role in transactivation mediated by C/EBPbeta, a conserved member of the C/EBP family. C/EBPbeta-dependent transactivation is strongly inhibited by adenovirus E1A but not by E1A mutants defective in p300 binding. Ectopic expression of p300 reverses the E1A-dependent inhibition and increases the transactivation potential of C/EBPbeta. C/EBPbeta and p300 interact with each other, and the sequences responsible for interaction map to the E1A binding region of p300 and the amino terminus of C/EBPbeta. Additionally, the minimal C/EBPbeta binding site of p300 acts as a dominant-negative inhibitor of C/EBPbeta. These observations identify p300 as a bona fide coactivator for C/EBPbeta. C/EBPbeta is highly expressed in the myelomonocytic lineage of the hematopoietic system and cooperates with Myb to activate mim-1, a gene specifically expressed during myelomonocytic differentiation. Recent evidence has shown that Myb recruits CBP (and presumably p300) as a coactivator and, in contrast to C/EBPbeta, interacts with the CREB binding site of p300-CBP. p300 not only stimulates the activity of Myb and C/EBPbeta individually but also increases the synergy between them. Thus, these results reveal a novel function of p300: in addition to linking specific transcription factors to the basal transcriptional machinery, p300 also mediates the cooperation between transactivators interacting with different domains of p300 (Mink, 1997).

The human papillomavirus type 18 (HPV-18) upstream regulatory region (URR) controls viral gene transcription in a cell-type-specific manner. The HPV-18 URR is active in HeLa cells but inactive in HepG2 cells. The activating activity of YY1 (Drosophila homolog: pleiohomeotic) in HeLa cells is dependent on its functional interactions with the switch region, which is critical for the HPV-18 URR activity in HeLa cells. A protein complex composed of C/EBP beta and YY1 binds the switch region, which is detected only in HeLa cells, not in HepG2 cells. Transfection of C/EBP beta into HepG2 cells restores the formation of the C/EBP beta-YY1-switch region complex, accompanied by increased transcription directed by the HPV-18 URR. Mutations in the switch region that abolish the complex formation also abrogate C/EBP beta-induced transcriptional activation. This provides a strong correlation between the binding of the C/EBP beta-YY1 complex to the switch region and cell-type-specific URR activity. Taken together, a novel C/EBP beta-YY1 complex has been identified that binds the switch region and contributes to cell-type-specific HPV-18 URR activity (Bauknecht, 1996).

C/EBPalpha and C/EBPbeta are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPbeta has been reported to produce four isoforms: full-length 38-kDa C/EBPbeta; 35-kDa LAP (liver-enriched transcriptional activator protein); 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. The mechanisms by which C/EBPbeta isoforms are generated in the liver and in cultured cells have been investigated. Using an in vitro translation system, it has been found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism-specific proteolytic cleavage of full-length C/EBPbeta. Studies of mice in which the C/EBPalpha gene has been deleted (C/EBPalpha-/-) show that the regulation of C/EBPbeta proteolysis is dependent on C/EBPalpha. The induction of C/EBPalpha in cultured cells leads to induced cleavage of C/EBPbeta to generate the LIP isoform. The cleavage activity in mouse liver extracts has been characterized and it is found that the proteolytic cleavage activity is (1) specific to prenatal and newborn livers; (2) is sensitive to chymostatin, and (3) is completely abolished in C/EBPalpha-/- animals. The lack of cleavage activity in the livers of C/EBPalpha-/- mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration shows that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. It is proposed that there are two mechanisms by which C/EBPbeta isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPalpha-dependent, specific proteolytic cleavage of full-length C/EBPbeta. The latter mechanism implicates C/EBPalpha in the regulation of posttranslational generation of the dominant negative C/EBPbeta isoform, LIP (Welm, 1999).

SB203580 and SB202190, pyridinyl imidazoles that selectively inhibit p38 mitogen-activated protein (MAP) kinase, are widely utilized to assess the physiological roles of p38. Treatment of 3T3-L1 fibroblasts with these p38 MAP kinase inhibitors prevents fibroblast differentiation into adipocytes as judged by an absence of lipid accumulation, a lack of expression of adipocyte-specific genes, and a fibroblastic morphological appearance. In 3T3-L1 fibroblasts and developing adipocytes, p38 is active. p38 activity decreases dramatically during later stages of differentiation. In accordance with the time course of p38 activity, p38 inhibitor treatment during only the early stages of differentiation is sufficient to block adipogenesis. In addition, a 3T3-L1 cell line harboring an inducible dominant negative p38 mutant was constructed. The induction of this dominant negative mutant of p38 prevents adipocyte differentiation. Thus, it is likely that the antiadipogenic activity of SB203580 and SB202190 is indeed due to inhibition of p38 MAP kinase. This study points out that CCAAT/enhancer-binding protein beta (C/EBPbeta), a transcription factor critical for the initial stages of 3T3-L1 adipogenesis, bears a consensus site for p38 phosphorylation and serves as a substrate for p38 MAP kinase in vitro. Although the induction of C/EBPbeta is not significantly altered in the presence of the p38 inhibitor, the amount of in vivo phosphorylated C/EBPbeta is reduced by SB203580. The transcriptional induction of PPARgamma, a gene whose expression is induced by C/EBPbeta, and a factor critically involved in terminal differentiation of adipocytes, is impaired in the presence of p38 inhibitors. Thus, transcription factors such as C/EBPbeta that promote adipocyte differentiation may be p38 targets during adipogenesis. Collectively, the data in this study suggest that p38 MAP kinase activity is important for proper 3T3-L1 differentiation (Engelman, 1998).

CHOP (GADD153) is a small nuclear protein that dimerizes avidly with members of the C/EBP family of transcription factors. Normally undetectable, it is expressed at high levels in cells exposed to conditions that perturb protein folding in the endoplasmic reticulum and induce an endoplasmic reticulum stress response. CHOP expression in stressed cells is linked to the development of programmed cell death and, in some instances, cellular regeneration. In this study, representational difference analysis was used to compare the complement of genes expressed in stressed wild-type mouse embryonic fibroblasts with those expressed in cells nullizygous for chop. CHOP expression, in concert with a second signal, was found to be absolutely required for the activation by stress of a set of previously undescribed genes referred to as DOCs (for downstream of CHOP). DOC4 is a mammalian ortholog of a Drosophila gene, Tenm/Odz, implicated in patterning of the early fly embryo, whereas DOC6 encodes a newly recognized homolog of the actin-binding proteins villin and gelsolin. These results reveal the existence of a novel CHOP-dependent signaling pathway, distinct from the known endoplasmic reticulum unfolded protein response, that may mediate changes in cell phenotype in response to stress (Wang, 1998).

