InteractiveFly: GeneBrief

Cryptocephal: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - cryptocephal

Synonyms - ATF4

Cytological map position-39C2-39C3

Function - transcription factor

Keywords - molting, metamorphosis, response to neuropeptides and peptide hormones, unfolded protein response

Symbol - crc

FlyBase ID: FBgn0000370

Genetic map position -

Classification - CREB/ATF superfamily bZIP domain proteins

Cellular location - nuclear

NCBI link: EntrezGene

crc orthologs: Biolitmine
Recent literature
Kang, M. J., Vasudevan, D., Kang, K., Kim, K., Park, J. E., Zhang, N., Zeng, X., Neubert, T. A., Marr, M. T., and Don Ryoo, H. (2016). 4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging. J Cell Biol. PubMed ID: 27979906
Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP (Thor) is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. This study shows that GCN2 and its downstream transcription factor, ATF4 (Cryptocephal), mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.
Celardo, I., Lehmann, S., Costa, A.C., Loh, S.H. and Miguel Martins, L. (2017). dATF4regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective. Cell Death Differ [Epub ahead of print]. PubMed ID: 28211874
Neurons rely on mitochondria as their preferred source of energy. Mutations in PINK1 and PARKIN cause neuronal death in early-onset Parkinson's disease (PD), thought to be due to mitochondrial dysfunction. In Drosophila pink1 and parkin mutants, mitochondrial defects lead to the compensatory upregulation of the mitochondrial one-carbon cycle metabolism genes by an unknown mechanism. This study uncovers that this branch is triggered by the activating transcription factor 4 (ATF4). ATF4 regulates the expression of one-carbon metabolism genes SHMT2 and NMDMC as a protective response to mitochondrial toxicity. Suppressing Shmt2 or Nmdmc causes motor impairment and mitochondrial defects in flies. Epistatic analyses show that suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. Conversely, the genetic enhancement of these one-carbon metabolism genes in pink1 or parkin mutants is neuroprotective. The study concludes that mitochondrial dysfunction caused by mutations in the Pink1/Parkin pathway engages ATF4-dependent activation of one-carbon metabolism as a protective response. These findings show a central contribution of ATF4 signalling to PD that may represent a new therapeutic strategy.
Malzer, E., Dominicus, C. S., Chambers, J. E., Dickens, J. A., Mookerjee, S. and Marciniak, S. J. (2018). The integrated stress response regulates BMP signalling through effects on translation. BMC Biol 16(1): 34. PubMed ID: 29609607
Developmental pathways must be responsive to the environment. Phosphorylation of eIF2alpha enables a family of stress-sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone morphogenetic proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. This study shows in Drosophila that GCN2 antagonises BMP signalling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2alpha phosphatase dPPP1R15 impairs developmental BMP signalling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signalling. Moreover, loss of d4E-BP, a target of crc, augments BMP signalling in vitro and rescues tissue development in vivo. These results identify a novel mechanism by which the ISR modulates BMP signalling during development.
Hunt, R. J., Granat, L., McElroy, G. S., Ranganathan, R., Chandel, N. S. and Bateman, J. M. (2019). Mitochondrial stress causes neuronal dysfunction via an ATF4-dependent increase in L-2-hydroxyglutarate. J Cell Biol. PubMed ID: 31645461
Mitochondrial stress contributes to a range of neurological diseases. Mitonuclear signaling pathways triggered by mitochondrial stress remodel cellular physiology and metabolism. How these signaling mechanisms contribute to neuronal dysfunction and disease is poorly understood. This study finds that mitochondrial stress in neurons activates the transcription factor ATF4 as part of the endoplasmic reticulum unfolded protein response (UPR) in Drosophila. ATF4 activation reprograms nuclear gene expression and contributes to neuronal dysfunction. Mitochondrial stress causes an ATF4-dependent increase in the level of the metabolite L-2-hydroxyglutarate (L-2-HG) in the Drosophila brain. Reducing L-2-HG levels directly, by overexpressing L-2-HG dehydrogenase, improves neurological function. Modulation of L-2-HG levels by mitochondrial stress signaling therefore regulates neuronal function.
Vasudevan, D., Katow, H., Huang, H. W., Tang, G. and Ryoo, H. D. (2021). A Protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development. Dis Model Mech. PubMed ID: 34919148
Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the Integrated Stress Response (ISR), an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. This study reports that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, these data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival.

