InteractiveFly: GeneBrief

Cyclic-AMP response element binding protein A : Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References

Gene name - Cyclic-AMP response element binding protein A

Synonyms - dCREB-A

Cytological map position - 71D1-71D2

Function - transcription factor

Keywords - salivary gland, a major and direct regulators of secretory capacity

Symbol - CrebA

FlyBase ID: FBgn0004396

Genetic map position - 3-[42]

Classification - basic leucine zipper

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Chung, C. G., Kwon, M. J., Jeon, K. H., Hyeon, D. Y., Han, M. H., Park, J. H., Cha, I. J., Cho, J. H., Kim, K., Rho, S., Kim, G. R., Jeong, H., Lee, J. W., Kim, T., Kim, K., Kim, K. P., Ehlers, M. D., Hwang, D. and Lee, S. B. (2017). Golgi Outpost Synthesis Impaired by Toxic Polyglutamine Proteins Contributes to Dendritic Pathology in Neurons. Cell Rep 20(2): 356-369. PubMed ID: 28700938
Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. This study shows that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, these results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.
Troha, K., Im, J. H., Revah, J., Lazzaro, B. P. and Buchon, N. (2018). Comparative transcriptomics reveals CrebA as a novel regulator of infection tolerance in D. melanogaster. PLoS Pathog 14(2): e1006847. PubMed ID: 29394281
Host responses to infection encompass many processes in addition to activation of the immune system, including metabolic adaptations, stress responses, tissue repair, and other reactions. The response to bacterial infection in Drosophila melanogaster has been classically described in studies that focused on the immune response elicited by a small set of largely avirulent microbes. Thus, there is surprisingly limited knowledge of responses to infection that are outside the canonical immune response, of how the response to pathogenic infection differs from that to avirulent bacteria, or even of how generic the response to various microbes is and what regulates that core response. This study addressed these questions by profiling the D. melanogaster transcriptomic response to 10 bacteria that span the spectrum of virulence. Each bacterium was found to trigger a unique transcriptional response, with distinct genes making up to one third of the response elicited by highly virulent bacteria. A core set of 252 genes was identified that are differentially expressed in response to the majority of bacteria tested. Among these, it was determined that the transcription factor CrebA is a novel regulator of infection tolerance. Knock-down of CrebA significantly increased mortality from microbial infection without any concomitant change in bacterial number. Upon infection, CrebA is upregulated by both the Toll and Imd pathways in the fat body, where it is required to induce the expression of secretory pathway genes. Loss of CrebA during infection triggered endoplasmic reticulum (ER) stress and activated the unfolded protein response (UPR), which contributed to infection-induced mortality. Altogether, this study reveals essential features of the response to bacterial infection and elucidates the function of a novel regulator of infection tolerance.
Ragheb, R., Chuyen, A., Torres, M., Defaye, A., Seyres, D., Kremmer, L., Fernandez-Nunez, N., Tricoire, H., Rihet, P., Nguyen, C., Roder, L. and Perrin, L. (2017). Interplay between trauma and Pseudomonas entomophila infection in flies: a central role of the JNK pathway and of CrebA. Sci Rep 7(1): 16222. PubMed ID: 29176735
In mammals, both sterile wounding and infection induce inflammation and activate the innate immune system, and the combination of both challenges may lead to severe health defects, revealing the importance of the balance between the intensity and resolution of the inflammatory response for the organism's fitness. Underlying mechanisms remain however elusive. Using Drosophila, this study showed that, upon infection with the entomopathogenic bacterium Pseudomonas entomophila (Pe), a sterile wounding induces a reduced resistance and increased host mortality. To identify the molecular mechanisms underlying the susceptibility of wounded flies to bacterial infection, the very first steps of the process were analyzed by comparing the transcriptome landscape of infected (simple hit flies, SH), wounded and infected (double hit flies, DH) and wounded (control) flies. Overexpressed genes in DH flies compared to SH ones are significantly enriched in genes related to stress, including members of the JNK pathway. The JNK pathway plays a central role in the DH phenotype by manipulating the Jra/dJun activity. Moreover, the CrebA/Creb3-like transcription factor (TF) and its targets were up-regulated in SH flies, and CrebA was shown to be required for mounting an appropriate immune response. Drosophila thus appears as a relevant model to investigate interactions between trauma and infection and allows unraveling of key pathways involved.

In Drosophila, two genes encoding the cAMP response element binding (CREB) protein family members have been cloned: CrebA and CrebB-17A. Of the two, CrebB-17A protein is more similar to mammalian CREB and CREM genes. CrebB-17A contains a consensus site for cAMP-dependent PKA phosphorylation and shows PKA-responsive transcriptional activation. CrebB-17A is also involved in long term memory in Drosophila (Yin, 1995).

CrebA shows less homology to the mammalian CREBs: it does not contain a PKA consensus phosphorylation site and its transcriptional activity is only mildly enhanced by cAMP (Smolik, 1992). CrebA is expressed in several embryonic tissues including the salivary gland and epidermis, structures affected by loss-of-function mutations in the gene.

In the salivary gland, CrebA transcription is activated by Sex combs reduced, the master regulator of salivary gland fate, and is repressed by Teashirt. CrebA is required for the the structural integrity of the salivary gland. Loss of CrebA results in a crooked salivary gland, one that exhibits significant and abnormal bends and kinks along the length of the organ (Andrew, 1997).

CrebA mutants exhibit severe defects in the larval cuticle. The most obvious defect is a weakening of the cuticle and a decrease in the overall length of mutant animals. There is an alteration in the cuticular pattern reminiscent of a segment polarity phenotype. Lacking are primary denticles, the ones associated with the segmental border row of cells. The dorsal hairs present in the CrebA mutants are most similar in size and morphology to the so-called quaternary hairs and the dorsolateral hairs of wild-type larvae (Andrew, 1997).

What developmental pathway is involved in the cuticular phenotype of CrebA mutants? One reasonable place to look is the signaling pathway involved in segment polarity. But CrebA fails to interact with loss-of-function alleles for those segment polarity genes that have been tested: engrailed, wingless, gooseberry, Cubitus interruptus and hedgehog. However, there is a genetic interaction with decapentaplegic and spitz, suggesting that CrebA is involved in dorso-ventral patterning. Such an involvment conforms with detailed aspects of the CrebA phenotype, one which results in a more lateral cuticular structure for both dorsal and ventral cuticles. Mammalian CREB is thought to be a target of the ras/mitogen-activated protein kinase (MAPK) pathway in cells from the renal medulla (Xing, 1996), for example, making it plausible that CrebA is activated by this pathway. It is equally likely that CrebA is a downstream target of Dpp (Andrew, 1997).

There are however, a few problems with these arguments. For example CrebA functions long after the involvement of Dpp and Spitz in the establishment of dorsoventral polarity. Then too, although segment polarity is well understood in Drosophila, there is still a possibility that CrebA could be targeted in that pathway without a demonstrated genetic interaction. For example, cAMP dependent protein kinase 1 (PKA) is involved in segment polarity, and PKA is known to target CREB in many contexts. There is even a suggestion that phosphorylation of Dorsal by PKA facilitates nuclear localization of Dorsal (Norris, 1995), thus pointing to a role for PKA in dorsal ventral polarity. Again this function takes place long before the involvement of CrebA in cuticular development. In chickens, Protein kinase A and Protein kinase C are involved in feather morphogenesis, thus serving as a model for involvement of PKA in epidermal morphogenesis (Noveen, 1995).

CrebA regulates secretory activity in the Drosophila salivary gland and epidermis

Understanding how organs acquire the capacity to perform their respective functions is important for both cell and developmental biology. This study examines the role of early-expressed transcription factors in activating genes crucial for secretory function in the Drosophila salivary gland. Expression of genes encoding proteins required for ER targeting and translocation, and proteins that mediate transport between the ER and Golgi is very high in the early salivary gland. This high level expression requires two early salivary gland transcription factors; CrebA is required throughout embryogenesis and Fkh is required only during late embryonic stages. Because Fkh is required to maintain late CrebA expression in the salivary gland, Fkh probably works through CrebA to affect secretory pathway gene expression. In support of these regulatory interactions, CrebA is shown to be important for elevated secretion in the salivary gland. Additionally, CrebA is required for the expression of the secretory pathway genes in the embryonic epidermis, where CrebA has been shown to be essential for cuticle development. Zygotic mutations in several individual secretory pathway genes result in larval cuticle phenotypes nearly identical to those of CrebA mutants. Thus, CrebA activity is linked to secretory function in multiple tissues (Abrams, 2005).