Upon activation by liver injury, hepatic stellate cells produce excessive fibrous tissue leading to cirrhosis. The hepatotoxin CCl4 induces activation of RSK (see Drosophila RSK), phosphorylation of C/EBPß on Thr217, and proliferation of stellate cells in normal mice, but causes apoptosis of these cells in C/EBPß-/- or C/EBPß-Ala217 (a dominant-negative nonphosphorylatable mutant) transgenic mice. Both C/EBPß-PThr217 and the phosphorylation mimic C/EBPß-Glu217, but not C/EBPß-Ala217, associate with procaspases 1 and 8 in vivo and in vitro and inhibit their activation. These data suggest that C/EBPß phosphorylation on Thr217 creates a functional XEXD caspase substrate/inhibitor box (K-Phospho-T217VD) that is mimicked by C/EBPß-Glu217 (KE217VD). C/EBPß-/- and C/EBPß-Ala217 stellate cells are rescued from apoptosis by the cell permeant KE217VD tetrapeptide or C/EBPß-Glu217. It is concluded that C/EBPß phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival (Buck, 2001).

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is a strong inhibitor of cell proliferation. C/EBPalpha directly interacts with cdk2 and cdk4 and arrests cell proliferation by inhibiting these kinases. A short growth inhibitory region of C/EBPalpha has been mapped between amino acids 175 and 187. This portion of C/EBPalpha is responsible for direct inhibition of cyclin-dependent kinases and causes growth arrest in cultured cells. C/EBPalpha inhibits cdk2 activity by blocking the association of cdk2 with cyclins. Importantly, the activities of cdk4 and cdk2 are increased in C/EBPalpha knockout livers, leading to increased proliferation. These data demonstrate that the liver-specific transcription factor C/EBPalpha brings about growth arrest through direct inhibition of cdk2 and cdk4 (Wang, 2001).

Chromatin remodeling is an important step in promoter activation during cellular lineage commitment and differentiation. The ability of the C/EBPalpha transcription factor to direct adipocyte differentiation of uncommitted fibroblast precursors and to activate SWI/SNF-dependent myeloid-specific genes depends on a domain, C/EBPalpha transactivation element III (TE-III), that binds the SWI/SNF chromatin remodeling complex. TE-III collaborates with C/EBPalpha TBP/TFIIB interaction motifs during induction of adipogenesis and adipocyte-specific gene expression. These results indicate that C/EBPalpha acts as a lineage-instructive transcription factor through SWI/SNF-dependent modification of the chromatin structure of lineage-specific genes, followed by direct promoter activation via recruitment of the basal transcription-initiation complex, and provide a mechanism by which C/EBPalpha can mediate differentiation along multiple cellular lineages (Pedersen, 2001).

The C/EBPalpha transcription factor has the capability to execute various differentiation programs. In hematopoiesis, both eosinophil and neutrophil lineage commitment can be induced by C/EBPalpha, and adipogenesis can be initiated by C/EBPalpha in uncommitted mesenchymal precursor cells or, in collaboration with PPARgamma, in myocytes. These observations suggest that C/EBPalpha provides a fundamental function generally required for the activation of specific differentiation programs, and that the collaborating factors (PPARgamma, GATA-1, Myb, PU.1) serve to direct this function to appropriate gene loci. The requirement for the SWI/SNF interacting TE-III domain of C/EBPalpha in both adipose conversion of NIH3T3 cells and in activation of SWI/SNF-dependent myeloid-specific gene expression (in the case of mim-1, in collaboration with c-Myb) provides evidence that the capacity to recruit SWI/SNF chromatin remodeling complexes is such a function. This is further supported by the previous demonstration that providing a 'selector' molecule such as c-Myb (which by itself does not recruit SWI/SNF) with an SWI/SNF recruiting domain renders it functionally independent of C/EBP activity for myeloid-specific gene activation. Together, these observations led to a proposal that SWI/SNF recruitment is an integral part of C/EBPalpha-dependent (and C/EBPß-dependent) differentiation processes. The inability of a C/EBPß molecule lacking the CR1 SWI/SNF binding domain to replace C/EBPalpha in adipogenesis, without affecting liver gene expression, provides in vivo evidence that SWI/SNF recruitment is indeed relevant for adipogenesis (Pedersen, 2001).

C/EBPalpha is one of the key transcription factors that mediate lineage specification and differentiation of multipotent myeloid progenitors into mature granulocytes. Although C/EBPalpha is known to induce granulopoiesis while suppressing monocyte differentiation, it has been unclear how C/EBPalpha regulates this cell fate choice at the mechanistic level. Inducers of monocyte differentiation inhibit the alternate cell fate choice, that of granulopoiesis, through inhibition of C/EBPalpha. This inhibition is mediated by extracellular signal-regulated kinases 1 and/or 2 (ERK1/2), which interact with C/EBPalpha through an FXFP docking site and phosphorylate serine 21. As a consequence of C/EBPalpha phosphorylation, induction of granulocyte differentiation by C/EBPalpha or retinoic acid is inhibited. This analysis of C/EBPalpha by fluorescent resonance energy transfer revealed that phosphorylation induces conformational changes in C/EBPalpha, increasing the distance between the amino termini of C/EBPalpha dimers. Thus, myeloid development is partly regulated by an ERK1/2-mediated change in the conformation of C/EBPalpha that favors monocyte differentiation by blocking granulopoiesis (Ross, 2004).

c-Myb, but not avian myeloblastosis virus (AMV) v-Myb, cooperates with C/EBPß to regulate transcription of myeloid-specific genes. To assess the structural basis for that difference, the crystal structures were determined for complexes comprised of the c-Myb or AMV v-Myb DNA-binding domain (DBD), the C/EBPß DBD, and a promoter DNA fragment. Within the c-Myb complex, a DNA-bound C/EBPß interacts with R2 of c-Myb bound to a different DNA fragment; point mutations in v-Myb R2 eliminate such interaction within the v-Myb complex. GST pull-down assays, luciferase trans-activation assays, and atomic force microscopy confirm that the interaction of c-Myb and C/EBPß observed in crystal mimics their long range interaction on the promoter, which is accompanied by intervening DNA looping (Tahirov, 2002).

