Interactive Fly, Drosophila

Tumor necrosis factor initiates a cytolytic signaling cascade that leads to increased levels of reactive oxygen intermediates and the subsequent apoptosis of some tumor cell lines and virally infected cells. To combat the lethal effect of reactive oxygen intermediates, eukaryote cells have evolved several reactive oxygen-scavenging enzymes, including superoxide dismutase. Manganese superoxide dismutase (MnSOD), a TNF-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identify a 238-bp region of intron 2 that is responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) has the properties of a traditional enhancer element that functions in an orientation- and position-independent manner. IVGF of the TNFRE reveals that TNF- and IL-1-induces factor occupancy of sites that can bind NF-kappaB and C/EBP. The 5' portion of the TNFRE binds C/EBP-beta in vitro and is both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE binds both NF-kappaB and C/EBP but is not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE is required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter (Jones, 1997).

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. The monocyte/macrophage-enriched nuclear transcription factor C/EBPbeta plays an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex have been analyzed. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which does not possess endogenous C/EBPbeta, expression of c-Jun by itself has relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun is synergistic, resulting in greater than 130-fold activation. This effect requires both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, an examination was made of U937 cells that possess endogenous C/EBPbeta, which were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67). U937 cells expressing either ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line, in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggests that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperates with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. These data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction is unique in that it does not require the c-Jun transactivation domain; it provides new insight into the cell-type-specific regulation of the TNF alpha gene (Zagariya, 1998).

Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which synergistic, tissue-specific activation can be studied. Myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors that are crucial for normal hematopoiesis: PU.1 (an ETS domain transcription factor), AML1 (the mammalian homolog of Drosophila Runt), and C/EBPalpha. These proteins interact in such a way as to demonstrate at least two examples of synergistic activation. AML1 and C/EBPalpha are shown to activate the M-CSF receptor promoter in a synergistic manner. AML1 also synergizes, and interacts physically, with PU.1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPalpha has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPalpha, but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPalpha. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPalpha, and PU.1 (Petrovick, 1998).

The cardiogenic homeodomain factor Nkx-2.5 and serum response factor (SRF) provide strong transcriptional coactivation of the cardiac alpha-actin (alphaCA) promoter in fibroblasts. Nkx-2.5 is shown to also cooperate with GATA-4, a dual C-4 zinc finger transcription factor expressed in early cardiac progenitor cells, to activate the alphaCA promoter. A similar cooperation also takes place on a minimal promoter, containing only multimerized Nkx-2.5 DNA binding sites (NKEs), in heterologous CV-1 fibroblasts. Transcriptional activity requires the N-terminal activation domain of Nkx-2.5 and Nkx-2.5 binding activity through its homeodomain but does not require GATA-4's activation domain. The minimal interactive regions were mapped to the homeodomain of Nkx-2.5 and the second zinc finger of GATA-4. Removal of Nkx-2.5's C-terminal inhibitory domain stimulates robust transcriptional activity, comparable to the effects of GATA-4 on wild-type Nkx-2.5, which in part facilitates Nkx-2.5 DNA binding activity. The following simple model is proposed: GATA-4 induces a conformational change in Nkx-2.5 that displaces the C-terminal inhibitory domain, thus eliciting transcriptional activation of promoters containing Nkx-2.5 DNA binding targets. Therefore, alphaCa promoter activity appears to be regulated through the combinatorial interactions of at least three cardiac tissue-enriched transcription factors, Nkx-2.5, GATA-4, and SRF (Sepulveda, 1998).

The transcription of the alpha1-acid glycoprotein gene (agp) is induced by inflammatory cytokines and glucocorticoids. C/EBPbeta is a major transcription factor involved in the induction of the agp gene by some cytokines. A novel transcriptional intermediary factor, TIF1beta, has been identified which can enhance the transcription of the agp gene by the glucocorticoid receptor (GR) and C/EBPbeta. TIF1beta belongs to a subgroup of RING (really interesting new gene) finger proteins that contain a RING finger preceding two B box-type fingers and a putative coiled-coil domain (RBCC domain). Immunoprecipitation experiments show that the interaction between GR and TIF1beta is ligand independent. The overexpression of the TIF1beta gene enhances GR-regulated expression in a ligand- and glucocorticoid-responsive element (GRE)-dependent manner. TIF1beta can also augment C/EBPbeta-mediated activity on wild-type and GRE-mutated agp genes, but this augmentation is diminished when all three C/EBPbeta-binding elements are mutated. Functional and biochemical characterizations indicated that the bZIP domain of C/EBPbeta, as well as the RBCC domain, PHD finger, and bromodomain of TIF1beta, are each crucial for the interactions of these two proteins. Taken together, these results suggest that TIF1beta serves as a converging mediator of signal transduction pathways for glucocorticoids and some inflammatory cytokines (Chang, 1998).