To test whether regulation of CrebA by Fkh is direct, a 2.8 kb fragment upstream of the CrebA transcription unit was identified that could drive salivary gland expression of a lacZ reporter gene (K. D. Henderson, PhD Thesis, Johns Hopkins University School of Medicine, 2000). Two smaller fragments from this enhancer resulted in salivary gland expression of the lacZ reporter gene either only after invagination had begun and later (CrebA-1100) or prior to invagination and later (CrebA-770). Since the later expression pattern fit the timeframe for Fkh-dependent salivary gland expression of CrebA, the CrebA-1100 construct, which contains six consensus Fkh-binding sites was further characterized. ß-Gal expression in the salivary glands with the CrebA-1100 construct was significantly reduced in fkh homozygotes although expression in the amnioserosa was unaffected, indicating that a Fkh-dependent salivary gland enhancer of CrebA had been identified. Flies were transformed with a CrebA-1100 reporter construct in which all six consensus Fkh-binding sites were mutated (CrebA-1100 fkh1-6 lacZ). Both lines carrying the mutated construct had significantly diminished salivary gland expression of ß-Gal, although ßGal expression in other tissues, including the amnioserosa and hemocytes was unaffected. It is conclude that Fkh functions directly to maintain late high-level expression of CrebA in the salivary gland (Abrams, 2005).

It was predicted that secretory pathway component encoding gene (SPCG) expression is controlled directly by CrebA. As a first step toward testing this possibility, lacZ reporter constructs for six of the 34 SPCGs were analyzed in this study. Each SPCG enhancer fragment spanned the 5' end of the most 5' cDNA for each gene and included ~1-2 kb of DNA further upstream. Transformant lines generated from five of the six constructs resulted in embryonic salivary gland expression. The srp68 lacZ enhancer construct did not express in the embryonic salivary gland. Salivary gland lacZ expression from the spase25 and sec61ß enhancer constructs wasdetected from early stage 12 and throughout embryogenesis. Salivary gland lacZ expression from the p24-1, zCop and SrpRa enhancer constructs was first detected during stage 13 and later. Expression of three of the constructs was examined in CrebA mutants: expression of ß-Gal from both the zCop-lacZ and sec61ß-lacZ constructs was completely absent in the salivary glands, whereas salivary gland ß-Gal expression from the spase25-lacZ construct was significantly reduced. Thus, CrebA-dependent salivary gland enhancers have been identified for at least three of the SPCGs (Abrams, 2005).

A search of the regions immediately upstream of the translation start sites of the SPCGs using MEME revealed a motif that is an excellent match for a mammalian Creb-binding site and that is present within 2 kb upstream of 32 of the 34 SPCGs. (The translation start site is used as a reference point since transcription start sites have not been mapped for any of the SPCGs.) Interestingly, of the two SPCGs that do not contain this consensus, one (sec62) is among the least affected by mutations in CrebA and the other, srp19, is one of only two genes examined that had ubiquitously high levels of expression in all tissues, including the salivary gland. Even more compelling is the finding that 13/32 have the site within 100 bp, another 7/32 have the site within 200 bp and another 5/32 have the site within 500 bp of the translation start site. All of the SPCG reporter gene constructs built contain this consensus site. Thus, not only is it predicted that the site is important for salivary gland expression of the SPCGs, but this could be the site through which CrebA acts to elevate transcription. The proximal location of these putative binding sites with respect to the start site of translation is consistent with the finding that mammalian Creb proteins bind close to the start of transcription. Also of relevance to these studies was the failure to discover consensus Fkh-binding sites conserved among the SPCGs through MEME analysis, further supporting an indirect role for Fkh in SPCG regulation (Abrams, 2005).

This study demonstrates that the Drosophila salivary gland prepares soon after specification to generate the machinery for its high-level secretory activity. The machinery includes components of the early secretory pathway crucial for targeting and translocating proteins into the ER and for vesicle transport between the ER and Golgi. Thus, one way the gland distinguishes itself from surrounding tissues is to greatly increase the relative transcriptional levels of the secretory pathway component genes (SPCGs). The leucine zipper transcription factor CrebA has a crucial and probably direct role in activating increased levels of SPCG expression not only in the salivary gland, but also in the epidermal cells, which secrete the larval cuticle. Fkh, the Drosophila FoxA/PHA-4 homolog, is required to maintain SPCG expression in the salivary gland, but acts indirectly, by maintaining CrebA expression. Hkb, the other early transcription factor examined in this study, is not required for elevated SPCG expression (Abrams, 2005).

CrebA is expressed at very high levels in the early salivary gland and this high level expression persists throughout larval life. Nonetheless, embryonic salivary glands in CrebA mutant embryos are relatively normal, showing only a mildly crooked phenotype when compared with the salivary glands of wild-type embryos. This study indicates a role for CrebA in mediating salivary gland secretory function through the transcriptional upregulation of genes encoding early components of the secretory pathway, supporting a physiological rather than morphogenetic role for this protein. Even so, in the CrebA mutants, where over 30 SPCGs are expressed at significantly reduced levels, effects on salivary gland secretion is not evident until late embryonic stages, when a significant reduction of secretory vesicles is observed. The late occurrence of overt defects in secretion in the CrebA mutants could reflect not only the increased secretory load on these cells that occurs only at the later embryonic stages, but also some level of maternal rescue of secretory function, since CrebA is provided maternally. Interestingly, loss-of-function mutations in single secretory pathway component genes does not show the same loss of secretory activity observed in CrebA mutant salivary glands. The residual function of each of the individual SPCGs, from either maternal supplies or the remaining function of the P-element insertional alleles, appears to suffice when all other components are present at wild-type levels, at least with regards to salivary secretion during late embryonic stages (Abrams, 2005).

CrebA mutants have major defects in cuticular development; the larval cuticles are smaller and weaker than the cuticles of their wild-type siblings, the mouthparts and filzkörper are poorly formed, and CrebA mutants frequently have large holes in the dorsal cuticle. In addition, there appears to be a general defect in patterning of the cuticle, with dorsal and ventral structures appearing more lateralized. Embryos mutant for individual SPCGs, whose epidermal expression is also dependent on CrebA, have nearly identical defects in the larval cuticle. The similarity in CrebA and the individual SPCG mutant cuticles suggests that CrebA defects could be entirely due to compromised secretory function in the epidermal cells that produce the cuticle. The lateralized appearance of the denticles and hairs could simply reflect compromised secretory function, which would limit the types of cuticular structures that form to the smaller, less pigmented structures that are characteristic of the lateral cuticle (Abrams, 2005).

Expression studies of the SPCGs indicate that CrebA could directly activate their high level expression. Moreover, a conserved motif was discovered upstream of the SPCGs that is not only a good fit with the mammalian Creb-consensus binding site (TGACGTG G/T C/A), but also matches the first six nucleotides of the sequence that was used to discover CrebA (TGACGTCAG). However, previously published experiments were designed to discover the Drosophila homolog of the cAMP-regulated Creb protein, which turns out to be what is now known as CrebB. Gel shift experiments (EMSAs) indicate that CrebA can bind to the TGACGTCAG consensus but not with the same high affinity and specificity as the mammalian cAMP-regulated Creb protein; thus, CrebA may bind instead with high affinity to the site discovered in the MEME motif search of the regions upstream of the SPCGs to regulate their expression. The CrebA-dependent SPCG enhancers characterized so far (for z-cop; sec61ß and spase25) contain at least two copies of the consensus motif (Abrams, 2005).

Fkh has several roles in salivary gland development and function, including mediating the cell shape changes of invagination, maintaining secretory cell viability and transcriptional activation of the sgs genes in late larval life. In addition to these positive roles, FKH also represses the expression of salivary duct-specific genes in the secretory cells. In this paper, yet another role for fkh in the salivary gland was discovered: the maintenance of SPCG expression (Abrams, 2005).

fkh is a direct transcriptional target of Scr and Exd and the temporal expression of CrebA and the presence of consensus Scr/Exd-binding sites upstream of the gene suggest that CrebA may also be directly controlled by Scr and its co-factors. Late expression of CrebA, however, requires fkh, as does late expression of fkh itself. This study shows that Fkh functions directly to maintain CrebA expression in the salivary gland. Based on the requirement for CrebA for expression of the SPCGs at all embryonic stages and the requirement for fkh only at late stages, these data support a model in which CrebA controls the expression of the SPCGs and Fkh is required only because of its role in maintaining CrebA expression. A direct test of this model would be to express CrebA in the salivary glands of embryos missing fkh function; this experiment, unfortunately, could not be carried out because Fkh-independent drivers capable of providing high-level salivary gland-specific expression of CrebA are not yet available (Abrams, 2005).

A subset of the SPCGs that encode proteins required for retrograde vesicle transport from the Golgi to the ER are still expressed at low levels in CrebA mutants. However, in late but not early fkh mutants, expression of these genes is not above levels in surrounding tissues. It is proposed that the residual expression of the genes observed in the CrebA mutants would be controlled through other early transcription factor genes that, like CrebA, would require Scr and its co-factors for their initial expression and would require Fkh for maintaining late expression. Taken together, these studies suggest that regulation of salivary gland genes does not fit the simple paradigm suggested by studies of the C. elegans pharynx. The genes that specify the salivary gland (Scr/Exd/Hth) are distinct from the genes that activate and maintain gene expression in the organ. Moreover, no single gene takes over for the organ-specifying genes as even Fkh, the homolog of C. elegans PHA-4, is not required for expression of every salivary gland gene. In cases where Fkh is required, it is often indirect, such as with the SPCGs. Fkh does appear to have direct roles, however, much later in development, as demonstrated by regulation studies involving the sgs glue genes. Thus, the involvement of Fkh in salivary gland development and function is complicated and more consistent with the complexity of gene regulation seen in the liver than that suggested for the C. elegans pharynx. The existence of a single 'organ-specifying gene' may be more the exception than the rule (Abrams, 2005).