The DBD of c-Myb consists of three imperfect tandem repeats of 51 or 52 amino acid residues, referred to as R1, R2, and R3 from the N terminus. Each repeat contains three helices (alpha1, alpha2, and alpha3) with the helix-turn-helix variant motif, and that R2 and R3 are involved in specific DNA recognition, while R1 loosely covers the DNA position next to the R2 binding site. The structure of R2 also contains a partially exposed hydrophobic patch; within the corresponding region of the AMV v-Myb DBD, this hydrophobic patch contains the three point mutations responsible for the failure of v-Myb to activate the mim-1 promoter, suggesting that this hydrophobic patch is important for the interaction of c-Myb and C/EBPß and for their synergistic activation of transcription (Tahirov, 2002 and references therein).

To establish the structural basis for the synergy between c-Myb and C/EBPß and its disruption by the mutations found in the AMV v-Myb DBD, the crystal structures were determined of ternary complexes containing the c-Myb or AMV v-Myb DBD, the C-terminal portion of C/EBPß, including the DBD, and a DNA fragment from the tom-1A promoter: c-Myb38-193-C/EBPß259-336-DNA [c-Myb38-193-C/EBPß273-336-DNA, and AMV v-Myb66-193-C/EBPß259-336-DNA are respectively named c-Myb complex I, c-Myb complex II (or generally c-Myb complex without discrimination of complexes I and II)], and v-Myb complex. It was found that the c-Myb complex shows specific intercomplex binding between c-Myb and C/EBPß: this is not shown by the v-Myb complex. This interaction between c-Myb and C/EBPß led to the speculation that, via DNA looping, c-Myb and C/EBPß are able to interact and cooperate despite the fact that they bind to natural promoters at some distance from one another. Atomic force microscopy (AFM) was used to confirm that c-Myb and C/EBPß could interact through looping of the mim-1 promoter and its functional relevance, in vivo, was shown using a luciferase trans-activation assay (Tahirov, 2002).

CCAAT/enhancer-binding proteins (C/EBP) are critical determinants for cellular differentiation and cell type-specific gene expression. Their functional roles in osteoblast development have not been determined. A key component of the mechanisms by which C/EBP factors regulate transcription of a tissue-specific gene during osteoblast differentiation was addressed. Expression of both C/EBPbeta and C/EBPdelta increases from the growth to maturation developmental stages and, like the bone-specific osteocalcin (OC) gene, is also stimulated 3-6-fold by vitamin D3, a regulator of osteoblast differentiation. A C/EBP enhancer element was characterized in the proximal promoter of the rat osteocalcin gene, which resides in close proximity to a Runx2 (Cbfa1) element, essential for tissue-specific activation. C/EBP and Runx2 factors interact together in a synergistic manner to enhance OC transcription (35-40-fold) in cell culture systems. It has been shown by mutational analysis that this synergism is mediated through the C/EBP-responsive element in the OC promoter and by a direct interaction between Runx2 and C/EBPbeta. Furthermore, a domain in Runx2 has been mapped that is necessary for this interaction by immunoprecipitation. A Runx2 mutant lacking this interaction domain does not exhibit functional synergism. It is concluded that, in addition to Runx2 DNA binding functions, Runx2 can also form a protein complex at C/EBP sites to regulate transcription. Taken together, these findings indicate that C/EBP is a principal transactivator of the OC gene and the synergism with Runx2 suggests that a combinatorial interaction of these factors is a principal mechanism for regulating tissue-specific expression during osteoblast differentiation (Gutierrez, 2002).

Regulation of C/EBP transcription

During adipocyte differentiation, C/EBPalpha expression is activated; in turn, this serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPalpha gene has led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, are found to contain a factor (as yet unsequenced), CUP (C/EBPalpha undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, it is shown that C/EBPalpha promoter-luciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPalpha gene. Mutation of the CUP-1 site in these constructs has little effect on reporter gene expression; however, when this mutation is combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes is dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) has been identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region have little effect on reporter gene transcription, mutation of both CUP elements markedly activates transcription. Thus, it appears that dual CUP regulatory elements, located on opposite sides of the C/EBP binding site regulating C/EBP transcription and the transcriptional start site, repress transcription of the C/EBPalpha gene prior to induction of the adipocyte differentiation program (Tang, 1997).

The putative transcriptional corepressor ETO/MTG8 (Drosophila homolog: Nervy) has been extensively studied due to its involvement in a chromosomal translocation causing the t(8;21) form of acute myeloid leukemia. Despite this, the role of ETO in normal physiology has remained obscure. ETO is highly expressed in preadipocytes and acts as an inhibitor of C/EBPbeta during early adipogenesis, contributing to its characteristically delayed activation. ETO prevents both the transcriptional activation of the C/EBPalpha promoter by C/EBPbeta and its concurrent accumulation in centromeric sites during early adipogenesis. ETO expression rapidly reduces after the initiation of adipogenesis, and this is essential to the normal induction of adipogenic gene expression. These findings define, for the first time, a molecular role for ETO in normal physiology as an inhibitor of C/EBPbeta and a novel regulator of early adipogenesis (Rochford, 2004).

The STAT3 transcription factor is an important initiator of mammary gland involution in the mouse. This work shows that the STAT3 target gene CCAAT/enhancer binding protein delta (C/EBPdelta) is a crucial mediator of pro-apoptotic gene expression events in mammary epithelial cells. In the absence of C/EBPdelta, involution is delayed and the pro-apoptotic genes encoding p53, BAK, IGFBP5 and SGP2/clusterin are not activated, while the anti-apoptotic genes coding for BFL1 and Cyclin D1 are not repressed. Consequently, p53 targets such as survivin, BRCA1, BRCA2 and BAX are not regulated appropriately and protease activation is delayed. Furthermore, expression of MMP3 and C/EBP during the second phase of involution is perturbed in the absence of C/EBP. In HC11 cells, C/EBP alone is sufficient to induce IGFBP5 and SGP2. It also suppresses Cyclin D1 expression and cooperates with p53 to elicit apoptosis. This study places C/EBP between STAT3 and several pro- and anti-apoptotic genes promoting the physiological cell death response in epithelial cells at the onset of mammary gland involution (Thangaraju, 2005).