The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. In steroidogenic tissues, StAR expression is acutely regulated by trophic hormones through a cAMP second messenger pathway, leading to increased StAR mRNA levels within 30 min, reaching maximal levels after 4-6 h of stimulation. The molecular mechanisms underlying such regulation remain unknown. The StAR promoter was examined for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormone-induced context. Two putative CCAAT/enhancer binding protein (C/EBP) DNA elements have been identified at -113 (C1) and -87 (C2) in the mouse StAR promoter. C/EBP beta binds with high affinity to C1, but C2 proves to be a low-affinity C/EBP site. Functional analysis of these sites in the murine StAR promoter show that mutation of one or both of these binding sites decreases both basal and (Bu)2cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation [(Bu)2cAMP-stimulated/basal] of the promoter. These two C/EBP binding sites are required for steroidogenic factor-1 (SF-1)-dependent transactivation of the StAR promoter in a nonsteroidogenic cell line. These data indicate that in addition to SF-1, C/EBP beta is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis (Reinhart, 1999).

The epidermis forms a vital barrier composed of stratified keratinocytes and their differentiated products. One of these products, keratin K10, is critical to epidermal integrity, because mutations in k10 lead to abnormal blistering. For the normal expression of k10, differentiation-associated transcription factors C/EBPalpha, C/EBPbeta, and AP-2 (see Drosophila AP-2) are well positioned to play an important role. Regulation of the k10 gene has been examined in keratinocytes in the skin of normal mice and in transgenic mice carrying targeted deletions of c/ebpbeta and ap-2alpha. In cultured cells, C/EBPalpha and C/EBPbeta are each capable of activating the k10 promoter via three binding sites, identified by site-directed mutagenesis. In a given epidermal cell in vivo, however, the selection of C/EBPalpha versus C/EBPbeta for k10 regulation is determined via a third transcription factor, AP-2. This novel regulatory scheme involves: (1) unique gradients of expression for each transcription factor, i.e., C/EBPbeta and AP-2 are most abundant in the lower epidermis, C/EBPalpha in the upper; (2) C/EBP-binding sites in the ap-2alpha gene promoter, through which C/EBPbeta stimulates ap-2alpha; and (3) AP-2 binding sites in the c/ebpalpha promoter, through which AP-2 represses c/ebpalpha. Promoter-analysis and gene-expression data presented herein support a regulatory model in which C/EBPbeta activates and maintains AP-2 expression in basal keratinocytes, whereas AP-2 represses C/EBPalpha in those cells. In response to differentiation signals, loss of AP-2 expression leads to derepression of the c/ebpalpha promoter and activation of k10 as cells migrate upward (Maytin, 1999).

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. Human beta1 sGC transcriptional regulation was investigated in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, this study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression (Sharina, 2003).

Odd-skipped related 2 (Osr2) gene is mouse homolog of Drosophila Odd-skipped gene. This study examined Osr2 expression regulation. The mouse Osr2 promoter region was cloned and characterized, and found to have two enhancer elements in the -1463/-1031 (distal) and -581/+3 (proximal) regions, and a repressor region (-4845/-1463, far distal). CCAAT/enhancer binding protein (C/EBP) binding sites were found in both the distal and proximal enhancer elements. Osr2 promoter activity was enhanced by C/EBPδ, a member of the C/EBP family, in a dose-dependent manner. Electrophoresis mobility shift assays showed that purified GST-C/EBPδ bound to distal (-1295/-1261) and proximal (-89/-55) C/EBP binding motifs. Chromatin immunoprecipitation demonstrated that acetylated histones H3, H4, and C/EBPδ in the proximal region (-280/-43), but not the distal region (-1438/-1196), indicating that the Osr2 promoter proximal region was transcriptionally activated in C3H10T1/2 cells. These results suggest that Osr2 expression is regulated by C/EBP regulatory elements (Kawai, 2006).