The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity

Secretion occurs in all cells, with relatively low levels in most cells and extremely high levels in specialized secretory cells, such as those of the pancreas, salivary, and mammary glands. How secretory capacity is selectively up-regulated in specialized secretory cells is unknown. This study found that the CrebA/Creb3-like family of bZip transcription factors functions to up-regulate expression of both the general protein machinery required in all cells for secretion and of cell type-specific secreted proteins. Drosophila CrebA directly binds the enhancers of secretory pathway genes and is both necessary and sufficient to activate expression of every secretory pathway component gene examined thus far. Microarray profiling reveals that CrebA also up-regulates expression of genes encoding cell type-specific secreted components. Finally, it was found that the human CrebA orthologues, Creb3L1 and Creb3L2, have the ability to up-regulate the secretory pathway in nonsecretory cell types (Fox, 2010).

This study provides evidence that the CrebA/Creb3-like bZIP transcription factors are direct and major regulators of secretory capacity. Drosophila CrebA directly activates high-level expression of secretory pathway component genes (SPCGs) through a site that is conserved among the enhancers of 34 CrebA-dependent SPCGs. Moreover, ectopic expression of CrebA in multiple tissues is sufficient to activate high-level expression of every SPCG tested. Microarray analysis indicates that CrebA is required for full expression of ~400 genes, including almost 200 implicated in secretion. The secretory target genes include general machinery required for secretion in all cells as well as cell type-specific secreted cargo, such as the cuticle proteins and mucins. Phenotypic characterization of CrebA mutant SGs revealed a range of expected secretory defects, including reduced luminal secretory content and a decrease in the size and frequency of apical secretory vesicles, as well as unexpected changes in organelle distribution. Active forms of the closest vertebrate orthologues Creb3L1 and Creb3L2 were found to activate the Drosophila SPCGs when expressed in embryos. Active Creb3L1 can also induce expression of multiple components of the secretory pathway when expressed in HeLa cells, a nonsecretory cell type (Fox, 2010).

CrebA is the single Drosophila member of the Creb3-like family of transcription factors that includes five different proteins in mammals (Creb3/Luman, Creb3L1/Oasis, Creb3L2/BBF2H7, Creb3L3/CrebH, and Creb3L4/Creb4) and two in worms (C27D6.4 and F57B10.1). This singularity means that the fly protein is likely to play a more pivotal role in the regulation of secretion because there is no possibility of compensation for its activity by other family members. Each member of the Creb3-like family has a unique expression pattern, with some overlap among family members. Creb3/Luman is most highly expressed in the brain, with expression detected in the liver, intestine, colon, and skeletal muscles. Creb3L1 is expressed in osteoblasts, prostate, pancreas, ovary, testis, the gut, lungs, kidney, and SGs. Creb3L2 is expressed in chondrocytes, heart, lung, liver, kidney, adrenal gland, bladder, submandibular gland, brain, ovary, pancreas, spleen, testis, and prostate. Creb3L3/CrebH is almost exclusively detected in the liver, whereas Creb3L4/Creb4 expression is elevated in the prostate, thymus, brain, pancreas, skeletal muscle, and peripheral leukocytes. Unlike the Drosophila and worm orthologues, all five members of the Creb3-like family are ER-bound transcription factors previously implicated as sensors in the unfolded protein response (UPR). Recently published phenotypes of the knockout mutations in each of the two genes most closely related to CrebA, Creb3L1, and Creb3L2 suggest a more physiological role for these genes during normal development, with a major defect being failure to secrete the extracellular matrix in the cell types expressing the highest levels of each gene (Murakami, 2009; Saito, 2009). These data support a model wherein one or more of the remaining members of the family may largely compensate for the loss of secretory capacity associated with the loss of any one family member. Indeed, findings that the expression of only a single Creb3-like family member in HeLa cells, a nonsecretory cell type, is sufficient to activate expression of multiple components of the secretory machinery further supports this hypothesis. Among the many secretory genes induced in HeLa cells by Creb3L1 are genes encoding multiple components of CopII vesicles: Sec16A, Sec23A, Sec24A, Sec24D, Sec31A, and Sar1A. The reduced expression of one or more of these genes could explain the ER trapping of ECM proteins observed with the loss of either Creb3L1 or Creb3L2 (Fox, 2010).

Microarray analysis of CrebA mutants revealed that CrebA up-regulates transcription of secretory cargo, specifically expression of multiple components of the insect cuticle, several mucin-like proteins (secreted highly-glycosylated proteins rich in serine and threonine), and multiple secreted proteins of unknown function. Although unexpected, this parallels the finding that mouse Creb3L1 directly up-regulates the type I collagen gene col1a1, a major secreted component of bone ECM (Murakami, 2009). The data also suggests that CrebA may function in parallel with tissue-specific regulators to control high-level expression of organ-specific cargo. An example is CG14756, which encodes an SG-specific secreted protein of unknown function. Loss of CrebA results in a 3.2-fold decrease in the expression of this gene based on the microarray analysis, but unlike the CrebA targets that show more general expression in all secretory tissues, expression of CG14756 could not be induced by CrebA in other cell types, which suggests the additional requirement for tissue-specific transcription factors for its activation. Indeed, expression of CG14756 is absolutely dependent on Fkh , and the region immediately upstream of CG14756 contains a good consensus Fkh binding site ~150 bp upstream of three clustered CrebA consensus binding sites. Thus, it is proposed that the CrebA/Creb3-like family enhances secretory capacity by coordinately up-regulating expression of the general secretory machinery and of tissue-specific secreted cargo, with the expression of cargo genes likely mediated through cooperation with tissue-specific factors (Fox, 2010).

More than 30% (116 of 383) of genes identified in the CrebA microarray experiments had GO terms associated with roles in the secretory pathway, and WoLF PSORT predictions suggested that more than half of the unknown targets are likely to have roles in secretion. Indeed, genes not implicated in the secretory pathway may, nonetheless, participate in secretion. Several of the ion channel/transporter genes have human orthologues known to function in secretory pathway organelles; for example, CG10449 (Drosophila catsup, human SLC39A7) encodes a Golgi-localized zinc transporter. Also, 26 of the target genes that did not have GO annotations have highly conserved human orthologues, several of which are involved in secretion. For example, CG4293 and CG7011 encode proteins similar to ERGIC2 and ERGIC3, respectively, which are proteins localized to the ER-Golgi intermediate compartment that function in protein folding and trafficking. Thus, it is likely that many of the newly identified CrebA target genes encode proteins that function in secretory organelles, highlighting the potential of the microarray studies to reveal new genes with key roles in the efficient production and delivery of products through the secretory pathway (Fox, 2010).

Altogether, these studies reveal that CrebA and its human orthologues Creb3L1 and Creb3L2 activate transcription of components that function at all steps in secretion. Coordinate up-regulation of secretory components by one (or a very few) transcription factors allows for easily adjustable levels of secretory capacity in a variety of cell types, as nicely exemplified in the Drosophila embryo, where levels of CrebA and corresponding SPCG expression correlate with the levels of secretory activity in the different tissues. Furthermore, microarray analysis combined with the recent studies of Creb3L1 and Creb3L2 in specialized cell types (osteoblasts and chondrocytes; Murakami, 2009; Saito, 2009) suggest that CrebA family proteins also up-regulate expression of tissue-specific secreted content, highlighting the significance of this protein family in secretory cell specialization and function (Fox, 2010).

Promoter Structure

The promoter lacks a TATA box. A canonical CRE sequence, 5'TGACGTCA3', is found 61 bp upstream from the 5'-most start suggesting that, like a number of other transcription factors, CrebA may autoregulate in vivo (Rose, 1997).

Transcriptional Regulation

Expression of CrebA in salivary gland depends on Sex combs reduced, since Scr mutants do not express CrebA in salivary glands and embryos expressing Scr in new places also express CrebA in new places. Activation is blocked by the trunk gene, teashirt and the posterior homeotic gene Abdominal-B. As with two other salivary gland genes, forkhead and trachealess, activation of CrebA in the salivary gland by Scr is blocked by dpp (Andrew, 1997).

Salivary gland formation in the Drosophila embryo is dependent on Scr. When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, SCR mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that Scr also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Two homeodomain-containing proteins encoded by the extradenticle and homothorax genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. Scr regulates the nuclear localization of Exd in the salivary gland primordia through repression of homothorax expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands (Henderson, 2000).

To determine if Exd cooperates with Scr to control salivary gland gene expression, the accumulation of two early salivary gland proteins, CrebA and Trh, was examined in embryos lacking exd function. Zygotic loss of exd function results in a reduction in the number of salivary gland cells expressing CrebA and Trh, as well as a decrease in the level of protein made in these cells. This reduced level of salivary gland protein expression is not as severe as the one seen in Scr mutant embryos. Unlike SCR, EXD mRNA is supplied maternally and, thus, the maternal contribution may partially compensate for the loss of zygotic function. To test this possibility, the maternal contribution of exd was removed using the FLP-FRT system. In embryos lacking maternal exd function, salivary gland expression of CrebA and Trh is at wild-type levels. However, salivary gland expression of CrebA and Trh is completely absent in embryos lacking both the maternal and the zygotic contributions of exd. Thus, exd is required for embryonic salivary gland gene expression. Moreover, zygotically provided exd can rescue the loss of maternally provided exd and maternally provided exd can partially compensate for zygotic loss of exd (Henderson, 2000).

