CrebA: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | 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
BIOLOGICAL OVERVIEW

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).


GENE STRUCTURE

Genomic length - 21 kb

cDNA clone length - 4478

Bases in 5' UTR - 988

Exons - 6

Bases in 3' UTR - 1942


PROTEIN STRUCTURE

Amino Acids - 515

Structural Domains

CrebA codes for a member of the bZIP superfamily of DNA binding proteins. The bZIP proteins include C/EBP (Drosophila homolog: SLBO), c-Fos (Drosophia homolog: Fos-related antigen), c-Jun (Drosophila homolog: Jun related antigen or JRA), ATF, and CREB (Drosophila homologs CrebA and CrebB-17A). All these proteins possess a basic region, which is responsible for DNA binding, followed by a heptad repeat of leucine residues, termed the leucine zipper, which mediates dimerization. The 40 amino acid basic region of CrebA is 58% similar to the corresponding region of c-Fos, 53% similar to that of the Drosophila FOS protein, 57% similar to the Drosophila JRA and 42% similar to the basic domain of C/EBF. The leucine repeat of CrebA contains five heptad repeats but is interrupted by the replacement of the third leucine residue by tyrosine. CrebA forms dimers in solution: it is known that an intact leucine zipper is required for dimer formation (Abel, 1992).

Like the mammalian CREB proteins, CrebA has a C-terminal leucine zipper, an adjacent, highly basic DNA-binding domain, an amino-terminal transcriptional-activation domain. The binding domain shows 48% homology with the corresponding region in mammalian CREB and includes the sequence RRKKKEY abutting the leucine zipper domain, which is exactly conserved among CREB, CREM and ATF-1. The leucine zipper of CrebA is unusual in that it has a tyrosine substitution at the third leucine position. Another unusual feature is the length of the leucine zipper. While the CREB zippers are defined by four leucine iterations, the CrebA zipper consists of six hydrophobic residue iterations. The CrebA protein does not contain a typical PKA site within its activation but does contain three possible CaM kinase II sites. Helical wheel analysis of the CrebA zipper suggests that it, like other basic-domain leucine zipper factors, should form dimers (Smolik, 1992).


CrebA: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 July 2013 

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