C/EBP, cell cycle and growth control

CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein (see Drosophila Dacapo). The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented in this paper. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life, when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers, which lack C/EBP alpha, suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Coimmunoprecipitation and a mammalian two-hybrid assay system reveals the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showsthat C/EBP alpha blocks proteolytic degradation of p21. These data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels (Timchenko, 1997).

The rate of hepatocyte proliferation in livers from newborn C/EBPalpha knockout mice is increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level is reduced in the knockout animals compared to that in wild-type littermates. Additional cell cycle-associated proteins are affected by C/EBPalpha. C/EBPalpha controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBPalpha mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPalpha knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction results in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses reveal an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals (Timchenko, 1999).

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. WA short growth inhibitory region of C/EBPalpha maps 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).

C/EBPalpha causes growth arrest via direct interaction with the cyclin-dependent kinases cdk2 and cdk4. Evidence is presented showing that C/EBPalpha enhances a proteasome-dependent degradation of cdk4 during growth arrest in liver of newborn mice and in cultured cells. Overexpression of C/EBPalpha in several biological systems leads to a reduction of cdk4 protein levels, but not mRNA levels. Experiments with several tissue culture models reveal that C/EBPalpha enhances the formation of cdk4-ubiquitin conjugates and induces degradation of cdk4 through a proteasome-dependent pathway. As a result, the half-life of cdk4 is shorter and protein levels of cdk4 are reduced in cells expressing C/EBPalpha. Gel filtration analysis of cdk4 complexes shows that a chaperone complex cdk4-cdc37-Hsp90, which protects cdk4 from degradation, is abundant in proliferating livers that lack C/EBPalpha, but this complex is weak or undetectable in livers expressing C/EBPalpha. These studies show that C/EBPalpha disrupts the cdk4-cdc37-Hsp90 complex via direct interaction with cdk4 and reduces protein levels of cdk4 by increasing proteasome-dependent degradation of cdk4 (Wang, 2002).

The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPalpha, normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). Evidence that aging switches the liver-specific pathway of C/EBPalpha growth arrest to repression of E2F transcription. An age-specific C/EBPalpha-Rb-E2F4 complex has been identified that binds to E2F-dependent promoters and represses these genes. The C/EBPalpha-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. These results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy, providing a possible mechanism for the age-dependent loss of liver regenerative capacity (Iakova, 2003).

Liver tumor cells arise from normal hepatocytes that escape negative control of proliferation. The transcription factor C/EBPalpha maintains quiescence of hepatocytes through two pathways: inhibition of cdks and repression of E2F. Nevertheless, liver tumors and cultured hepatoma cell lines proliferate in the presence of C/EBPalpha. Evidence suggests that the activation of the PI3K/Akt pathway in liver tumor cells blocks the growth inhibitory activity of C/EBPalpha through the PP2A-mediated dephosphorylation of C/EBPalpha on Ser 193, leading to a failure of C/EBPalpha to interact with and inhibit cdks and E2F. Mutation of Ser 193 to Ala also abolishes the ability of C/EBPalpha to cause growth arrest because of a lack of interactions with cdk2 and E2F-Rb complexes. These data provide a molecular basis for the development of liver tumors in which the activation of PI3K/Akt pathway neutralizes C/EBPalpha growth inhibitory activity (Wang, 2004).

C/EBP and neural development

Mammalian neurogenesis is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues such as growth factors. A signaling cascade, a MEK-C/EBP pathway, has been defined that is essential for cortical progenitor cells to become postmitotic neurons. Inhibition of MEK or of the C/EBP family of transcription factors inhibits neurogenesis while expression of a C/EBPß mutant that is a phosphorylation-mimic at a MEK-Rsk site enhances neurogenesis. C/EBP mediates this positive effect by direct transcriptional activation of neuron-specific genes such as Talpha1 alpha-tubulin. Conversely, inhibition of C/EBP-dependent transcription enhances CNTF-mediated generation of astrocytes from the same progenitor cells. Thus, activation of a MEK-C/EBP pathway enhances neurogenesis and inhibits gliogenesis, thereby providing a mechanism whereby growth factors can selectively bias progenitors to become neurons during development (Ménard, 2002).