Fork head and Sage maintain a uniform and patent salivary gland lumen through regulation of two downstream target genes, PH4αSG1 and PH4αSG2; Fkh acts largely indirectly to regulate salivary gland genes through maintenance of CrebA

Forkhead (Fkh) is required to block salivary gland apoptosis, internalize salivary gland precursors, prevent expression of duct genes in secretory cells and maintain expression of CrebA, which is required for elevated secretory function. This study characterized two new Fkh-dependent genes: PH4αSG1 and PH4αSG2. In vitro DNA-binding studies and in vivo expression assays show that that Fkh cooperates with the salivary gland-specific bHLH protein Sage to directly regulate expression of PH4αSG2, as well as sage itself, and to indirectly regulate expression of PH4αSG1. PH4αSG1 and PH4αSG2 encode α-subunits of resident ER enzymes that hydroxylate prolines in collagen and other secreted proteins. Salivary gland secretions are altered in embryos missing function of PH4αSG1 and PH4αSG2; secretory content is reduced and shows increased electron density by TEM. Interestingly, the altered secretory content results in regions of tube dilation and constriction, with intermittent tube closure. The regulation studies and phenotypic characterization of PH4αSG1 and PH4αSG2 link Fkh, which initiates tube formation, to the maintenance of an open and uniformly sized secretory tube (Abrams, 2006).

The diverse activities of Fkh support a major role for this protein in controlling many of the tissue specific functions of the salivary gland. Indeed, the long list would support a model wherein Fkh could be viewed as an organ-specifying gene, much like the role proposed for its C. elegans homologue, PHA-4, in pharynx development (Gaudet, 2002). Nonetheless, these studies reveal notable differences in the roles of the two genes in organ formation. PHA-4 is proposed to directly regulate expression of all pharynx-specific genes (Gaudet, 2002), whereas Fkh is required for the expression of only about one-third of the salivary gland-specific genes tested so far (>200 genes). Moreover, many downstream genes are indirect targets of Fkh. For example, although Fkh is required for high-level expression of 34 secretory pathway component genes, its role in their activation is largely indirect through maintenance of CrebA, which is more directly involved in the expression of these genes (Abrams, 2005). As observed with the secretory pathway genes, more than half of the Fkh targets require Fkh only for maintenance, not initiation, suggesting that regulation is mediated by Fkh-dependent downstream transcription factors. Even with SG1 and SG2, which absolutely require Fkh for all stages of expression, only SG2 appears to be regulated directly by Fkh. Fkh regulation of SG1 appears to be through an intermediate, currently unidentified transcription factor, even though the SG1 enhancer contains two sites that bind Fkh protein in vitro. Indeed, detailed enhancer analyses have revealed only a small number of direct transcriptional targets of Fkh, including both sage and CrebA, which encode transcription factor genes that, in turn, either function downstream of or in parallel with Fkh to regulate gene expression. Thus, although Fkh functions as a key regulator of salivary gland development, it does so in collaboration with other early expressed transcription factors (Abrams, 2006).

When this analysis of Fkh regulation of SG1 and SG2 was undertaken, every potential direct binding site was sought within the SG1 and SG2 enhancers using data from three different studies, that revealed a seven bp core consensus Fkh-binding site. Although four such consensus sites were found within each enhancer, not all of the sites were bound by Fkh protein in vitro. Three sites showed strong binding, two sites showed moderate or weak (non-specific) binding and three sites were not bound at all. Subsequently, Takiya (2003) reported Fkh DNA binding to be strongly influenced by negative cooperativity among neighboring bases. The binding data from this study are consistent with those findings. The three strong Fkh-binding sites in the SG1 and SG2 enhancers matched the optimal sequences for binding determined by Takiya (2003) in all positions. The moderate Fkh-binding site had a C in position 11, which was shown to reduce binding affinity. The weak Fkh-binding site had an inhibitory T9A10 dinucleotide, and the three sites that did not bind Fkh at all each had an unfavorable A7T8 dinucleotide as well as nucleotides unfavorable for binding at positions 9 and/or 11. The demonstration that the Fkh-binding sites within both the SG2 and sage enhancers are required for their full level salivary gland expression indicates a direct correlation between in vitro studies and site occupancy in vivo; sites that function in vivo are bound by the Fkh protein in vitro. These studies of SG1 regulation, however, indicate that not all sites bound by Fkh protein in vitro are necessary in vivo, as SG1 reporter gene expression was unaltered when the Fkh sites were disrupted (Abrams, 2006).

Functional analysis of SG1 and SG2 suggests a role for apical secretion in the maintenance of uniform open salivary gland tubes. This finding supports studies in the Drosophila trachea, demonstrating the importance of apical secretions during tracheal remodeling showed that two apical proteins, Piopio (Pio) and Dumpy (Dp), are required during secondary branch formation. In this process, cells that are arrayed side-by-side in a multicellular tube rearrange to an end-to-end configuration to form unicellular tubes, while maintaining tube integrity with uniform lumenal space. In pio or dp mutants, tracheal cells detach from the main tracheal artery just as they complete their rearrangements to form unicellular tubes. It has been suggested that this occurs because the formation of the autocellular junctions of the unicellular tubes continues to completion instead of stopping at the point where the cells contact their proximal neighbors. It was further suggested that Pio and Dp contribute to an apical ECM that prevents reduction in lumen diameter as the secondary branches form. A role for an apical ECM in maintaining tube diameter has also been discovered in the multicellular tubes of the dorsal trunk. Genetic or pharmacological disruptions in chitin synthesis lead to regions of tube constriction and dilation. The existence has been demonstrated of a transient chitin network in the tracheal lumen that ensures that as the different segments of the dorsal trunk fuse to form a continuous tube, uniform tube diameter is maintained (Abrams, 2006 and references therein).

These studies demonstrate a correlation between apical ECM volume/morphology and lumen size uniformity, even in tubes not undergoing extensive cell rearrangements. The phenotypes of SG1/SG2-deficient salivary glands suggest that the apical ECM has both a barrier function that prevents cells from contacting one another and closing the tube, as well as a scaffolding function that prevents lumenal dilation. Similar defects were observed with mutations in pasilla (ps), which encodes a splicing factor homologous to the mammalian proteins Nova1 and Nova2 (Seshaiah, 2001). At the TEM level, ps mutants exhibit a decrease in secreted lumenal content and a reduction in the number and size of apical secretory granules. Although, as a splicing factor, Pasilla must be acting indirectly to affect secretion levels, its phenotype demonstrates a direct correlation between secretory volume and lumen size uniformity, a correlation supported by the defects in SG1/SG2-deficient glands. As SG1 and SG2 encode enzymes that could modify proteins in apical secretions, their role in this process is likely to be more direct (Abrams, 2006).

The apical matrix of wild-type glands forms a fibrillar network structure that is not apparent in salivary glands of embryos missing SG1 and SG2. This phenotype suggests that protein modification by the SG1 and SG2 prolyl-4-hydroxylases (PH4s) alters the character of secreted apical proteins to allow them to form fibrillar structures that maintain an expanded network of ECM. A role for prolyl hydroxylation in the formation of fibrillar collagen has been known for decades. Although canonical collagens are not expressed in the Drosophila salivary gland, a large number of uncharacterized genes encoding secreted proteins that contain the Pro-X-Gly repeats exist, which could be substrates for SG1/SG2 prolyl hydroxylation. Collagens are the major protein components of the ECM, where they serve key structural roles as exemplified by mutations in the human genes that lead to fragile bones, bone and joint deformities, as well as fragile skin and blood vessels. A 'structural' role for a collagen-related protein(s) in the apical matrix of salivary glands is consistent with these observations. Interestingly, formation of collagen fibrils occurs post-secretion, where enzymes outside the cell remove the propeptides from procollagen to allow fibrillar collagen formation. Similarly, the fibrillar nature of the lumenal secretions of the wild-type Drosophila salivary gland is not visible in the subapical secretory vesicles, suggesting that the fibrillar structures found in the apical matrix also form post-secretion. It is proposed that the denser apical matrix with reduced volume is the basis for the defects observed in SG1/SG2-deficient salivary glands. In areas where there is little to no apical content, the opposing sides of the tubes meet to either close or form very small lumena lined with small apical surfaces and closely arrayed adherens junctions. The similarity of the SG1/SG2 deficiency phenotypes to those seen with mutations affecting the Drosophila trachea suggests the potential for shared mechanisms for maintaining lumen size uniformity in epithelial tubes (Abrams, 2006).