The cryptocephal (crc) mutation causes pleiotropic defects in ecdysone-regulated events during Drosophila molting and metamorphosis. crc encodes a Drosophila homolog of vertebrate ATF4, a member of the CREB/ATF family of basic-leucine zipper (bZIP) transcription factors. Three putative protein isoforms were identified. CRC-A and CRC-B contain the bZIP domain, and CRC-D is a C-terminally truncated form. Seven new crc alleles were generated. Consistent with the molecular diversity of crc, these alleles show that crc is a complex genetic locus with two overlapping lethal complementation groups. Alleles representing both groups were rescued by a cDNA encoding CRC-B. One lethal group (crc1, crcR6, and crcRev8) consists of strong hypomorphic or null alleles that are associated with mutations of both CRC-A and CRC-B. These mutants display defects associated with larval molting and pupariation. In addition, they fail to evert the head and fail to elongate the imaginal discs during pupation, and they display variable defects in the subsequent differentiation of the adult abdomen. The other group (crcR1, crcR2, crcE85, crcE98, and crc929) is associated with disruptions of CRC-A and CRC-D; except for a failure to properly elongate the leg discs, these mutants initiate metamorphosis normally. Subsequently, they display a novel metamorphic phenotype, involving collapse of the head and abdomen toward the thorax. The crc gene is expressed throughout development and in many tissues. In third instar larvae, crc expression is high in targets of ecdysone signaling, such as the leg and wing imaginal discs, and in the ring gland, the source of ecdysone. Together, these findings implicate CREB/ATF proteins in essential functions during molting and metamorphosis. In addition, the similarities between the mutant phenotypes of crc and the ecdysone-responsive genes indicate that these genes are likely to be involved in common signaling pathways (Hewes, 2000).

The development of Drosophila and other insects is punctuated by several molts, during which the animal produces a new external cuticle and sheds the old one. The larval molts are initiated and coordinated by steroid hormones, the ecdysteroids (hereafter called ecdysone). At the onset of metamorphosis, a high titer pulse of ecdysone triggers pupariation, which is followed ~12 hr later by a brief ecdysone pulse that causes head eversion and the prepupal-pupal transition. Subsequently, a large, prolonged surge of ecdysone directs adult development. These remarkable developmental changes involve the programmed cell death of most larval tissues, extensive remodeling of other larval tissues, and the generation of adult tissues from islands of undifferentiated imaginal cells (Hewes, 2000).

Detailed studies of the responses to ecdysone in the larval salivary glands provided key insights into the gene regulatory pathways controlling molting and metamorphosis. Expression of a small set of about six early genes is triggered rapidly and directly by ecdysone. Together with ecdysone, these genes regulate numerous late genes. Four early genes have been molecularly characterized. E74 encodes a family of ETS proteins, E75 encodes a family of orphan nuclear receptors, the Broad-Complex (BR-C) encodes a family of zinc-finger proteins, and E63-1 encodes a novel Ca2+-binding protein. E74 and the BR-C control developmental responses to ecdysone in diverse larval and imaginal tissues at least in part through direct transcriptional regulation of the late genes. Thus, the early genes are near the top of a complex gene regulatory hierarchy (Hewes, 2000).

Mutations in several ecdysone-responsive genes reveal distinctive phenotypes that reflect important developmental functions. For example, an E74B mutant displays incomplete differentiation of the adult abdomen and failed gas bubble translocation at pupation. A mutation of ßFTZ-F1, which functions as a competence factor for pupal stage-specific responses to ecdysone, displays similar translocation defects. Mutations in E74, ßFTZ-F1, and two other ecdysone-response genes, crol and DHR3, each display several additional common features. These include defective eversion of the adult head and (with the exception of DHR3) incomplete leg disc elongation. The head eversion defect is called the 'cryptocephal' phenotype, named after cryptocephal (crc1), a mutation that displays all of the above-mentioned defects (Hadorn, 1943). These phenotypic parallels indicate that crc and the ecdysone-response genes are likely to be involved in common regulatory pathways (Hewes, 2000).

Fristrom (1965) examined chitin biosynthesis in the crc1 mutant and concluded that the head eversion defect is due to excess chitin deposition in (and increased stiffness of) the cuticle. Sparrow (1982) tested this hypothesis with a different crc1 strain and found normal chitin deposition. At pupation, crc1 mutants display contractions of the abdomen that are slower, more irregular, and weaker than in wild-type animals, indicating that behavioral abnormalities may cause at least some of the phenotypic defects observed in these mutants (Chadfield, 1985). However, behavioral abnormalities likely do not explain other aspects of the crc1 mutant phenotype, such as incomplete abdominal differentiation. In a discussion of the similar phenotypic defects displayed by E74B mutants, Fletcher (1995) hypothesized that premature muscle death accounts for the full range of defects observed in these cryptocephalic mutants. To distinguish among these and other competing models, it will be important to characterize crc gene function at the molecular and cellular level (Hewes, 2000).

