CrebB-17A

Targets of Activity

Decapentaplegic (Dpp) is an extracellular signal of the transforming growth factor-beta family with multiple functions during Drosophila development. For example, it plays a key role in the embryo during endoderm induction. During this process, Dpp stimulates transcription of the homeotic genes Ultrabithorax in the visceral mesoderm and labial in the subjacent endoderm. A cAMP response element (CRE) from an Ultrabithorax enhancer mediates Dpp-responsive transcription in the embryonic midgut, and endoderm expression from a labial enhancer depends on multiple CREs. The enhancer, called Ubx B confers Wingless- and Decapentaplegic-dependent expression in the visceral mesoderm. Staining mediated by Ubx B is in two stripes of cells in the visceral mesoderm, a wide prominent one in parasegments 6-9 and a narrow weak one in parasegment 3. The Drosophila CRE-binding protein dCREB-2 binds to the Ultrabithorax CRE. Binding is at a palindromic sequence TGGCGTCA that resembles a typical cAMP response element (CRE) (TGACGTCA). Mutation of this site results in the elimination of response to Dpp, but a maintenance of response to Wg. This residual expression is in parasegment 8 and 9 coinciding with the main source of wg expression in the middle midgut. The Ubx CRE can also mediate response to Dpp signaling in the endoderm. Other transcription factors act through the Ubx B enhancer to confer its tissue-specific response to Dpp in the visceral mesoderm. CRE needs to cooperate with a LEF-1 binding site to respond to the Dpp signal in the visceral mesoderm. Schnurri, a transcription factor implicated in Dpp signaling, fails to interact with Ubx B. Adjacent to the CRE is another palindromic sequence that antagonizes the activating effects of Dpp and Wg signaling on the Ubx B enhancer. Ubiquitous expression of a dominant-negative form of dCREB-2 suppresses CRE-mediated reporter gene expression and reduces labial expression in the endoderm. Therefore, a dCREB-2 protein may act as a nuclear target, or as a partner of a nuclear target, for Dpp signaling in the embryonic midgut (Eresh, 1997).

Long term memory requires de novo gene expression mediated by CREB family genes. Using an inducible transgene that expresses CrebB-17A, the dominant negative member of the CREB family, long term memory has been specifically and completely blocked while short term memory (anasthesia-resistent memory) remains unaffected (Yin 1995b).

Induced expression of a CrebB-17A activator isoform enhances long term memory in Drosophila, so that maximum learning is achieved after only one training session. Memory requires phosphorylation of the activator isoform (Yin 1995b).

Fasciclin2 mutants lead to an increase in number of boutons at neuromuscular synapses without affecting quantal content. Increased cAMP in dunce mutants increases both synaptic structure and quantal content. Thus there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with Fas2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, CrebB-17A-a, in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, CrebB-17A-a, increases synaptic strength, by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of CrebB-17A-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release and expression of CrebB-17A-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity. It is concluded that cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, G. W 1996).

Drosophila CREB genes are implicated in regulation of the Drosophila homolog of mammalian JUN. A 43-bp 5' proximal promoter region is necessary for the transcription activity of DJUN (Perkins, 1988a). Deletion of this fragment decreases transcriptional activity 67-fold. This 43-bp sequence alone, containing a Drosophila transcription factor DTF-1 binding site and TATA box, however, is not sufficient for transcription activity. An 80-bp sequence including the start of transcription has considerable basal activity. This intragenic region containing an AP-1 site and a CRE site (presumably binding a Drosophila CREB) modulates or fine tunes activity of the promoter. An extragenic region containing two AP-1 sites similarly affects promoter activity (Wang, 1994).

The responsiveness of DJun to CREB suggests a role for DJun in the preservation of long term memory in the fly. To date, such a role has not been documented.

Protein Interactions

In the cyclic AMP signal transduction pathway, protein kinase A (PKA) activates CREB by phosphorylation (Drain, 1991). The isoform CrebB-17A-a is a PKA dependent activator of transcription (Yin 1994a). The isoform CrebB-17A-b does not function as a PKA dependent activator, but works as a direct antagonist of PKA-dependent activation by CrebB-17A-a (Yin 1995a).

Interactions of Creb-binding Protein

Drosophila CBP (Nejire) is a co-activator of cubitus interruptus in hedgehog signaling. Drosophila CBP predicts a protein of relative molecular mass 332,000; the gene maps to position 8F/9A on the X chromosome. Mutants for dCBP gene, nejire, die at stages 9 or 10 during embryogenesis, although some embryos survive to hatching. The most severe phenotype of the nej hemizygotes is the twisting of the embryo at germband elongation. The expression of wingless is strikingly reduced at the posterior margin of each parasegment in mutants. In addition, engrailed expression, which is maintained by WG protein, is significantly lower than in wild type. These observations suggest the Drosophila CBP might contribute to the functioning of some transcription factors involved in the wingless-engrailed signaling pathway. Cubitus interruptus protein physically interacts with Drosophila CBP (dCBP). A series of deletion mutants of ci indicate that a region of CI between amino acids 1020 and 1160 is required for phosphorylation independent interaction with dCBP. This region is part of the CI transactivation domain, C-terminal to five putative PKA sites. dCBP expression augments transactivation by CI up to a maximum of 62 fold. The dominant gain-of-function ciD mutant phenotype in which the longitudinal vein 4 of the adult wing is shortened, some posterior row hairs are missing, and the posterior wing margin is flattened, can be explained by the inappropriate expression of ci in the posterior compartment of the wing imaginal disc, where it is usually repressed by Engrailed. A subset of the ciD wing defects is suppressed by haploinsufficiency of dCBP. Thus dCBP is required for the activation of Cubitus interruptus target genes such as patched, and CBP is required for the activator function of CI but not for the repressor function. dCBP binds to dCREB2, the Drosophila homolog of CREB, in a phosphorylation-dependent manner, whereas the dCBP-CI interaction is phosphorylation-independent. These findings raise the possiblilty that a limited amount of dCBP might be recruited to PKA-phosphorylated dCREB2, resulting in a decrease in CI activity, explaining the antagonistic actions of PKA and Hedgehog (Akimaru, 1997a).

Attempts to demonstrate trans-activation activity by the Drosophila Myb gene product have been unsuccessful so far. Co-transfection of Schneider cells with a plasmid expressing the Drosophila homolog of transcriptional co-activator CBP (dCBP) results in transactivation by Myb. Using this assay system, the functional domains of Myb have been analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution (Hou, 1997).

Although CREB-binding protein (CBP) functions as a co-activator of many transcription factors, relatively little is known about the physiological role of CBP. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haplo-insufficiency disorder characterized by abnormal pattern formation. Drosophila CBP is maternally expressed, suggesting that it plays a role in early embryogenesis. Mesoderm formation is one of the most important events during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal Dorsal protein, a transcription factor that is homologous to the NF-kappa B family of proteins. Drosophila CBP mutants fail to express twi and generate twisted embryos. This is explained by results showing that dCBP is necessary for Dorsal-mediated activation of the twi promoter (Akimaru, 1997b).

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein (dCBP) binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, dCBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of dTCF, and this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF (Waltzer, 1998).

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