Sustained CPEB-dependent local protein synthesis is required to stabilize synaptic growth for persistence of long-term facilitation in Aplysia

The time course of the requirement for local protein synthesis in the stabilization of learning-related synaptic growth and the persistence of long-term memory was examined using Aplysia bifurcated sensory neuron-motor neuron cultures. Following repeated pulses of serotonin (5-HT), the local perfusion of emetine, an inhibitor of protein synthesis, or a TAT-AS oligonucleotide directed against ApCPEB blocks long-term facilitation (LTF) at either 24 or 48 hr and leads to a selective retraction of newly formed sensory neuron varicosities induced by 5-HT. By contrast, later inhibition of local protein synthesis, at 72 hr after 5-HT, has no effect on either synaptic growth or LTF. These results define a specific stabilization phase for the storage of long-term memory during which newly formed varicosities are labile and require sustained CPEB-dependent local protein synthesis to acquire the more stable properties of mature varicosities required for the persistence of LTF (Miniaci, 2008).

To explore how local protein synthesis is regulated, the role of a translational regulator, CPEB, was examined. The stabilization of 5-HT-induced synaptic growth and the persistence of LTF rely upon the local synthesis of ApCPEB as well as protein synthesis. These results reinforce the finding that ApCPEB plays a critical role in the long-term maintenance of LTF and suggest that the activity of ApCPEB as a translational regulator is required during a specific temporal window when the 5-HT-induced synaptic changes are labile and must be stabilized to persist. Interestingly, CPEB appears to be more active and, therefore, more capable of regulating the synthesis of other mRNAs when it is in the prion-like state (Si, 2003). Since the conversion of CPEB molecules to prion-like states requires high levels of the proteins, as observed following 5-HT stimulation, it is likely that several peak periods of protein synthesis occur during this stabilization phase. It is also possible that CPEB stimulates synthesis of itself, thus creating a positive feedback loop. Such sustained synthesis of CPEB might be necessary to compensate the short half-life of the protein, as demonstrated in the pleural ganglia of Aplysia where the induced level of CPEB following 5-HT treatment returns almost to the basal levels within 5-6 hr (Si, 2003). After this stabilization phase is over, ApCPEB as well as local protein synthesis are either not required or may still be necessary, but perhaps at lower levels of activity, which then could serve to maintain the total population of sensory neuron varicosities, i.e., both preexisting and newly formed, for the duration of the memory (Miniaci, 2008).

The existence of this later, critical period for local translation at the sensory to motor neuron synapse during LTF is consistent with the idea that the requirement of protein synthesis during long-term memory is likely to consist of multiple time-dependent phases, perhaps each with its own molecular requirements, that encompass the initiation, expression, and maintenance of memory storage. The relatively brief time window of these protein synthesis-dependent phases compared to the duration of the memory further suggests that high levels of translation are not continuously required over the entire life span of the memory and raises the question of how the molecular memory at the synapse is maintained. The findings that local protein synthesis selectively stabilizes 5-HT-induced synaptic growth suggest that activity-dependent regulation of the proteome at the synapse is one attractive candidate for the molecular mechanisms that underlie the persistence of memory storage (Miniaci, 2008).

C/EBP and long-term memory

Long-term facilitation of the connections between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia requires five repeated pulses of serotonin (5-HT). The repeated pulses of 5-HT initiate a cascade of gene activation that leads ultimately to the growth of new synaptic connections. Several genes in this process have been identified, including the transcriptional regulators apCREB-1, apCREB-2, apC/EBP, and the cell adhesion molecule apCAM, which is thought to be involved in the formation of new synaptic connections. The transcriptional regulators apCREB-2 and apC/EBP, as well as a peptide derived from the cytoplasmic domain of apCAM, are phosphorylated in vitro by Aplysia mitogen-activated protein kinase (apMAPK). The cDNA encoding apMAPK (see Drosophila MAPK) has been cloned and apMAPK activity is increased in sensory neurons treated with repeated pulses of 5-HT and by the cAMP pathway. These results suggest that apMAPK may participate with cAMP-dependent protein kinase during long-term facilitation in sensory cells by modifying some of the key elements involved in the consolidation of short- to long-lasting changes in synaptic strength. How might the PKA and MAPK pathways converge in Aplysia sensory neurons? In the simplest case, the MAPK pathway would be downstream from the PKA pathway. In many cells, PKA negatively regulates the MAPK pathway by phosphorylating Raf-1. However, in B-Raf-containing cells, PKA activates the MAPK pathway by signaling through Rap1, a member of the Ras family of small G proteins. PKA may thus activate MAPK by phosphorylating the Aplysia homolog of Rap1, therby activating B-Raf, MEK, and MAPK (Martin, 1998).