This study shows that crc encodes multiple proteins in the activating transcription factor 4 (ATF4) subfamily of CREB/ATF basic-leucine zipper (bZIP) transcription factors. ATF4 proteins have been implicated in several important developmental and disease processes, including wound healing (Estes, 1995), long-term synaptic facilitation (Bartsch, 1995), stress responses (Fawcett, 1999), apoptosis (Kawai, 1998), and cancer (Mielnicki, 1996). The Drosophila ATF4 homologs play critical roles in molting and metamorphosis. Seven new crc alleles have been isolated, that reveal multiple functions of the gene in larval molting, pupariation, pupation, and adult differentiation. These tissues include several targets of ecdysone signaling as well as the endocrine source of ecdysone, the ring gland. These findings implicate CREB/ATF transcription factors for the first time in the hormonal regulation of molting and metamorphosis. Moreover, these results indicate that there are likely to be important interactions between signaling by crc and the ecdysone-response genes (Hewes, 2000).

crc is a complex locus encoding multiple mRNA and protein isoforms. The two most abundant forms are CRC-A and CRC-B; on the basis of their representation among ESTs, the transcripts encoding CRC-A outnumber those encoding CRC-B by approximately nine to one. Consistent with this observation, most of the in situ hybridization signal observed with a probe for both isoforms was attributable to the transcript encoding CRC-A. CRC-A and CRC-B differ at the N terminus, while a common C-terminal region contains identical bZIP protein dimerization and DNA-binding domains. Therefore, CRC-A and CRC-B likely share dimerization partners and show identical DNA-binding properties (Hewes, 2000).

In addition to the two major mRNA isoforms, there were three uncommon transcripts, crc-d-f, which may serve regulatory functions. crc-d encodes CRC-D, a C-terminally truncated form of CRC-A. Therefore, CRC-D lacks the bZIP domain and could function as a dominant negative regulator by competing with CRC-A (or other factors) for protein-binding sites. The expression of CRC-D may be essential for viability; the crcE85 mutation, which partially deletes a CRC-D-specific exon, displays significant lethality. The crc-e and crc-f transcripts have very small open reading frames that are preceded by suboptimal translational start sites, indicating that they may not be efficiently translated. Rather, these transcripts may participate in the regulation of the crc gene. For mammalian CREB, the expression of truncated forms has been proposed to interrupt a positive feedback loop involving autoactivation of the gene. Therefore, similar mechanisms may be involved in the regulation of crc expression (Hewes, 2000).

Genetic analysis demonstrated that crc is a complex locus consisting of at least two overlapping lethal complementation groups. These complementation groups correlate with the molecular structure of the crc gene, indicating that the different CRC protein isoforms have overlapping, but distinct functions. The following hypothesis is proposed to explain the correlation between the molecular and genetic results. The 3' complementation group phenotypes reflect the functions of both CRC-A and CRC-B. Consistent with this prediction, the one observed sequence alteration in crc1 (Q171R) was found in a region common to CRC-A and CRC-B. The 5' group phenotypes reflect CRC-A- and/or CRC-D-specific functions that do not require CRC-B (Hewes, 2000).

Nevertheless, some overlap is anticipated in the functions of the different CRC proteins. The lethal phenotypes of both crc complementation groups were rescued by ectopic expression of a single RNA isoform encoding CRC-B. Furthermore, the C-terminal 288 amino acids of CRC-A and CRC-B are identical, and both lethal complementation groups displayed similar defects in leg disc elongation. R6, which deleted this common region, failed to complement both 5' and 3' group alleles (Hewes, 2000).

crc mutant alleles displayed several defects associated with molting and metamorphosis. The mutant phenotypes associated with the two lethal complementation groups are distinct, although there was some overlap. Therefore, these mutations define multiple roles for crc during development (Hewes, 2000).

In insects, the molts between successive larval stages are initiated and coordinated by pulses of ecdysone. This process appears to require crc. As previously described for the crc1 allele (Chadfield, 1985), both crc1 and R6 displayed larval lethality associated with failure to shed the old larval mouthparts. These alleles comprise the 3' complementation group and involve disruptions common to the crc-a, crc-b, and crc-c transcripts. Therefore, CRC-A and/or CRC-B perform an important role(s) in the regulation of larval molting. Similar larval phenotypes have been described for mutations in the dare gene, which encodes an adrenodoxin reductase likely to be involved in ecdysone biosynthesis. Likewise, mutants in EcR-B, which is a component of heterodimeric ecdysone receptors, and PHM, an enzyme involved in neuropeptide biosynthesis, both displayed this larval molting phenotype. These similarities indicate that CRC-A and CRC-B may perform necessary functions in the peptidergic neurons that stimulate ecdysone biosynthesis, in the ecdysone-producing prothoracic gland cells, and/or in the tissues that respond to the ecdysone signal (Hewes, 2000).

During the third larval instar, pulses of ecdysone trigger the onset of metamorphosis. A late high titer pulse of ecdysone triggers puparium formation. Approximately 12 hr later, a subsequent brief pulse of ecdysone directs pupation. crc mutants displayed defects in pupariation and pupation, indicating that both of these developmental transitions require crc. The pupariation defects seen in R6 hemizygotes -- retention of the larval shape, failure to form the abdominal gas bubble, and incomplete eversion of the anterior spiracles -- are reminiscent of similar defects described for late-arrested EcR-B mutants and for mutations in E74B and DHR3 (Hewes, 2000).