Targets of Activity

The sequences required for tissue-specific and temporal expression of the Adh genes of D. melanogaster (Dme) and D. mulleri (Dmu) have been characterized. The Dme Adh gene is expressed from two promoters: the proximal promoter primarily active in larvae, and the distal promoter primarily active in adults. In contrast the Dmu Adh locus contains two functional genes: one that is expressed in larvae and another that is expressed in adults. The Dme Adh adult fat body element (AAE) acts to simulate transcription from the distal promoter of adults. The Dmu adult enhancer is located about 2500 bp upstream of the Adh-2 promoter. The Dmu larval fat body enhancer, box B, is a 90 bp regulatory element located about 200 bp upstream of the Adh-1 gene. Box B drives expression of Adh-1 gene in larvae. Both box B and AAE act as fat body-specific enhancers when linked to a heterologous promoter. CrebA binds to the Dmu box B (the larval fat body enhancer of the Adh-1) and also binds to the AAE of Dme adult fat body element. A CrebA binding site is also located within the Dmu adult enhancer, which directs expression of the Adh-2 gene, although CrebA binds about threefold less well to this sequence than to box B and the AAE. CrebA also binds to a region of the Yolk protein gene fat body enhancer. dCREB-A acts as a transcriptional activator from box B, and binds to two mammalian liver-specific regulatory elements, the hepatocyte-specific enhancer of rat tyrosine aminotransferase and the mammalian Adh (Abel, 1992).

Although shown to bind fat-body and liver-specific regulatory elements, CrebA is not expressed in the fat body during any developmental stage (Andrew, 1997).

Protein Interactions

Fasting launches CRTC to facilitate long-term memory formation in Drosophila

Canonical aversive long-term memory (LTM) formation in Drosophila requires multiple spaced trainings, whereas appetitive LTM can be formed after a single training. Appetitive LTM requires fasting prior to training, which increases motivation for food intake. However, this study found that fasting facilitates LTM formation in general; aversive LTM formation also occurred after single-cycle training when mild fasting was applied before training. Both fasting-dependent LTM (fLTM) and spaced training-dependent LTM (spLTM) requires protein synthesis and cyclic adenosine monophosphate response element-binding protein (CREB) activity. However, spLTM requires CREB activity in two neural populations--mushroom body and dorsal-anterior-lateral (DAL) neurons--whereas fLTM required CREB activity only in mushroom body neurons. fLTM uses the CREB coactivator CREB-regulated transcription coactivator (CRTC), whereas spLTM uses the coactivator CBP. Thus, flies use distinct LTM machinery depending on their hunger state (Hirano, 2013).

In Drosophila, canonical aversive long-term memory (LTM), which is dependent on de novo gene expression and protein synthesis, is generated after multiple rounds of spaced training. In contrast, appetitive LTM can be formed by single-cycle training. Because both aversive and appetitive LTM require protein synthesis and activation of CREB, it is likely that both types of LTM are formed by similar mechanisms. Appetitive and aversive LTM are known to differ (i.e., octopamine is specifically involved in appetitive but not aversive memory formation). However, it remains unclear why single-cycle training is sufficient for appetitive but not aversive LTM formation. Appetitive LTM cannot form unless fasting precedes training. Although fasting increases motivation for food intake (a requirement for appetitive memory) it was suspected that fasting may activate a second, motivation-independent, memory mechanism that facilitates LTM formation after single-cycle training (Hirano, 2013).

Flies were deprived of food for various periods of time and then subjected to aversive single-cycle training. Fasting prior to training significantly enhanced 1-day memory, with a peak at 16 hours of fasting and a return to nonfasting levels at 20 to 24 hours of fasting. In contrast, 16 hours of fasting did not increase short-term memory (STM, measured 1 hour after training). In this protocol, flies were returned to food vials after training, raising a possibility that the perception of food as a reward after training may enhance the previous aversive memory. This possibility was tested by inserting refeeding periods between food deprivation and training. Although fasting followed by a 4-hour refeeding period failed to induce appetitive LTM, it significantly enhanced aversive 1-day memory; this finding suggests that enhancement of aversive memory occurs through a mechanism unrelated to increased motivation or perception of food as a reward. A 6-hour refeeding period was sufficient to prevent aversive memory enhancement. Continuous food deprivation after training suppressed aversive memory enhancement, which indicates that both fasting before training and feeding after training are required to enhance aversive memory (Hirano, 2013).

Administration of the protein synthesis inhibitor cycloheximide (CHX) abolished 1-day memory enhancement but had no effect on 1-hour memory, supporting the idea that memory enhancement consists of an increase of LTM. Memory remaining after CHX treatment is likely to be protein synthesis-independent, anesthesia-resistant memory (ARM). Fasting for 16 hours neither enhanced protein synthesis-independent memory nor canonical aversive LTM generated by spaced training (spLTM). Furthermore, fasting-dependent memory decayed within 4 days, and food deprivation did not enhance 4-day spLTM, indicating that fasting-dependent memory is physiologically different from spLTM (Hirano, 2013).

Fasting-dependent memory was blocked by acute, dose-dependent, expression of CREB2-b, a repressor isoform of CREB, in the mushroom bodies (MBs). Expression of the repressor from two copies of UAS-CREB2-b under control of the MB247-Switch (MBsw) GAL4 driver, which induces UAS transgene expression upon RU486 feeding, significantly suppressed fasting-dependent memory upon RU486 feeding, whereas expression from one copy of UAS-CREB2-b did not. Defects in LTM formation are highly correlated with CREB2-b amounts. Significantly higher MBsw-dependent expression of CREB proteins was found in flies carrying two copies of UAS-CREB2-b relative to flies carrying one copy. MBsw-dependent CREB2-b expression did not affect STM in either fed or food-deprived conditions. Because the aversive memory enhanced by fasting is mediated by protein synthesis and CREB, this memory is referred to as fasting-dependent LTM (fLTM). Similar to the results in aversive fLTM, MBsw-dependent CREB2-b expression also decreased appetitive LTM but not appetitive STM (Hirano, 2013).

A recent study concluded that CREB activity in MB neurons is not required for spLTM. In that study, CREB2-b was expressed using the OK107 MB driver and GAL80ts was used to restrict CREB2-b expression to 30°C. However, this study found that the GAL80ts construct still inhibited expression of CREB considerably at 30°C. When higher amounts of CREB2-b were acutely expressed in MBs using MBsw, a significant decrease was observed in 1-day spLTM, indicating that CREB activity in the MBs is likely to be required for spLTM (Hirano, 2013).

Consistent with the previous results expression of CREB2-b in two dorsal-anterior-lateral (DAL) neurons impaired aversive spLTM. In contrast, expression of CREB2-b in DAL neurons did not affect aversive fLTM. Moreover, appetitive LTM was also not affected by expression of CREB2-b in DAL neurons. MBsw did not express GAL4 in DAL neurons (Hirano, 2013).

CREB requires coactivators, including CBP (CREB-binding protein), to activate transcription needed for LTM formation. Acute expression of an inverted repeat of CBP (CBP-IR) in MBs significantly impaired spLTM without affecting either STM or 1-day memory after multiple massed trainings, which do not lead to LTM formation. However, neither aversive fLTM nor appetitive LTM was impaired by CBP-IR expression, indicating that an alternative coactivator may be required for fasting-dependent memory (Hirano, 2013).

Recent studies demonstrate the involvement of a cAMP-regulated transcriptional coactivator (CRTC) in hippocampal plasticity. In metabolic tissues, phosphorylated CRTC is sequestered in the cytoplasm while dephosphorylated CRTC translocates to the nucleus to promote CREB-dependent gene expression. Fasting causes CRTC dephosphorylation and activation. In line with this, significant accumulation of hemagglutinin (HA)-tagged CRTC (CRTC-HA) was found within MB nuclei after 16 hours of food deprivation. Subcellular fractionation indicated that food deprivation causes CRTC-HA nuclear translocation without affecting total CRTC-HA amounts (Hirano, 2013).

To examine the role of CRTC in fLTM and spLTM, a CRTC inverted repeat (CRTC-IR) was acutely expressed using MBsw, and suppression of aversive fLTM was observed but no effect was seen on STM. CHX treatment did not further decrease 1-day aversive memory, and CRTC-IR expression from a second MB driver, OK107, also impaired fLTM formation. CRTC-IR expression from MBsw also impaired appetitive LTM without affecting appetitive STM. In contrast, CRTC-IR expression from MBsw did not impair spLTM. CRTC-IR expression in DAL neurons had no effect on either aversive fLTM or appetitive LTM. Consistent with these results showing lack of fLTM after 24-hour fasting, 1-day aversive memory after 24-hour fasting did not decrease upon CRTC-IR expression in MBs (Hirano, 2013).

To examine the effects of spaced training on fLTM and the effects of fasting on spLTM, fed or fasted flies expressing either CBP-IR or CRTC-IR were space-trained. When CBP-IR was expressed to impair spLTM, 1-day memory after spaced training was impaired in fed conditions but not in fasting conditions, which suggested that spaced training protocols do not block fLTM. When CRTC-IR was expressed to impair fLTM formation, 1-day memory after spaced training was not affected by fasting, which suggested that mild fasting does not impair spLTM formation (Hirano, 2013).