Granulocyte-colony-stimulating factor (G-CSF) stimulates the activation of multiple signaling pathways, leading to alterations in the activities of transcription factors. Gfi-1 is a zinc finger transcriptional repressor that is required for granulopoiesis. How Gfi-1 acts in myeloid cells is poorly understood. The expression of Gfi-1 is up-regulated during G-CSF-induced granulocytic differentiation in myeloid 32D cells. Truncation of the carboxyl terminus of the G-CSF receptor, as seen in patients with acute myeloid leukemia evolving from severe congenital neutropenia, disrupts Gfi-1 up-regulation by G-CSF. Ectopic expression of a dominant negative Gfi-1 mutant, N382S, which is associated with severe congenital neutropenia, results in premature apoptosis and reduces proliferation of cells induced to differentiate with G-CSF. The expression of neutrophil elastase (NE) and CCAAT enhancer-binding protein epsilon (C/EBPepsilon) is significantly increased in 32D cells expressing N382S. In contrast, overexpression of wild type Gfi-1 abolishes G-CSF-induced up-regulation of C/EBPepsilon but has no apparent effect on NE up-regulation by G-CSF. Notably, G-CSF-dependent proliferation and survival are inhibited upon overexpression of C/EBPepsilon but not NE. These data indicate that Gfi-1 down-regulates C/EBPepsilon expression and suggest that increased expression of C/EBPepsilon as a consequence of loss of Gfi-1 function may be deleterious to the proliferation and survival of early myeloid cells (Zhuang, 2006).

C/EBP DNA binding specificity

PAR (see Drosophila Par-domain protein 1) and C/EBP family proteins are liver-enriched basic leucine zipper (bZip) transcription factors that bind similar sites on the promoters of albumin and cholesterol 7 alpha hydroxylase genes. However, C/EBP proteins have a more relaxed binding specificity than PAR proteins, in that they recognize many sites within promoter or randomly selected rat genomic DNA sequences that are ignored by PAR proteins. Thus, DNAse I protection experiments suggest that C/EBP recognizes a binding site every 200 to 300 bp with an affinity similar to that of the cholesterol 7 alpha hydroxylase gene promoter. The frequency of PAR protein binding sites with comparable affinities is about 20-fold lower in the rat genome. By using a PCR-based amplification assay, high affinity DNA-binding sites were selected for C/EBP beta and the PAR protein DBP from a pool of oligonucleotides. Both proteins indeed recognize similar sequences with the optimal core binding sequences 5'RTTAY.GTAAY3'. However, as expected, DBP, is considerably less tolerant to deviations from the consensus site. A single amino acid substitution mutant of C/EBP beta that increases its target site specificity has been characterized. This protein, C/EBP beta V to A, contains a valine to alanine substitution at position 13 of the basic domain (residue 216 of C/EBP beta). C/EBP beta V to A selectively binds only the subset of C/EBP sites that are also DBP sites, both as oligonucleotides and within the natural contexts of the albumin and cholesterol hydroxylase promoters (Falvey, 1996).

Transcriptional regulation of C/EBP

Overexpression of the nuclear form of sterol regulatory element-binding protein-1c (nSREBP-1c/ADD1) in cultured 3T3-L1 preadipocytes promotes adipocyte differentiation. Transgenic mice were produced that overexpress nSREBP-1c in adipose tissue under the control of the adipocyte-specific aP2 enhancer/promoter. A syndrome with the following features was observed: (1) disordered differentiation of adipose tissue -- white fat failed to differentiate fully, and the size of white fat depots was markedly decreased; brown fat was hypertrophic and contained fat-laden cells resembling immature white fat; levels of mRNA encoding adipocyte differentiation markers (C/EBPalpha, PPARgamma, adipsin, leptin, UCP1) were reduced, but levels of Pref-1 and TNFalpha were increased; (2) marked insulin resistance with 60-fold elevation in plasma insulin; (3) diabetes mellitus with elevated blood glucose (>300 mg/dl) that failed to decline when insulin was injected; (4) fatty liver from birth and elevated plasma triglyceride levels later in life. These mice exhibit many of the features of congenital generalized lipodystrophy (CGL), an autosomal recessive disorder in humans (Shimomura, 1998).

Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPbeta genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPbeta and C/EBPalpha mRNAs by differential initiation of translation. These isoforms retain different parts of the amino terminus and therefore display different functions in gene regulation and proliferation control. PKR and mTOR signaling pathways control the ratio of C/EBP isoform expression through the eukaryotic translation initiation factors eIF-2alpha and eIF-4E, respectively. An evolutionary conserved upstream open reading frame in C/EBPalpha and C/EBPbeta mRNAs is a prerequisite for regulated initiation from the different translation initiation sites and integrates translation factor activity. Deregulated translational control leading to aberrant C/EBPalpha and C/EBPbeta isoform expression or ectopic expression of truncated isoforms disrupts terminal differentiation and induces a transformed phenotype in 3T3-L1 cells. These results demonstrate that the translational controlled ratio of C/EBPalpha and C/EBPbeta isoform expression determines cell fate (Calkhoven, 2000).

Initiation of translation is affected by a number of pathways that control the activity and level of eukaryotic translation initiation factors (eIFs). The RNA-dependent protein kinase (PKR) affects translation initiation by phosphorylation-induced inactivation of the rate-limiting eIF-2alpha, a component of the eIF-2 holocomplex. It was asked whether interference with PKR or eIF-2 function would modify C/EBPalpha and C/EBPß protein isoform ratios. For this purpose the pre-adipocyte 3T3-L1 cell line was used; this line undergoes C/EBPalpha and C/EBPß dependent differentiation upon hormone treatment. In these cells the eIF-2 pathway is constitutively activated by retroviral introduction of a kinase-inactive and dominant-negative PKR mutant (PKRDelta6) or the eIF-2alpha mutant S52A, which cannot be phosphorylated and thus resists inactivation by PKR. Transgene-expressing and control (empty vector) 3T3-L1 cultures were subjected to a standard differentiation protocol and analyzed for C/EBPalpha and C/EBPß protein isoform expression by immunoblotting. Ectopic expression of either PKRDelta6 or eIF-2alphaS52A, shifts C/EBPalpha and C/EBPß isoform expression toward the truncated isoform. A similar shift in C/EBP isoform expression is obtained by treating differentiated 3T3-L1 adipocytes with the PKR inhibitor 2-aminopurine (2-AP). Concomitantly with 2-AP-induced dephosphorylation and inactivation of PKR, expression of truncated C/EBPalpha isoform is enhanced. These results show that high eIF-2 activity shifts the ratio of C/EBP isoform expression toward a more truncated isoform (Calkhoven, 2000).