At pupation, crc1 and R6 both displayed the cryptocephal phenotype as well as defects in imaginal disc elongation. Similar pupation defects are associated with mutations in several ecdysone-response genes, including E74B, crol, ßFTZ-F1, DHR3, and the BR-C. Unlike crc1 and R6, the leg and wing discs in the 5' group mutants remained bulbous and undifferentiated, and often discolored. A phenotype similar to that of 5' group mutants has been reported for ßFTZ-F1. Therefore, lesions in crc and the ecdysone-response genes generate common defects in the larval, prepupal, and pupal responses to ecdysone signaling. These similarities indicate that crc has a central role in the regulation of ecdysone biosynthesis/secretion or in determining the responses of target tissues to the steroid signals. As an important next step in the analysis of crc function, whether crc is also an ecdysone-response gene will be examined (Hewes, 2000).

After comparing aspects of the E74B pupal phenotype and the phenotypes of mutations affecting larval muscle development, it has been proposed that premature death of the larval muscles might account for the defects observed at pupariation and pupation in those mutants. Due to similarities in phenotype between E74B and crc, this model could also account for the pupariation and pupation defects observed in crc mutants, but it probably does not explain the crc larval molting and adult fecundity defects. Moreover, as is true for E74B, most crc1 and R6 mutants display normal larval locomotion, indicating that the larval muscles develop and function normally prior to metamorphosis (Hewes, 2000).

Transgenic UAS-crc lines rescued the lethal phenotype of both the 5' and 3' lethal complementation groups (929 and crc1), confirming the identification of crc. The rescue was partial, and some aspects of the mutant phenotype, such as the reduction in female fecundity and the defects in adult wing expansion and tanning, showed no improvement. Several factors may have contributed to the incomplete rescue. These include requirements for expression of the CRC-A and CRC-D isoforms, or for more precise temporal and/or spatial regulation of CRC expression (Hewes, 2000).

One aspect of the rescue experiments did not fit simple predictions but may be explained by technical details of the transgene expression. The rank order of potency for the rescue of crc1 by the different UAS-crc lines was reversed for the rescue of 929. The variation in the degree of crc1 rescue was likely due to position effects that led to constitutive, low level expression of the transgene. By contrast, c929 is an enhancer trap P element that drives heterogeneous GAL4 reporter gene expression in several tissues. Thus, to explain the second observation, c929 may rescue the wild-type pattern of crc expression to a significant degree, while minimizing the negative effects of crc misexpression in other tissues (Hewes, 2000).

By analogy to other CREB/ATF proteins, the roles of crc during molting and metamorphosis are likely to involve heterodimerization with other bZIP proteins and competition with them for DNA-binding sites. Similarly, it is hypothesized that ecdysone-responsive signaling pathways include crc. For example, by convergence on the transcriptional coactivator, CREB-binding protein (CBP), CREB/ATF proteins can antagonize the activity of members of the nuclear receptor superfamily. This family includes several ecdysone-response genes. Therefore, further analysis of crc may elucidate several points of interaction between crc and these hormonal signaling pathways (Hewes, 2000).

Drosophila melanogaster Activating Transcription Factor 4 regulates glycolysis during endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response (UPR), which plays important roles in development and disease. This study shows that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the TCA cycle and respiratory chain complexes were down-regulated. The UPR transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells (Lee, 2015).

As the flux of proteins through the ER varies considerably among cell types and in different conditions, cells maintain a balance between the load on the ER and its protein folding capacity. However, a number of biochemical, physiological, and pathological stimuli can disrupt this balance, resulting in ER stress. To re-establish ER homeostasis, the unfolded protein response is activated. This network of pathways up-regulates genes encoding ER-specific chaperones and other proteins involved in protein secretion, while also attenuating protein translation and degrading certain ER-associated mRNAs. The UPR is broadly conserved across eukaryotes and is essential for normal development in several model organisms, particularly for professional secretory cells, where it is thought to be important for the establishment and maintenance of high levels of protein secretion . It is also induced during many metabolic conditions including diabetes, hyperlipidemia, and inflammation, and has been implicated in various cancers, especially in the growth of large tumors that rely on an effective response to hypoxia (Lee, 2015).

