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).

GENE STRUCTURE

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).

date revised: 28 August 97 

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