The consolidation of long-term memory requires protein and mRNA synthesis. A similar requirement has been demonstrated for learning-related synaptic plasticity in the gill-withdrawal reflex of Aplysia. The monosynaptic component of this reflex can be reconstituted in vitro, where it undergoes both short- and long-term increases in synaptic strength in response to serotonin (5-HT), a neurotransmitter released during behavioral sensitization, a simple form of learning. As with sensitization, the long-term synaptic modification is characterized by a brief consolidation period during which gene expression is required. During this phase, the transcription factor Aplysia CCAAT enhancer-binding protein (ApC/EBP) is induced rapidly by 5-HT and by cAMP, even in the presence of protein synthesis inhibitors. Blocking the function of ApC/EBP blocks long-term facilitation selectively without affecting the short-term process. These data indicate that cAMP-inducible immediate-early genes have an essential role in the consolidation of stable long-term synaptic plasticity in Aplysia (Alberini, 1994).

Long-term facilitation (LTF) of the sensory-to-motor synapses that mediate defensive reflexes in Aplysia requires induction of the transcription factor Aplysia CCAAT/enhancer binding protein (ApC/EBP) as an early response gene. The time course of ApC/ EBP DNA binding during the induction of LTF was examined: binding activity is detected within 1 h of the sensitization treatment with serotonin, reaches a maximum at 2 h, and decreases after 6 h. How are DNA binding and the turnover of ApC/EBP regulated? Phosphorylation of ApC/EBP by mitogen-activated protein (MAP) kinase is essential for binding. MAP kinase appears to be activated through protein kinase C. ApC/EBP is degraded through the ubiquitin-proteasome pathway but phosphorylation by MAP kinase renders it resistant to proteolysis. Thus, phosphorylation by MAP kinase is required for ApC/EBP to act as a transcription activator as well as to assure its stability early in the consolidation phase, when genes essential for the development of LTF begin to be expressed (Yamamoto, 1999).

Patterns of gene expression in human tumors have been deconvoluted to reveal a mechanism of action for the cyclin D1 oncogene. Computational analysis of the expression patterns of thousands of genes across hundreds of tumor specimens suggest that a transcription factor, C/EBPß/Nf-Il6, participates in the consequences of cyclin D1 overexpression. Functional analyses confirmed the involvement of C/EBPß in the regulation of genes affected by cyclin D1 and established this protein as an indispensable effector of a potentially important facet of cyclin D1 biology. This work demonstrates that tumor gene expression databases can be used to study the function of a human oncogene in situ (Lamb, 2003).

It has been assumed that the established ability of cyclin D1 to activate cdk4/6, leading to phosphorylation of pRb with consequent derepression of E2F-mediated transcription, and the resulting promotion of cell cycle progression, underlies the tumorigenic consequences of cyclin D1 overexpression. However, conventional analyses of human tumor material have frequently failed to find correlative evidence in support of this model. For example, breast cancers overexpressing cyclin D1 do not show correspondingly high levels of the canonical E2F target gene cyclin E. Similarly, hyperphosphorylation of pRb is not observed in B cell lymphomas overexpressing cyclin D1. Such data have led some to suggest that the oncogenic activity of cyclin D1 must be exerted through pathways other than cdk-dependent and E2F-mediated acceleration of the cell cycle (Lamb, 2003).

The similarity of the transcriptional consequences of ectopic overexpression of cyclin D1 and the cyclin D1 KE mutant (which is unable to activate cdk4), the paucity of E2F target genes in this cyclin D1 expression signature, and the absence of a correlation between the expression patterns of these E2F target genes and cyclin D1 argue directly against activation of cdk4 or sequestration of cdk inhibitor proteins by catalytically inactive cyclin D1/cdk4 complexes as the mechanism of cyclin D1 action in human tumors. Therefore, a data-mining strategy (KSS) was developed to exploit the experimentally determined expression signature for the identification of potential participants in the mechanism of cyclin D1 oncogenicity ab initio. A transcription factor previously unconnected with cyclin D1 was ranked very highly across a number of independent tumor gene expression databases. Subsequent functional analyses confirmed that C/EBPβ is indeed a direct effector of the activity of cyclin D1 encoded in its expression signature. These findings indicate that the pathways connecting cyclin D1 with E2F-mediated transcription are perhaps not so germane as previously thought and instead implicate modulation of C/EBPβ function as a major mechanism of cyclin D1 action in human cancer (Lamb, 2003).

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