The UPR is carried out by three main signaling branches. One of these is initiated by the ER transmembrane protein Inositol-requiring enzyme 1 (Ire1). When activated by ER stress, the cytosolic endoribonuclease domain of Ire1 cleaves the mRNA encoding the transcription factor Xbp1, thereby initiating an unconventional splicing event that produces the mRNA template encoding a highly active form of Xbp1. Ire1 also cleaves other mRNAs associated with the ER membrane, through a pathway that is particularly active in Drosophila cells and that may reduce the load on the ER. A second sensor of ER stress, Activating transcription factor 6 (Atf6), is activated by proteolysis, which releases it from the ER membrane and allows it to travel to the nucleus and regulate gene expression. Finally, Protein kinase RNA (PKR)- like Pancreatic ER kinase (Perk) (see Drosophila pancreatic eIF-2α kinase or Pek) phosphorylates eukaryotic initiation factor 2 alpha, leading to a general attenuation of protein synthesis as well as the translational up-regulation of certain mRNAs that contain upstream open reading frames (ORFs) in their 5' untranslated regions. Activating transcription factor 4 (Atf4)/Cryptocephal is among those proteins that are up-regulated translationally during ER stress, and regulates genes involved in protein secretion as well as amino acid import and resistance to oxidative stress (Lee, 2015).

In addition to its direct effects on the protein secretory pathway, the UPR influences several other cellular pathways including apoptosis, inflammation, and lipid synthesis. Furthermore, the UPR (particularly the Perk/Atf4 branch) appears to have close ties to mitochondrial function. For example, knockout of Mitofusin 2, a key mitochondrial fusion protein, activates Perk, leading to enhanced reactive oxygen species (ROS) production and reduced respiration. Atf4 also increases expression of Parkin, which mediates degradation of damaged mitochondria, protecting cells from ER stress-induced mitochondrial damage. Despite clear links between ER stress and mitochondria, the mechanistic relationship between the UPR and mitochondrial metabolism is not well-understood (Lee, 2015).

This study reports that the UPR in Drosophila S2 cells triggers a coordinated change in the expression of genes involved in carbon metabolism. The metabolism of glucose as an energy source produces pyruvate, which can then enter the mitochondria and the tricarboxylic acid (TCA) cycle to produce reducing equivalents for oxidative phosphorylation (OXPHOS). For most cells in normal conditions, the majority of ATP is produced through OXPHOS. However, in hypoxic conditions when OXPHOS is limited, cells rely heavily on glycolysis to compensate for the decrease in ATP production, and convert the excess pyruvate to lactate, which then leaves the cel. This shift from OXPHOS to glycolysis is seen in a variety of cancers even when cells have access to oxygen, an effect known as aerobic glycolysis or the Warburg effect, and is thought to be a hallmark of cancer cells. Aerobic glycolysis is also becoming increasingly recognized as a metabolic signature of other cell types as well, including stem cells and activated immune cells (Lee, 2015).

In Drosophila, the Estrogen-related receptor (dERR) is the only transcription factor known to regulate glycolytic genes (Li et al. 2013). Its activity is temporally regulated during mid-embryogenesis to support aerobic glycolysis during larval growth (Tennessen, 2011). Moreover, a recent study found that glycolytic gene expression under hypoxic conditions in larvae is partially dependent on dERR (Li, 2013). This study shows that the UPR transcription factor Atf4 also regulates glycolytic genes, contributing to a broad regulation of metabolic gene expression during ER stress that is reminiscent of the Warburg effect (Lee, 2015).

This study has shown that Drosophila S2 cells subjected to ER stress up-regulate glycolytic genes and Ldh and down-regulate genes involved in the TCA cycle and respiratory chain complex. Furthermore, Atf4 is responsible for the up-regulation of glycolytic genes and Ldh. How TCA cycle and respiratory chain complex genes are down-regulated during ER stress requires further investigation, although the lack of effect of Atf4 depletion suggests that these are not regulated as an indirect consequence of glycolysis up-regulation (Lee, 2015).

Despite a highly coordinated change in gene expression for metabolic genes during ER stress, this study did not detect any changes in actual metabolism in S2 cells. Because these cells have been in culture for decades and have likely been selected for rapid proliferation, it is possible that they are already undergoing some version of aerobic glycolysis, such that the underlying gene regulation during ER stress is preserved but any metabolic changes are masked. Others have also noted that S2 cells are resistant to hypoxia, and do not produce more lactate except in extreme conditions. The increase in lactate observed through in vivo studies in flies subjected to ER stress, however, suggests that in a more physiological setting, the gene expression changes shown here do mediate a metabolic shift toward aerobic glycolysis (Lee, 2015).

Up-regulation of glycolytic genes during ER stress has not been observed in genome-wide studies of mammalian cells. However, several lines of evidence suggest that mammalian cells subjected to ER stress may undergo a glycolytic shift. For example, a recent study examining human gliomas found coordinated up-regulation of UPR targets and glycolysis, which correlated with poor patient prognosis; and both ER stress and overexpresson of Perk have been shown to reduce mitochondrial respiration in cultured mammalian cells (Lee, 2015).