Is activation of CRTC sufficient to generate fLTM in the absence of fasting? HA-tagged constitutively active CRTC (CRTC-SA-HA) was expressed from MBsw, and its nuclear accumulation was observed in the absence of fasting. Acute expression of CRTC-SA-HA from MBsw increased 1-day aversive memory after single-cycle training in fed flies, and this increase was not further enhanced by fasting. In contrast, expression of control CRTC-HA did not alter the fasting requirement for memory enhancement. CRTC-SA-HA expression did not affect feeding itself, which suggested that the memory enhancement is not due to impaired feeding. Taken together, CRTC activity in MBs is necessary and sufficient to form fLTM. Similar to the effects of fasting, CRTC-SA-HA expression did not affect STM or 4-day spLTM (Hirano, 2013).

In mammalian metabolic tissues, CRTC is phosphorylated by insulin signaling, which is suppressed by fasting. CRTC phosphorylation is also regulated by insulin signaling in flies. To determine whether reduced insulin signaling activates CRTC and promotes fLTM formation, heterozygous mutants for chico, which encodes an adaptor protein required for insulin signaling, were tested. Although chico1 null mutants are semilethal and defective for olfactory learning, heterozygous chico1/+ mutants are viable and display normal learning (Hirano, 2013).

CRTC accumulated in MB nuclei in chico1/+ mutants in the absence of food deprivation. Under conditions where flies were fed, chico1/+ flies had significantly greater 1-day memory after single-cycle training relative to control flies, whereas 1-hour memory was unaffected. Enhanced 1-day memory in chico1/+ flies was not further enhanced by fasting. Because the chico1/+ mutation does not affect feeding itself, the memory enhancement would not seem to be attributable to impaired feeding. The increased 1-day memory in chico1/+ mutants was suppressed by CHX treatment and CRTC-IR expression using MBsw, which suggests that reduced insulin signaling mimics fLTM through activation of CRTC in MBs (Hirano, 2013).

Single-cycle training after mild fasting generates both appetitive and aversive LTM, and CRTC in the MBs plays a key role in both types of LTM. A CRTC-dependent LTM pathway is unlikely to be involved in increasing motivation required to form appetitive memory, because CRTC knockdown did not affect appetitive STM and because CRTC-SA expression was not sufficient to form appetitive LTM without prior fasting. Although mild 16-hour fasting induced aversive fLTM, longer 24-hour fasting impaired aversive fLTM but not appetitive LTM. Thus, although aversive and appetitive fLTM share mechanistic similarities, they may be regulated by different inputs controlling motivation and fasting time courses. Because nuclear translocation of CRTC was sustained even after 24 hours of food deprivation, prolonged fasting may suppress a CRTC-independent step in aversive fLTM formation. spLTM was not affected by 24-hour fasting prior to training, which suggests that the unknown inhibitory effect of 24-hour fasting does not occur after spaced training. Continuous food deprivation after training suppressed aversive fLTM. In another study, it was reported that continuous food-deprivation after spaced training suppresses spLTM as well (Hirano, 2013).

Suppression of aversive LTM by prolonged fasting may ensure that starving flies pursue available food, with less concern for safety. Although the biological importance of aversive fLTM in natural environments is currently unclear, the current results indicate that different physiological states may induce different types of LTM in flies (Hirano, 2013).

Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance

The starvation-inducible coactivator cAMP response element binding protein (CREB)-cAMP-regulated transcription coactivator (Crtc) has been shown to promote starvation resistance in Drosophila by up-regulating CREB target gene expression in neurons, although the underlying mechanism is unclear. This study found that Crtc and its binding partner CREB enhance energy homeostasis by stimulating the expression of short neuropeptide F (sNPF), an ortholog of mammalian neuropeptide Y, which was shown to be a direct target of CREB and Crtc. Neuronal sNPF was found to promote energy homeostasis via gut enterocyte sNPF receptors, which appear to maintain gut epithelial integrity. Loss of Crtc-sNPF signaling disrupts epithelial tight junctions, allowing resident gut flora to promote chronic increases in antimicrobial peptide (AMP) gene expression that compromised energy balance. Growth on germ-free food reduces AMP gene expression and rescues starvation sensitivity in Crtc mutant flies. Overexpression of Crtc or sNPF in neurons of wild-type flies dampens the gut immune response and enhances starvation resistance. These results reveal a previously unidentified tolerance defense strategy involving a brain-gut pathway that maintains homeostasis through its effects on epithelial integrity (Shen, 2016).

Disruptions in energy balance are a component of the collateral damage associated with mounting an immune response. In addition to regulating the magnitude of an immune response, energy allocation must be properly regulated to minimize physiological damage during infection. This study found that Drosophila sNPF, a mammalian NPY homolog, is regulated by CrebB/Crtc within the CNS, where it promotes energy balance by maintaining epithelial integrity and thereby attenuating overexuberant immune activation in the gut. The effects of sNPF were unexpected, given its role in food-seeking behavior. Indeed, food intake appears comparable between Crtc mutants and control flies (Shen, 2016).

The effects of sNPF are mediated by enterocyte sNPF-Rs, suggesting that the sNPF brain-gut signal is released by a subset of the sNPF+ neurons that directly innervate the gut. Although neuronal activity is known to contribute to energy homeostasis, the results suggest that the modulation of the gut immune system by CrebB/Crtc is a critical component in this process (Shen, 2016).

Epithelial tissues are typically colonized by both commensal and invasive microbes. sNPF appears to be actively expressed and released from the CNS in times of stress, providing nonautonomous control of gut immunity from the brain. Based on its widespread expression in the midgut, sNPF-R may provide ubiquitous attenuation of the innate immune response. Consistent with observations in Drosophila, activation of the NPY receptor ortholog (NPR-1) in Caenorhabditis elegans also down-regulates inflammatory gene expression. The current studies extend these findings by showing how a neuronal fasting-inducible pathway modulates energy balance via its effects on the gut immune system (Shen, 2016).

Following their activation, sNPF-Rs appear to promote energy balance by enhancing epithelial integrity. Although the mechanism underlying these effects is unclear, it is noted that disruption of the tight junction protein Bbg in flies also causes constitutive up-regulation of innate immunity genes. Future studies should reveal whether sNPF-R modulates the activity of Bbg or related proteins in enterocytes (Shen, 2016).

In mammals, inflammatory bowel diseases, such as ulcerative colitis, are often associated with profound weight loss, due, in part, to the chronic activation of the immune system. By reducing inflammatory gene expression and enhancing energy homeostasis, gut neuropeptides, such as NPY, may provide therapeutic benefit in this setting (Shen, 2016).



The CrebA protein is expressed in the nuclei of the embryonic salivary gland, proventriculus and stomodeum. CREBA's mRNAs are first seen at germ band extension (about 7 h into embryogenesis) in the salivary-gland placodes, and continue to be expressed in the salivary gland up to the 16th hour of development (Smolik, 1992).

The highest level of CREB-A mRNA is detected in the salivary gland primordia, initially in both presecretory cells and a subset of the duct cells (embryonic stages 9 through early 11), and later in only the secretory cells (Andrew, 1997).

CrebA transcripts are first seen at germ band extention in the salivary-gland placodes and continue to be expressed in the salivary gland up to the 16th hour of development. Low levels of CrebA mRNA are detected in the cell bodies of the brain and the optic lobe. CrebA mRNA is detected in midgut epithelial cells. CrebA is also expressed at lower levels in other tissues, including the trachea, a subset of neuroblasts, the proventriculus, the amnioserosa, the epidermis, and the foregut and its derivatives. The protein first appears in the foregut primordia by embryonic stage 6 and persists in the foregut derivatives until the end of embryogenesis. Expression in the amnioserosa begins during stage 8 and disappears during stage 13. Transient expression is observed in a subset of neuroblasts from stage 9 through stage 11 and in the proventriculus from stage 13 to 17. Tracheal expression is first detected at the time of tracheal pit formation (stage 11) and persists in the dorsal trunk tracheal cells throughout embryogenesis. The epidermal cells, which secrete the larval cuticle just before hatching, begin to express CrebA during stage 11 in a subset of cells in each segment, with accumulation of protein in all epidermal cells by stage 13. Staining is in the epithelial cell nuclei of the segmental boundaries (Rose, 1997). Protein persists for the remainder of embryogenesis (Andrew, 1997).

CREB-A mRNA is present throughout the life cycle of the fly, albeit at varying levels. The highest levels are seen during embryogenesis and in adult males. mRNA levels in third-instar larvae are approximately equivalent in the trachea, fat body and gut, but only the gut and fat body express Adh. CREB-A mRNA and protein are also found in the ovaries, an adult tissue that expresses Adh (Abel, 1992). Another study shows that although shown to bind fat-body and liver-specific regulatory elements, CrebA is not expressed in the fat body during any developmental stage (Andrew, 1997).


CrebA is found in the adult salivary gland, the columnar but not the squamous follicle cells in the ovary, and in the male seminal vesicle, anterior ejaculatory duct, and ejaculatory bulb. CrebA is initially expressed in stage 9 follicle cell nuclei as they migrate posteriorly toward and around the oocyte. In stages 10A and 10B, CrebA is expressed uniformly in the nuclei of the columnar follicle cells surrounding the oocyte. This expression pattern lasts until stage 11, when only a few nuclei expressing the CrebA protein can be seen over the reduced nurse cell chamber. By the onset of stage 12, CrebA protein is no longer detected (Rose, 1997).