The FKBP12-rapamycin-associated protein (FRAP)/mammalian target of rapamycin (mTOR) enhances the level of accessible eIF-4E: mTOR phosphorylates and inhibits phosphatase PP2A, which keeps the inhibitory 4E-binding protein 1 (4E-BP1, also called PHAS1) in an active unphosphorylated state. 3T3-L1 cells that overexpress eIF-4E were generated by retroviral transfer and were subjected to the standard differentiation protocol. Enhanced levels of eIF-4E shift C/EBPalpha isoform expression toward the truncated isoform. In contrast, when mTOR is inhibited by rapamycin, expression of the truncated C/EBPalpha isoform is reduced concomitantly with the dephosphorylation of 4E-BP1. Similar results were obtained with C/EBPß. In conclusion, two rate-limiting translation initiation factors control the ratio of C/EBP isoforms: At high eIF-2 and eIF-4E activity, relatively more truncated C/EBP isoforms are expressed, whereas at lower eIF activity, expression of the full-length isoforms dominates (Calkhoven, 2000).

To investigate the role of the conserved upstream open reading frames (uORF) in conjunction with eIF activity for the regulation of C/EBPalpha and C/EBPß isoform expression, undifferentiated 3T3-L1 control cells and 3T3-L1 cells expressing eIF-2alphaSA or eIF-4E transgenes were transiently transfected with C/EBPalpha wild-type or the corresponding mutated uORF (DeltaD) expression constructs. The expression of truncated C/EBP isoforms depends on the uORF under conditions of enhanced eIF activity as well. eIF-4E has been implicated in the selection of upstream initiation sites in bicistronic messengers. The effect of increased eIF-4E levels on translation initiation from the uORF site D was examined. To do so the uORF of C/EBPalpha was fused in frame to the C/EBPalpha coding sequence and the effect of eIF-4E on translation initiation from D and B1 was tested. Overexpression of eIF-4E enhances translation initiation from the uORF initiation site D at the expense of initiation from site B1. These results indicate that the uORF is crucial for the modulation of C/EBPalpha isoform ratio through eIF activity (Calkhoven, 2000).

Terminal differentiated 3T3-L1 adipocytes form monolayers of contact-inhibited fat cells that store large amounts of lipids. It was noticed that interference with translational control pathways alters the differentiation program of 3T3-L1 cells. Increased eIF activity reduces contact inhibition and results in foci formation in differentiating cultures. To determine whether deregulated expression of C/EBP isoforms might account for the altered growth properties, truncated C/EBPalpha or C/EBPß isoforms were stably introduced by retroviral gene transfer and adipogenesis was induced by the standard protocol. Sustained ectopic expression of truncated C/EBPalpha or truncated C/EBPß isoform give rise to a heterogeneous population of predominantly small and spindle-shaped cells that are poorly differentiated. The cells display reduced adherence to the culture dish, form foci, and continue to multiply during differentiation. Thus, up-regulation of truncated C/EBP isoforms prevents proliferation arrest and contact inhibition, interferes with terminal adipogenic differentiation, and induces a transformed phenotype. Apparently, proper translational regulation of C/EBP isoform expression is a prerequisite for terminal fat cell differentiation and proliferation arrest (Calkhoven, 2000).

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of ß-catenin to the proteasome. In fact, sustained activation of ß-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. The mechanisms regulating the balance between ß-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes has been investigated. Specifically, it has been shown that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of ß-catenin, which depends on glycogen synthase kinase (GSK) 3ß activity. Mutation of serine 37 (a target of GSK3ß) to an alanine renders ß-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3ß phosphorylation-defective S37A-ß-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-ß-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant ß-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-ß-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKß is a key regulator of the balance between ß-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes (Liu, 2004).

Calreticulin interacts with C/EBPalpha and C/EBPbeta mRNAs and represses translation of C/EBP proteins

An RNA binding protein, CUGBP1, has been identified that binds to GCN repeats located within the 5' region of C/EBPbeta mRNAs and regulates translation of C/EBPbeta isoforms. To further investigate the role of RNA binding proteins in the posttranscriptional control of C/EBP proteins, additional RNA binding proteins were identified that interact with GC-rich RNAs and that may regulate RNA processing. In HeLa cells, the majority of GC-rich RNA binding proteins are associated with endogenous RNA transcripts. The separation of these proteins from endogenous RNA identified several proteins in addition to CUGBP1 that specifically interact with the GC-rich 5' region of C/EBPbeta mRNA. One of these proteins was purified to homogeneity and was identified as calreticulin (CRT). CRT is a multifunctional protein involved in several biological processes, including interaction with and regulation of rubella virus RNA processing. The data demonstrate that both CUGBP1 and CRT interact with GCU repeats within myotonin protein kinase and with GCN repeats within C/EBPalpha and C/EBPbeta mRNAs. GCN repeats within these mRNAs form stable SL structures. The interaction of CRT with SL structures of C/EBPbeta and C/EBPalpha mRNAs leads to inhibition of translation of C/EBP proteins in vitro and in vivo. Deletions or mutations abolishing the formation of SL structures within C/EBPalpha and C/EBPbeta mRNAs led to a failure of CRT to inhibit translation of C/EBP proteins. CRT-dependent inhibition of C/EBPalpha is sufficient to block the growth-inhibitory activity of C/EBPalpha. This finding further defines the molecular mechanism for posttranscriptional regulation of the C/EBPalpha and C/EBPbeta proteins (Timchenko, 2002).

Mutation of C/EBP

C/EBP alpha binds to specific promoter sequences and directs transcription of many genes expressed in the liver. Overexpression of C/EBP alpha in established cell lines inhibits cell proliferation. Primary hepatocytes from newborn C/EBP alpha(-/-) mice and normal littermates were used to determine whether the absence of C/EBP alpha increased proliferation and/or transformation of these cells in vitro. DNA synthesis, as measured by bromodeoxyuridine (BrdU) incorporation 24 hours postharvest, was fourfold higher in cells from C/EBP alpha(-/-) pups. Established cell lines were derived from 7 of 8 hepatocyte cultures initiated from null mutants, 4 of 23 cultures from heterozygotes, and 0 of 12 cultures from wild-type animals. C/EBP alpha(-/-) cultures had epithelial morphology, showed bile canaliculi, and expressed albumin messenger RNA (mRNA). When cultured on Matrigel, which promotes differentiation, cell lines derived from C/EBP alpha(-/-) mice formed cords and increased albumin mRNA expression by 1.7- to 3.8-fold. C/EBP alpha(-/-) cell lines exhibited rapid growth and rapid accumulation of chromosomal abnormalities, and were capable of forming nodules when inoculated into the abdominal subcutaneous tissue of nude mice. These data show that C/EBP alpha is an important regulator of hepatocyte proliferation and participates in the maintenance of the nontransformed hepatic phenotype in vitro (Soriano, 1998).