The link between ER stress and metabolism can be rationalized by the need to generate building blocks for biosynthesis of glycoproteins and lipids. Early intermediates of glycolysis are necessary for production of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), an important donor molecule for N-glycosylation of proteins in the ER. Both fructose-6-phosphate and dihydroxyacetone phosphate are also required for synthesis of glycolipids. An increased flux through glycolysis may therefore be important to support the increased production of glycerophospholipids and glycoproteins that are associated with the UPR. In support of this view, glucose deprivation or inhibition of glycolysis with 2-deoxy-D-glucose induces the UPR, which contributes to cell death, especially in cancer cells, and this effect can be rescued by UDP-GlcNAc. The hexosamine biosynthetic pathway generating UDP-GlcNAc is also directly activated by Xbp1, and stimulates cardioprotection during ischemia/reperfusion injury and increases longevity in worms (Lee, 2015).

A second, non-mutually exclusive explanation for a shift to glycolysis during ER stress is the need to limit production of ROS. Along with mitochondrial respiration, protein folding in the ER is one of the main sources of ROS, which are produced by the normal process of disulfide bond-coupled folding. If allowed to accumulate, these ROS can cause oxidative stress and damage to cells, eventually leading to apoptosis. Several studies have confirmed that ROS are produced during ER stress, when protein folding is inefficient and more rounds of oxidation and reduction are required to fold proteins. Limiting other sources of oxidative stress, such as by down-regulating the TCA cycle and thereby restricting the flux through OXPHOS (the main source of ROS in the mitochondria), may be a way to mitigate the damage and allow cells to recover more effectively (Lee, 2015).

Finally, the advantage of the Warburg effect for tumor growth may arise from the increased rate of ATP production by glycolysis compared to OXPHOS, despite its lower efficiency of conversion. By analogy, a metabolic shift during ER stress could rapidly supply ATP necessary for protein folding and processing. Indeed, cancer cells showing elevated levels of ENTPD5, an ER UDPase, promotes aerobic glycolysis to increase ATP for protein N-glycosylation and refolding (Lee, 2015).

Overall, these results identify Atf4 as a transcriptional regulator of glycolysis during ER stress. As Atf4 is expressed throughout fly development (Hewes, 2000), it may regulate glycolysis in other situations as well: notably, Atf4 mutant flies are lean and have reduced circulating carbohydrates, suggesting a role in metabolism. Furthermore, because the Perk-Atf4 branch of UPR is activated during hypoxia, it will be interesting to see whether Atf4 contributes to regulation of glycolysis in other developmental, physiological (hypoxia), or pathological process during which glycolysis regulated. More broadly, since the UPR is activated in many types of cancer, its ability to regulate glucose metabolism may play a contributing role in the Warburg effect (Lee, 2015).

The GCN2-ATF4 signaling pathway induces 4E-BP to bias translation and boost antimicrobial peptide synthesis in response to bacterial infection

Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. This study used a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). 4E-BP was found to be induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5' UTRs of AMPs score positive in cap-independent translation assays, and this cap-independent activity is enhanced by 4E-BP. These results are corroborated in vivo using transgenic 5' UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis (Vasudevan, 2017).

Much research has focused on the transcriptional response to infection mediated by the innate immune response pathways. Although translational inhibitors such as GCN2 and 4E-BP have been implicated in the antibacterial response, the incongruence of translation inhibition and the need for AMP synthesis had not been addressed experimentally. This study provides results that resolve this discrepancy and establish that AMPs have evolved mechanisms to bypass translation inhibition imposed by pathogenic bacteria via 4E-BP activation. This study has shown that the ISR pathway promotes innate immunity against pathogenic bacteria by mediating 4e-bp induction, which in turn biases cellular translation toward cap-independent mechanisms that favor AMP synthesis. The newly identified role of the ISR pathway in the innate immune response is likely coordinated with the established roles of other innate immune response pathways, such as those mediated by FOXO, IMD, and Dorsal/Dif (Vasudevan, 2017).

In addition to regulation by GCN2/ATF4 and FOXO, 4E-BP is also famously regulated post-translationally by another amino acid-sensitive kinase, TOR. While under steady-state conditions, TOR phospho-inactivates 4E-BP, and TOR itself is inactivated in response to amino acid deprivation. Thus, 4E-BP newly synthesized in response to infection-mediated amino acid deprivation will likely not be subject to inactivation by TOR (Vasudevan, 2017).

These observations are consistent with the positive effects of 4e-bp induction against a non-lethal pathogen such as Ecc15. However, infection by a severe pathogen such as Pseudomonas entomophila results in a GCN2-mediated translation block in the gut and is detrimental to the host immune response (Chakrabarti, 2012). Why GCN2 mediates such different outcomes remains unresolved, but it could be because of factors other than ATF4 that are downstream of GCN2 (Vasudevan, 2017).