In the epidermis, CrebA is required for patterning cuticular structures on both dorsal and ventral surfaces since CrebA mutant larvae have only lateral structures around the entire circumference of each segment. The most obvious defect is a weakening of the cuticle and a decrease in the overall length of homozygous mutants. Mutants average 40% the length of heterozygous sibling. Cuticular weakening is evident by the frequent 'blow-outs' or 'holes' which occur at random positions in mutants. The cuticular weakening and the decrease in larval length could be related. At all early embryonic stages, mutants are the same length as heterozygous siblings, suggesting that the length differences arise during the formation of the cuticle. Both weaker and smaller larval cuticles might be expected if cuticle protein synthesis were impaired. Alternatively, mutant larvae could be shorter because they have patterning defects (Andrew, 1997).

The dorsal hairs of CrebA mutants are most similar in size and morphology to the so-called quaternary hairs but these hairs are neither as orderly or as densely packed as the dorsal quaternary hairs of wild-type embryos. Rather, the loose irregular spacing of these hairs is more reminiscent of those seen in the dorsolateral position in wild-type embryos. CrebA ventral denticles are, however, indistinguishable from those found at a ventrolateral position in wild-type larvae. Because the morphology and arrangement of the ventral denticles and dorsal hairs in CrebA mutants accurately represent structures normally found on the lateral surface of wild-type larvae, it is believed that the altered cuticle patterns is not due to a failure to differentiate denticles or hairs, but rather arises because wild-type CrebA is required for correct patterning of the epidermis along the dorsal-ventral axis. Double mutants of CrebA and segment polarity genes give simple additive phenotypes, and do not demonstrate genetic interaction that would suggest that segment polarity genes and CrebA function in the same pathway (Andrew, 1997).

CrebA is thought to be epistatic to known dorsal/ventral patterning genes. Epistasis tests were done with the decapentaplegic gene and the spitz gene. In dpp/CrebA double mutants the entire cuticle is lateralized, while in dpp mutants the cuticle is ventralized. In spitz/CrebA double mutants narrower denticle bands are seen with some fusion of denticles between segments; however the denticles have the same morphology found in CrebA mutants alone. It is thought that near the end of both the Dpp- and Spi-signaling cascades, CrebA functions to translate the corresponding extracellular signals into changes in gene expression. The only determinant tested that shows altered expression patterns in CrebA mutants was Dsc73, a secreted protein expressed at late embryonic stages in the epidermal cells that produce denticles and hairs. There was a decrease in the levels of Dsc73 on the dorsal and ventral surfaces compared to levels of Dsc73 in lateral positions, which appear unchanged (Andrew, 1997).

The only defect observed in salivary glands is that they are 'crooked,' showing significant bends or kinks where normally there would be none (Andrew, 1997).

CrebA mutation is lethal during late embryonic development. One possible explanation for the late embryonic lethality is that the salivary gland is unable to secrete factors necessary for hatching. To test this possibility, CrebA was deliberately expressed in the salivary glands of CrebA mutants. Such expression does not rescue the embryonic lethality (Rose, 1997).


For information on the involvement of mammalian CREB in learning and the interaction of CREB with Creb binding protein (CBP) see CrebB-17A. For information on the targeting of CREB by cAMP dependent Protein kinase A, see in cAMP dependent Protein kinase 1 site.

An orchestrated program regulating secretory pathway genes and cargos by the transmembrane transcription factor CREB-H

CREB3 proteins comprise a set of ER-localized bZip transcription factors defined by the presence of a transmembrane domain. They are regulated by inter-compartmental transport, Golgi cleavage and nuclear transport where they promote appropriate transcriptional responses. Although CREB3 proteins play key roles in differentiation, inflammation and metabolism, a general framework relating their defining features to these diverse activities is lacking. This study identified unique features of CREB3 organization including the ATB domain, which was shown to be essential for transcriptional activity. This domain is absent in all other human bZip factors, but conserved in Drosophila CREBA, which controls secretory pathway genes (SPGs). Furthermore, each of the five human CREB3 factors was capable of activating SPGs in Drosophila, dependent upon the ATB domain. Expression of the CREB3 protein, CREB-H, in 293 cells, upregulated genes involved in secretory capacity, extracellular matrix formation and lipid metabolism and increased secretion of specific cargos. In liver cells, which normally express CREB-H, the active form specifically induced secretion of apolipoproteins, including ApoA-IV, ApoAI, consistent with data implicating CREB-H in metabolic homeostasis. Based on these data and other recent studies, a general role is proposed for the CREB3 family in regulating secretory capacity, with particular relevance to specialized cargos (Barbosa, 2013).

CREB-H is a member of the CREB3 family, a specialised set of ER-anchored bZip transcription factors related to ATF6, a bZip transmembrane factor involved in the unfolded protein response in the ER. Despite this similarity, several lines of evidence indicate that CREB3 family members are regulated in a distinct manner and are likely to be involved in promoting distinct responses to ER stress or demands. However a unifying hypothesis providing a framework for comparative analysis and understanding of the CREB3 family has been lacking. A model is proposed wherein the core function of these proteins (and their sequence orthologues in other species) is to modulate secretory pathways, coordinating production of specific cargos (which will be distinct in distinct cell types or organisms) with efficient secretory capacity for these cargos and that the ultimate physiological roles of CREB3 proteins are underpinned by these activities. Several lines of evidence provide support for this general hypothesis (Barbosa, 2013).

CREB3 DNA binding domains are more closely related to the Drosophila melanogaster factor CREBA than to ATF6 or any other bZip factor. A distinct domain, termed the ATB domain, is shared exclusively between CREBA and the CREB3 family, and it to be critical for transactivation by CREB-H. With respect to specific ATB-bZip organisation, there is no homologue of CREBA in mammalian genomes other than the CREB3 family, increasing further the significance of this feature. This relationship is reinforced by the results indicating that CREB-H target genes are enriched for secretory pathway components. Indeed several individual genes regulated by CREBA in Drosophila (Fox, 2010), such as Sec31a, Sec13, KDELR, ARFGAP3, were also identified in microarray and qPCR analysis of CREB-H target genes in human cells (Barbosa, 2013).

It is likely, notwithstanding the similarity between the CREB3 ATB-bZip domains and the overall themes in target gene induction, that specificity will operate either directly, via differential DNA binding and/or indirectly via selective function of additional cooperative transcription factors. On the other hand, the conservation of the ATB domain, in a region which in other bZip factors is not required for DNA binding, is strongly indicative of conservation of some aspect of gene targeting or the mechanism of activation. There is an interesting parallel to the ATB domain in the Maf subset of bZip factors, which contain a region termed the EH, flanking the bZip domain (but with no similarity to the ATB domain identified in this study). The EH domain contributes to DNA binding, probably in a stabilising role rather than conferring specificity. The precise role of the ATB domain in DNA binding and/or transactivation will be the subject of separate biochemical investigation and side-by-side comparisons with other family members (Barbosa, 2013).

In previous work, certain individual target genes of CREB-H have been reported but with different general conclusions. It was reported that CREB-H could activate certain gluconeogenic target genes though this was not supported in other studies which instead reported activation of a subset of acute phase response genes, or upregulation of the iron-binding hormone hepcidin. More recent studies of knock-out mice, have revealed CREB-H to be a key factor in metabolic homeostasis of lipids and triglycerides. The current results are consistent with these latter studies. In particular, in CREB-H knock-out animals, there is a significant defect in liver expression of several genes which were identified as being upregulated by overexpression of the active form of CREB-H, in particular ApoA-IV and ApoAI. Moreover, in addition to a main phenotype of imbalances in metabolism, several secretory pathway genes including, for example, ADP-ribosylation factor 4a, RAB, RABGAP1l, and Secs including Sec24d, Sec23a and Sec22a were also significantly downregulated as a consequence of CREB-H loss. The current data add to these previous findings with the demonstration that CREB-H can induce and orchestrate the secretion of high levels of specific cargos in human liver cells, in this case a series of apolipoproteins, and is capable of inducing secretion of at least some of these cargos in cells that otherwise never secrete such proteins. Although not necessary for the general conclusions, it will be very interesting to examine the composition and density profile of apolipoprotein secretion induced by CREB-H in liver cells and to examine any role for CREB-H in secretion in the small intestine, its other main site of expression. For example it may be that CREB-H is involved in chylomicron secretion, whose large particles make unusual demands on secretory capacity (Barbosa, 2013).