A transgenic mouse was generated with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. The liver is engorged with lipid, and the internal organs are enlarged. The mice are diabetic, with reduced leptin (20-fold) and elevated serum glucose (3-fold), insulin (50- to 400-fold), free fatty acids (2-fold), and triglycerides (3- to 5-fold). The A-ZIP/F-1 phenotype suggests a mouse model for the human disease lipoatrophic diabetes (Seip-Berardinelli syndrome), indicating that the lack of fat can cause diabetes. The myriad of consequences of having no fat throughout development can be addressed with this model (Moitra, 1998).

The C/EBPalpha transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, C/EBPalpha mutants defective in repression of E2F-dependent transcription were identified and they were found to be impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, E2F repression-deficient C/EBPalpha alleles were shown to fail to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation (Porse, 2001).

The transcription factor C/EBPalpha is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Heterozygous mutations have been identified in CEBPA in ten patients with acute myeloid leukemia (AML). Five mutations in the amino terminus truncate the full-length protein, but do not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPalpha DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fail to induce granulocytic differentiation. This is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML (Pabst, 2001).

The CCAAT/enhancer binding proteins C/EBPalpha and C/EBPbeta are related transcription factors that are important for the function of various organs in the postnatal mouse. Gene replacement and tissue culture experiments have suggested partial redundancy of both transcription factors. Mouse embryos deficient for both C/EBPalpha and C/EBPbeta (C/EBPalphabeta-/-) die between embryonic day 10 (E10) and E11 and display defective placentas. In situ hybridization revealed that C/EBPalpha and C/EBPbeta are coexpressed in the chorionic plate at E9.5 and later in the trophoblasts of the labyrinthine layer. In C/EBPalphabeta-/- placentas, allantoic blood vessels invaded the chorion; however, vessel expansion and development of the labyrinthine layer was impaired. Furthermore, a single copy of either C/EBPalpha in the absence of C/EBPbeta or C/EBPbeta in the absence of C/EBPalpha is sufficient to complete development, suggesting complementation of these C/EBPs during embryogenesis. A single copy of C/EBPalpha in the absence of C/EBPbeta, however, fails to rescue survival after birth, suggesting haploinsufficiency of C/EBPalpha in newborns. These data thus reveal novel essential, redundant, and dosage dependent functions of C/EBPs (Begay, 2004).

Inducible enhancement of memory storage and synaptic plasticity in transgenic mice expressing an inhibitor of ATF4 (CREB-2) and C/EBP proteins

To examine the role of C/EBP-related transcription factors in long-term synaptic plasticity and memory storage, the tetracycline-regulated system was used and a broad dominant-negative inhibitor of C/EBP (EGFP-AZIP) was expressed in the forebrain of mice. This inhibitor preferentially interacts with several inhibiting isoforms of C/EBP. EGFP-AZIP also reduces the expression of ATF4, a distant member of the C/EBP family of transcription factors that is homologous to the Aplysia memory suppressor gene ApCREB-2. Consistent with the removal of inhibitory constraints on transcription, an increase was found in the pattern of gene transcripts in the hippocampus of EGFP-AZIP transgenic mice and both a reversibly enhanced hippocampal-based spatial memory and LTP. These results suggest that several proteins within the C/EBP family including ATF4 (CREB-2) act to constrain long-term synaptic changes and memory formation. Relief of this inhibition lowers the threshold for hippocampal-dependent long-term synaptic potentiation and memory storage in mice (Chen, 2003; full text of article).

C/EBP and Gonads

The transcription factor C/EBP beta (CCAAT/enhancer-binding protein beta) is expressed in ovaries and testes, as well as many other tissues of adult mice. Mice carrying a targeted deletion of the C/EBP beta gene exhibit reproductive defects. Although these animals develop normally and males are fertile, adult females are sterile. Transplantation of normal ovaries into mutant females restores fertility, thus localizing the primary reproductive defect to the ovary proper. In normal ovaries, C/EBP beta mRNA is specifically induced by luteinizing hormone (LH/hCG) in the granulosa layer of preovulatory antral follicles. C/EBP beta-deficient ovaries lack corpora lutea and fail to down-regulate expression of the prostaglandin endoperoxidase synthase 2 and P450 aromatase genes in response to gonadotropins. These findings demonstrate that C/EBP beta is essential for periovulatory granulosa cell differentiation in response to LH. C/EBP beta is thus established as a critical downstream target of G-protein-coupled LH receptor signaling and one of the first transcription factors, other than steroid hormone receptors, known to be required for ovarian follicle development in vivo (Sterneck, 1997).

C/EBP and Liver

The liver-enriched transcription factor C/EBP alpha has been implicated in the regulation of numerous liver-specific genes. Mice carrying a homozygous null mutation at the c/ebp alpha locus die as neonates due to the absence of hepatic glycogen and the resulting hypoglycemia. However, the lethal phenotype precludes further analysis of the role of C/EBP alpha in hepatic gene regulation in adult mice. To circumvent this problem, a conditional knockout allele of c/ebp alpha was constructed by using the Cre/loxP recombination system. Homozygous c/ebp-loxP mice are indistinguishable from their wild-type counterparts. However, when subject to conditional knockout, more than 80% of the conditional c/ebp genes are deleted specifically in liver and C/EBP alpha expression is reduced by 90%. This condition results in a reduced level of bilirubin UDP-glucuronosyltransferase expression in the liver. After several days, the knockout mice develop severe jaundice due to an increase in unconjugated serum bilirubin. The expression of genes encoding phosphoenolpyruvate carboxykinase, glycogen synthase, and factor IX is also strongly reduced in adult conditional-knockout animals, while the expression of transferrin, apolipoprotein B, and insulin-like growth factor I genes is not affected. These results establish C/EBP alpha as an essential transcriptional regulator of genes encoding enzymes involved in bilirubin detoxification and gluconeogenesis in adult mouse liver (Lee, Y.-H., 1997b).