It is worth noting that GCN2 engages two different translation inhibition mechanisms: (1) phospho-eIF2α, which induces ATF4; and (2) 4e-bp, which is transcriptionally induced by ATF4. This study primarily focused on 4E-BP because the effect of phospho-eIF2α on translation may be temporary because ATF4 induces the expression of an eIF2α phosphatase subunit, GADD34, thereby stimulating eIF2α dephosphorylation. It is speculated that eIF2α phosphorylation may not serve as a long-term deterrent for AMP translation, while the second translational block by 4E-BP may persist longer. In addition, the data show that 4e-bp itself is synthesized favorably by cap-independent translation conferred by its 5' UTR, suggesting that this inhibition mechanism is self-sustaining (Vasudevan, 2017).

Intriguingly, the 5' UTRs of Drosomycin and Attacin A, which confer cap-independent translation capabilities to their respective mRNA, are relatively small at 63 and 29 bases, respectively. These are the smallest characterized cap-independent translation elements known. Although there are no known consensus sequences for IRESs, scoring positively in biochemical assays such as bicistronic assays and cap competition assays has been recognized to be a reliable indicator for them. Although both Drosomycin and Attacin A are likely translated cap-independently via IRES-like elements in their 5' UTRs, they may utilize slightly different mechanisms. Although both 5' UTRs confer cap-independence, which is enhanced by 4E-BP, the Attacin A 5' UTR reporter shows basal translation of the reporter even in the absence of 4E-BP, whereas the Drosomycin 5' UTR relies heavily on 4E-BP. Additionally, eliminating competing transcripts in the presence of excess m7GpppG also had a small but significant effect on Attacin A 5' UTR reporter mRNA, suggesting that there is an additional layer of regulatory mechanisms acting on these 5' UTRs (Vasudevan, 2017).

Because suppression of cap-dependent translation by 4E-BP appears to stimulate the synthesis of AMPs, it is speculated that the AMP 5' UTRs do not compete well with other mRNAs for ribosomes and initiation factors. Thus, in addition to the established role of 4E-BP in the inhibition of cap-dependent translation, the data show a more nuanced role for 4E-BP as a promoter of cap-independent translation required for driving the synthesis of essential immune response proteins. Interestingly, it had been previously suggested that eIF2α-kinases such as GCN2 could promote cap-independent translation, although the effectors of such regulation were unknown. Based on the curren work showing that 4E-BP downstream of GCN2/ATF4 signaling regulates AMP translation, it can now be surmised that these previously observed instances of cap-independent translation downstream of phospho-eIF2α are also likely mediated by ATF4-induced 4E-BP. Additionally, given the conservation of translation inhibition during infection in mammals, it would be interesting to examine whether the mammalian innate immune response is aided by any of the three known mammalian 4E-BPs and if first-response cytokines are synthesized cap-independently (Vasudevan, 2017).

ATF4-induced Warburg metabolism drives over-proliferation in Drosophila

The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. This study shows that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. This stress adaptation is found to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. These results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity (Sorge, 2020).

Controlling cell proliferation is one of the major challenges of multicellular life, both during phases of growth in developing organisms and phases of homeostatic cell replenishment essential in adult animals. Lack of appropriate control can lead to severe disorders, including cancer, at any stage of life. While over-proliferation of transformed, cancerous cells is usually caused by inactivation of tumor suppressors and/or activation of oncogenes, it has long been noted that tumors exhibit altered cellular characteristics such as a glycolytic metabolism. While this metabolic switch has been shown to be caused by oncogene signaling, cellular metabolism is also controlled at multiple levels under normal physiological (non-transformed) conditions, including at the transcriptional level through diverse stress-response pathways. One of these is the activating transcription factor 4 (ATF4), which is known to activate a transcriptional (integrated) stress response (ISR) under various stress conditions that trigger phosphorylation of eIF2α. The ATF4 transcriptional program consists of a diverse set of genes with cytoprotective function, but chronic activation induces apoptosis indirectly through transcription of the mammalian ATF4 target CHOP. Yet, ATF4 activation has been detected in several human tumors, especially in hypoxic or nutrient-deprived regions, where ATF4 has been attributed with pro-survival and pro-proliferative effects. Interestingly, a recent study showed that melanoma cells respond to inhibition of their glycolytic metabolism by activating an ATF4 response, whose metabolic reconfiguration allows these cells to continue oncogenic growth, together arguing that ATF4 can provide cancer cells with a metabolic flexibility that allows them to tolerate hypoxic and nutritional stress or cancer therapy aimed at metabolism. Among the many conditions activating ATF4, recent work with cultured cells showed that inhibition of mitochondrial function is linked to ATF4 translation and activity. However, from these and other studies, both the mechanistic basis and the in vivo implications of this response remain to be elucidated (Sorge, 2020).