The proposal for a general secretory role of CREB3 proteins is also supported by data on other family members including the phenotype of knock-out animals lacking CREB3 factors OASIS or BBF2H7 and of mutations in the BBF2H7 homologue in zebrafish. Although expressed widely in adult mammalian tissues, the effect of BBF2H7 knock-out in mice was most obvious in cartilage and bone tissue. Particularly in chondrocytes, the ER was abnormally expanded with accumulations of ECM components and defects in collagen secretion and ECM composition. Loss of BBF2H7 was accompanied by down regulation of a number of secretory pathway genes, again including e.g., Sec23a, Sec24d and KDELR3. Intriguingly in cell culture, the effects of BBF2H7 loss on ER function and collagen secretion were rescued by Sec23a indicating that a prime function was in remodelling the secretory pathway for ECM cargo secretion and that at least some phenotypic defects were pleiotropic to this role. This conclusion is reinforced by identification of the feelgood mutation in zebrafish, which results in defects in collagen secretion and skeletal formation, as being due to mutation in the zebrafish BBF2H7 homologue. Again Sec23a and Sec24d were found to be targets of the zebrafish factor. The main defect reported for OASIS knock-out animals was in bone formation and ECM secretion, in this case reflecting an abnormal ER and reduced ECM and collagen secretion in osteoblasts. It is also likely that both these factors function in additional tissues and indeed OASIS has been shown to induce secretory pathway genes such as KDLER3 and Copzeta2 and ECM remodelling in pancreatic cells where it is also highly expressed. Recent data also indicate that OASIS plays a role in secretion and differentiation of intestinal goblet cells. Transcription profiling of CREB3L4, whose expression is high in secretory tissues such as prostate, pancreas and small intestine again identified target genes with a common theme including Sec24d, KDLR2, KDLR3, and ECM components with significant overlap to those identified for CREB-H. In addition to a strong weighting in secretory pathway genes, there is a notable trend where ECM components are also targets of all of the CREB3 family members, including in this work our demonstration at the protein level of secretion of Col12a, Nidogen 1 and SPARC. These components are known to play a role in cell adhesion and presumably help explain the increased adhesion observed after CREB-H induction in 293 cells (Barbosa, 2013).

With increased numbers of CREB3-like proteins in this analysis, it was clear that the TM class could be segregated into two subclasses. With regard to the MP1 region between the end of the leucine zipper and start of the TM domain, the difference in the two subclasses was not a continuum; rather CREB3 species with a TM domain were either one class or the other. This precise region encompasses major determinants of ER retention in CREB-H. Furthermore, within TM itself, at position 15, class A proteins always had a serine and class B always a proline (with a single exception). In CREB-H, a class B protein, the proline at this position is critical for cleavage and release of the active form to the nucleus (Llarena, 2010). Although the serine presumably permits cleavage in class A proteins, nevertheless the strict distinction strongly suggests potential differences in cleavage mechanism, whether in the enzymes involved or presentation to S2P. Taken together, the distinct subgrouping, firstly in a region shown to influence ER retention/transport and secondly in the TM region, indicate potential differences in regulation and processing between the two classes. Although not the subject of the current work, specific predictions based on these proposals will be tested in future analysis (Barbosa, 2013).

In certain species including Drosophila, the class C proteins, which lack a TM domain, activity must be regulated in a distinct manner. Thus, although CREBA, the only class C protein for which any information is available, does indeed regulate secretory pathway genes, it cannot be regulated by pathways dependent upon integral membrane localisation in the ER. It is also possible that CREBA plays multiple roles, not only in responsive, homeostatic controls, but also in other pathways such as differentiation, involving SPGs, or other pathways not limited to SPGs. While the reason for the lack of a TM domain is unclear, the most evolutionary ancient family which was found to contain ATB-BZip proteins was the sponges; in this case the two sponge proteins both contained TM domains. Presumably certain lineages lost the TM domain and although the Drosophila CREBA protein clearly controls secretory function it may have acquired additional roles as a consequence of distinct nuclear localisation (Barbosa, 2013).

There is pronounced overlap in the spectrum of genes in the secretion pathway under CREB3 protein control; nonetheless there are also differences in subsets of genes targeted by individual members. It is likely that distinct signals and mechanisms are involved in relaying demand or stress to distinct CREB3 members, though there is currently limited information in this regard. Also whereas most target genes identified to date are either secretory pathway components, secreted cargos or modifying enzymes, CREB3 proteins including TM-containing members may play additional roles outside these activities. This possibility notwithstanding, taking the following considerations together: 1, that CREB3 factors are anchored in the ER; 2, that they share with CREBA a unique feature of their DNA binding domains, only seen in CREB3/CREBA; 3, that they all function in SPG induction in Drosophila; 4, that independent studies of different family members reveal a consistent theme on target gene involvement in the secretory apparatus and extra-cellular matrix formation; 5, that CREB-H directly induces increased secretion rates and specialised cargo secretion, it is reasonable to propose a general framework for the CREB3 family where their primary role is to orchestrate the secretory apparatus for increased flux though the secretory pathway, potentially with specific regard to specialised cargos or modifying components. It is proposed that this function underpins their roles in diverse physiological pathways such as promoting differentiation, responding to metabolic fluctuation or potential inflammatory signalling (Barbosa, 2013).


Search PubMed for articles about Drosophila Cyclic-AMP response element binding protein A

Abel, T., Bhatt, R. and Maniatis, T. (1992). A Drosophila CREB/ATF transcriptional activator binds to both fat body- and liver-specific regulatory elements. Genes Dev 6: 466-80. PubMed Citation: 1532159

Abrams, E. W. and Andrew, D. J. (2005). CrebA regulates secretory activity in the Drosophila salivary gland and epidermis. Development 132(12): 2743-58. 15901661

Abrams, E. W., Mihoulides, W. K. and Andrew, D. J. (2006). Fork head and Sage maintain a uniform and patent salivary gland lumen through regulation of two downstream target genes, PH4αSG1 and PH4αSG2. Development 133(18): 3517-27. PubMed Citation: 16914497

Andrew, D. J., et al. (1997). The Drosophila dCREB-A gene is required for dorsal/ventral patterning of the larval cuticle. Development 124: 181-193. PubMed Citation: 9006079

Barbosa, S., Fasanella, G., Carreira, S., Llarena, M., Fox, R., Barreca, C., Andrew, D. and O'Hare, P. (2013). An orchestrated program regulating secretory pathway genes and cargos by the transmembrane transcription factor CREB-H. Traffic 14: 382-398. PubMed ID: 23279168

Fox, R. M., Hanlon, C. D. and Andrew, D. J. (2010). The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity. J Cell Biol 191: 479-492. PubMed ID: 21041443

Gaudet, J. and Mango, S. E. (2002). Regulation of organogenesis by the Caenorhabditis elegans FoxA protein PHA-4. Science 295: 821-825. PubMed Citation: 11823633

Henderson, K. D. and Andrew, D. J. (2000). Regulation and function of Scr, exd, and hth in the Drosophila salivary gland. Dev. Biol. 217: 362-374. PubMed ID: 10625560

Hirano, Y., Masuda, T., Naganos, S., Matsuno, M., Ueno, K., Miyashita, T., Horiuchi, J. and Saitoe, M. (2013). Fasting launches CRTC to facilitate long-term memory formation in Drosophila. Science 339: 443-446. PubMed ID: 23349290

Llarena, M., Bailey, D., Curtis, H. and O'Hare, P. (2010). Different mechanisms of recognition and ER retention by transmembrane transcription factors CREB-H and ATF6. Traffic 11: 48-69. PubMed ID: 19883396

Mair, W., Morantte, I., Rodrigues, A. P., Manning, G., Montminy, M., Shaw, R. J. and Dillin, A. (2011). Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB. Nature 470: 404-408. PubMed ID: 21331044

Murakami, T., et al. (2009). Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation. Nat Cell Biol 11: 1205-1211. PubMed ID: 19767743

Norris, J. L. and Manley, J. L. (1995). Regulation of dorsal in cultured cells by Toll and tube: tube function involves a novel mechanism. Genes Dev. 9: 358-369. PubMed ID: 7867932

Rose, R. E., et al. (1997). The CRE-Binding protein dCREB-A is required for Drosophila embryonic development. Genetics 146: 595-606. PubMed ID: 9178009

Saito, A., Hino, S., Murakami, T., Kanemoto, S., Kondo, S., Saitoh, M., Nishimura, R., Yoneda, T., Furuichi, T., Ikegawa, S., Ikawa, M., Okabe, M. and Imaizumi, K. (2009). Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis. Nat Cell Biol 11: 1197-1204. PubMed ID: 19767744

Seshaiah, P., Miller, B., Myat, M. M. and Andrew, D. J. (2001). pasilla, the Drosophila homologue of the human Nova-1 and Nova-2 proteins, is required for normal secretion in the salivary gland. Dev. Biol. 239: 309-322. PubMed Citation: 11784037

Shen, R., Wang, B., Giribaldi, M.G., Ayres, J., Thomas, J.B. and Montminy, M. (2016). Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance. Proc Natl Acad Sci U S A 113(23):E3307-14. PubMed ID: 27208092

Smolik, S. M., Rose, R. E. and Goodman, R. H. (1992). A cyclic AMP-responsive element-binding transcriptional activator in Drosophila melanogaster, dCREB-A, is a member of the leucine zipper family. Mol Cell Biol 12: 4123-31. PubMed ID: 1508208

Takiya, S., Gazi, and Mach, V. (2003). The DNA binding of insect Fork head factors is strongly influenced by the negative cooperation of neighboring bases. Insect Biochem. Mol. Biol. 33: 1145-1154. PubMed Citation: 14563365

Yin, J.C., Del Vecchio, M., Zhou, H. and Tully, T. (1995). CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long term memory in Drosophila. Cell 81(1): 107-15. PubMed ID: 7720066

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date revised: 20 August 2016

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