Hepatocyte growth factor (HGF) is an inducible cytokine; it is essential for the normal growth and development of various tissues, such as the liver. To decipher the molecular mechanisms that regulate HGF gene induction at the transcriptional level, in vitro and in vivo studies were carried out on the mouse HGF gene promoter. A novel regulatory element, located between -6 and +7 bp (from the transcription start site) in the HGF basal promoter region has been identified, which binds to inducible transcription factors and dictates responsiveness to extracellular stimuli that activate this gene. The core binding sequence for the inducible cis-acting factors is TTTGCAA (-4 to +3 bp) within the HGF promoter. Competition and gel mobility supershift assays show that these binding complexes are composed of C/EBPbeta (CCAAT/enhancer-binding protein beta) and C/EBPdelta. DNA binding analysis also revealed that the binding site for the C/EBP family of transcription factors in the HGF promoter region overlaps that of another binding protein (complex C1), which binds specifically to a novel sequence with a core binding site of ACCGGT located adjacent to the C/EBP site (-9 to -4 bp). C1 binds to this region of the promoter and represses the inducible upregulation by C/EBP through direct competition for C/EBP's individual binding sites. Partial hepatectomy, known to activate HGF gene expression in the liver, increases C/EBP (especially C/EBPbeta) binding activity to this region of the HGF promoter. Thus, a mechanistic explanation is provided for the transcriptional induction of the HGF gene by extracellular signals (i.e., cytokines) that induce tissue growth and regeneration (Jiang, 1997).

The ability of C/EBPbeta but not C/EBPalpha to synergize with an SP1 (Drosophila homolog: Buttonhead) protein is specified by the leucine zipper and activation domain. The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. Liver-specific transcription fo the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5 that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBPalpha and C/EBPbeta are expressed abundantly in liver, only C/EBPbeta is capable of stimulating the 2D5 promoter in HepG2 cells. In addition, activation of the 2D5 promoter by C/EBPbeta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBPbeta proteins containing either the leucine zipper or the activation domain of C/EBPalpha are unable to stimulate the 2D5 promoter. The serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperativity with C/EBPbeta. These findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein (Y.-H. Lee, 1997a).

LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrates that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. Two sites have been identified that are important for specific complex formation within this region. CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that shown for the CREB consensus sequence. These sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. The protein kinase A pathway not only stimulates the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. After two-thirds hepatectomy a functional link exists between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. Since several pathways control CREB phosphorylation, these results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions (Niehof, 1997).

Knockout of C/EBPalpha causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, a new C/EBPalpha-null mouse strain was generated in which C/EBPß, in addition to its own expression, substituted for C/EBPalpha expression in tissues. The homozygous mutant mice C/ebpalpha(ß/ß) are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPalpha-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebpalpha(ß/ß) and wild-type mice. However, despite their normal growth rate, C/ebpalpha(ß/ß) mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebpalpha(ß/ß) mice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. This study demonstrates that when expressed from the C/ebpalpha gene locus, C/EBPß can act for C/EBPalpha to maintain liver functions during development. Moreover, these studies with the C/ebpalpha(ß/ß) mice provide new insights into the nonredundant functions of C/EBPalpha and C/EBPß on gene regulation in WAT (Chen, 2000).

C/EBP and mammary cell development

Studies of C/EBPbeta-deficient mice have demonstrated a pivotal role for this transcription factor in hematopoiesis, adipogenesis, and ovarian function. C/EBPbeta is also essential for normal development and function of the mammary gland. Ductal morphogenesis in virgin C/EBPbeta-deficient mice is disrupted, with ducts displaying reduced growth and branching. To distinguish whether the effect of C/EBPbeta deficiency on mammary epithelium is indirect or cell autonomous, ovarian and mammary gland transplants were performed. Transplants of wild-type ovaries into mutant females partially restore ductal morphogenesis during puberty but fail to support mammopoiesis during pregnancy. At term, mutant mice harboring wild-type ovaries exhibit reduced alveolar proliferation and impaired epithelial cell differentiation, including a complete absence of milk protein expression. Mammary gland transplant experiments demonstrate that development of C/EBPbeta-deficient epithelium is defective within a wild-type stroma and host background. Cell proliferation during pregnancy is reduced and differentiation, as measured by the activity of milk protein genes, is inhibited. However, wild-type epithelium develop in a C/EBPbeta-deficient stroma. Thus, C/EBPbeta plays an essential, cell autonomous role in the proliferation and differentiation of mammary secretory epithelial cells and is required for the activation of milk protein genes (Robinson, 1998).

The CCAAT/enhancer binding proteins (C/EBPs) are differentially expressed throughout mammary gland development and interact with binding sites within the promoter of a milk protein gene, beta-casein. The specific roles of C/EBPbeta and C/EBPalpha in mouse mammary gland development and differentiation have been investigated in mice that carry targeted deletions of these genes. C/EBPbeta-/- virgin mice exhibit cystic, enlarged mammary ducts with decreased secondary branching. Transplantation of C/EBPbeta-/- mammary epithelium into the cleared mammary fat pads of nude mice confirms that this defect in ductal morphogenesis is intrinsic to the epithelium. When treated with estrogen/progesterone (E+P) to simulate pregnancy, C/EBPbeta-/- mammary glands display only limited lobuloalveolar development and ductal side branching. Primary mammary epithelial cells obtained from E+P-treated C/EBPbeta-/- mice that are cultured on extracellular matrix gels do not functionally differentiate in response to lactogenic hormones despite their organization into three-dimensional structures. Expression of beta-casein protein is inhibited 85%-100% and whey acidic protein (WAP) is undetectable. In contrast, no detectable alterations in mammary development or beta-casein expression are observed in mammary outgrowths derived from newborn C/EBPalpha-/- mammary epithelium transplanted into the cleared mammary fat pads of syngeneic hosts. These results demonstrate that C/EBPbeta, but not C/EBPalpha, is required for ductal morphogenesis, lobuloalveolar development, and functional differentiation of mammary epithelial cells (Seagroves, 1998).

C/EBP and hematopoiesis

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) regulates a number of myeloid cell-specific genes. To delineate the role of C/EBPalpha in human granulopoiesis, its expression and function was studied in human primary cells and bipotential (granulocytic/monocytic) myeloid cell lines. The expression of C/EBPalpha initiates with the commitment of multipotential precursors to the myeloid lineage, is specifically upregulated during granulocytic differentiation, and is rapidly downregulated during the alternative monocytic pathway. Conditional expression of C/EBPalpha alone in stably transfected bipotential cells triggers neutrophilic differentiation, concomitant with upregulation of the granulocyte-specific granulocyte colony-stimulating factor receptor and secondary granule protein genes. Induced expression of C/EBPalpha in bipotential precursors blocks their monocytic differentiation program. These results indicate that C/EBPalpha serves as a myeloid differentiation switch acting on bipotential precursors and directing them to mature to granulocytes (Radomska, 1998).

Hematopoietic differentiation involves the commitment of multipotent progenitors to a given lineage, followed by the maturation of the committed cells. To study the transcriptional events controlling these processes, the role of C/EBP proteins has been investigated in lineage choice of multipotent hematopoietic progenitors (MEPs) transformed by the E26 virus. Forced expression in MEPs of either the alpha or beta isoforms of C/EBP induces eosinophil differentiation; in addition, C/EBPbeta can induce myeloid differentiation. Conversely, dominant-negative versions of C/EBPbeta inhibit myeloid differentiation. C/EBP-induced eosinophil differentiation can be separated into two distinct events: lineage commitment and maturation. Thus, eosinophils induced by transactivation-deficient C/EBPbeta alleles are found to be blocked in their maturation, whereas those expressing wild-type C/EBP proteins are not. Likewise, a 1-day activation of a conditional C/EBPbeta allele in multipotent progenitors leads to the formation of immature eosinophils, whereas sustained activation produces mature eosinophils. These results show that C/EBP can induce both myeloid and eosinophil lineage commitment and that transactivation independent and dependent C/EBP functions are required during eosinophil lineage commitment and maturation, respectively (Nerlov 1998).

The commitment of multipotent cells to particular developmental pathways requires specific changes in their transcription factor complement to generate the patterns of gene expression characteristic of specialized cell types. The role of the GATA cofactor Friend of GATA (FOG: Drosophila homolog U-shaped) in the differentiation of avian multipotent hematopoietic progenitors has been examined. Multipotent cells express high levels of FOG mRNA, which are rapidly down-regulated upon their C/EBP-mediated commitment to the eosinophil lineage. Expression of FOG in eosinophils leads to a loss of eosinophil markers and the acquisition of a multipotent phenotype, and constitutive expression of FOG in multipotent progenitors blocks activation of eosinophil-specific gene expression by C/EBPbeta. These results show that FOG is a repressor of the eosinophil lineage, and that C/EBPbeta-mediated down-regulation of FOG is a critical step in eosinophil lineage commitment. Furthermore, the results indicate that maintenance of a multipotent state in hematopoiesis is achieved through cooperation between FOG and GATA-1. A model is presented in which C/EBPbeta induces eosinophil differentiation by the coordinate direct activation of eosinophil-specific promoters and the removal of FOG, a promoter of multipotency as well as a repressor of eosinophil gene expression (Querfurth, 2000).

The CCAAT boxes found in the promoters of beta-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. The NFE-6 complex contains C/EBPgamma. In mice, FNE-6's role in globin gene regulation is addressed by erythroid overexpression of C/EBPgamma, and a dominant-negative form C/EBPgammaDeltaB. Elevated levels of C/EBPgamma, but not C/EBPgammaDeltaB, increase expression of the (fetal) gamma-globin relative to the (adult) beta-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPgamma and C/EBPgammaDeltaB levels are further increased in homozygous transgenics. It is suggested that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice (Zafarana, 2000).

C/EBP and the myeloid lineage

Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. The temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta has been determined in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While C/EBP alpha, C/EBP beta, and C/EBP delta have been detected in multiple myeloid leukemia cell lines, and C/EBP alpha has been detected in normal myeloid cells and in de novo human myeloid leukemias, none of these C/EBP isoforms have been detected in either erythroid or lymphoid cells. C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes (Scott, 1992).

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBPepsilon) 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/EBPepsilon activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. 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/EBPepsilon-/- mice and in SGD patients. Based on these observations, potential interactions between C/EBPepsilon and CDP/cut during neutrophil maturation were investigated. Inducible expression of C/EBPepsilon in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells has revealed the absence of C/EBPepsilon mRNA. It is therefore hypothesized that C/EBPepsilon positively regulates SGP gene expression, and that C/EBPepsilon is itself negatively regulated by CDP/cut during neutrophil maturation. The C/EBPepsilon promoter has been shown to be regulated by CDP/cut during myeloid differentiation (Khanna-Gupta, 2001).

It has been suggested that CDP/cut activity is restricted to proliferating cells, and that CDP/cut target genes are repressed in proliferating cells and are up-regulated as cells undergo cell cycle arrest and terminal differentiation. Target genes of CDP/cut include c-myc, c-mos, thymidine kinase (TK), cyclin-dependent kinase (cdk) inhibitor p21WAF1/CIP1, CFTR, TGFß-type II receptor, gp91phox, MHC class 1 locus, and the neutrophil SGP genes. Recently, CDP/cut has been shown to function as a repressor of transcription involving chromatin modification through recruitment of histone deacetylases (HDAC), consistent with the notion that transcriptional silencing is associated with hypoacetylated histones. Both acetylation of CDP/cut via p300/CBP and phosphorylation of CDP/cut are posttranscriptional modifications that have been postulated to regulate CDP/cut function. It is speculated that CDP/cut uses a different combination of its four binding elements to bind to the CDP/cut motifs in the promoters of C/EBPepsilon and the SGP genes, respectively, during neutrophil maturation. Differential modification, involving either phosphorylation and/or acetylation, of CDP/cut-DNA complexes in the promoters of C/EBPepsilon and SGP genes could result in the differential repression exerted by CDP/cut during neutrophil development (Khanna-Gupta, 2001 and references therein).

C/EBPalpha is a transcription factor required for differentiation of myeloid progenitors. In addition to specific DNA binding, C/EBPalpha is also involved in protein-protein interactions, some of which (p21, Cdk2/Cdk4, E2F) appear to be required for inhibition of proliferation and possibly differentiation. To investigate the mechanisms of C/EBPalpha-induced granulocytic differentiation, C/EBPalpha mutants were generated that were reportedly defective in DNA binding, transactivation, and Cdk2/Cdk4 and E2F interaction, and their effects were assessed in a myeloid precursor cell line, primary bone marrow and C/EBPalpha knockout fetal liver precursor cells. The DNA binding-deficient Lys298Glu mutant, the E2F binding-deficient basic region mutant 2 (BRM-2) carrying the Ile294Ala and Arg297Ala substitutions, and the transactivation-deficient N-terminus truncated p30 mutant all fail to promote differentiation on ectopic expression in myeloid precursor cells. By contrast, ectopic expression of the Cdk2/Cdk4 interaction-deficient Delta177-191 mutant promotes differentiation and induces gene expression as effectively as wild-type C/EBPalpha. Thus, the integrity of the transactivation and DNA binding domains, but not of the Cdk2/Cdk4 interaction region, is necessary for C/EBPalpha-induced differentiation. Since the E2F binding-deficient BRM-2 mutant interacts with E2F-1 but fails to activate gene expression, these results lend support to the hypothesis that activation of gene transcription is the determining factor in C/EBPalpha-dependent differentiation (Keeshan, 2003).

C/EBP and adipocytes

Continued: see slow border cells Evolutionary homologs part 2/3 | part 3/3

slow border cells: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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