This study shows that in the fruit fly, Drosophila melanogaster, genetic perturbation of the electron transport chain (ETC), which induces a Warburg-like metabolism, activates a transcriptional stress response mediated through the eIF2α-kinase PERK and ATF4 in eye progenitor cells of Drosophila larvae. Importantly, this in vivo stress response is activated under ETC knockdown conditions, in the absence of obvious mitochondrial dysfunction. Interestingly, these results show that the ATF4 transcriptional response, which by itself causes reduced fitness of progenitor cells, is hijacked by growth-promoting pathways like Notch or Ras, leading to increased cellular fitness and enhanced proliferation. The data furthermore suggest that the pH changes associated with ETC impairment resulting in a switch of the metabolism to aerobic glycolysis play an important role in progenitor over-proliferation. In sum, this study shows that ATF4-mediated transcriptional adaptation provides a cell-autonomous response to ETC defects, altering cellular behavior through metabolic adaptation (Sorge, 2020).

Genetically induced disturbance of ETC complex assembly resultrd in a metabolic shift typical for mitochondrial impairment and activated an ATF4-dependent stress response. The in vivo transcriptional adaptation presented in this study confirmed the regulation of LDH and glycolytic enzymes, as shown in Drosophila cultured cells, and further includes several targets shown to be ATF4 target genes in mammalian models. The results showed that the eIF2α-kinase PERK, so far only described for its role in mediating one branch of the unfolded protein response of the endoplasmic reticulum (UPRER), is the upstream kinase phosphorylating eIF2α, thereby inducing ATF4 translation in response to mitochondrial ETC disturbance. Mitochondrial ETC disturbance specifically activated PERK, while other branches of the UPRER were non-responsive. PERK activation upon mitochondrial defects was recently observed in Drosophila models of Parkinson's disease and was explained by the authors by its preferential localization to mitochondria-associated ER membranes, which might make PERK more susceptible to a local stress signal. ROS (reactive oxygen species) released by mitochondria have been suggested to mediate mitochondrial retrograde signaling. While this study observed an attenuation of Delta overexpression (DlOE), COX7RNAi-induced over-proliferation upon overexpression of either cytoplasmic catalase or GPx (but not mitochondrial catalase), this study failed to detect increased ROS levels in the larval eye disc. A possible scenario to explain these observations is that ROS are generated locally in the cytoplasm or ER in response to ETC disturbance, thereby triggering PERK activation. Importantly, Drosophila PERK isoform B contains a potential mitochondrial signal peptide, which is not found in mammalian PERK isoforms. Although no evidence for this has been found, Drosophila PERK could reside in the mitochondrial membrane and sense the folding status of mitochondrial complexes. This hypothesis could explain the evolutionary difference between mitochondrial defects and ATF4 induction, as this appears to require GCN2 but not PERK in mammals or to be triggered independently of a single eIF2α-kinase. In addition to canonical ATF4 target genes, ATF4-dependent upregulation of mitochondrial chaperones, a response classically referred to as the mitochondrial UPR (UPRmt) was observed. In C. elegans, mitochondrial chaperone induction upon stress is mediated by ATF4-like transcription factor Atfs-1, while the mammalian UPRmt has been shown to be regulated by another evolutionary-related transcription factor, ATF5. The current data now showed that Drosophila ATF4 is required cell autonomously for the induction of mitochondrial chaperones upon ETC subunit knockdown, implying that Drosophila might represent the evolutionary ancestral ISR-UPRmt regulation through a single ATF4-like transcription factor (Sorge, 2020).

The cooperation between ATF4 target genes and the Notch or Ras pathways in Drosophila imaginal progenitors raised the intriguing possibility that these or other oncogenic pathways could benefit from ATF4 activity in human cancers. Over the last decades, it had been demonstrated that human cancer cells are exposed to several stresses, including hypoxia, ROS, or limitations in nutrient availability. In order to survive these conditions and maintain their growth capacity, tumor cells activate responses like the HIF1α transcription factor axis. Though less well studied, an involvement of ATF4 in cancer has been suggested mostly through work with cultured cells. This study analyzed gene expression in human cancer samples of The Cancer Genome Atlas (TCGA) datasets using Cancer-RNaseq-Nexus and the human protein pathology atlas and found that many of the well-characterized direct ATF4 targets are upregulated in a variety of cancer types. Most strikingly, transcriptomes of kidney renal clear cell carcinoma showed progressive induction of ATF4 and many of its direct targets (EIF4EBP1, ASNS, TRIB3, and VEGFA) on the transcriptional level, which strongly correlated with a poor prognosis in this type of cancer. These data suggest that the ATF4-mediated ISR is used by cancer cells to adapt their metabolic repertoire, thereby sustaining fast growth under increasingly unfavorable conditions (Sorge, 2020).

A novel finding presented in this study was the discovery that ATF4-mediated transcriptional adaptation due to ETC impairment allowed eye progenitors to increase their proliferation in response to signals from the Notch and Ras pathways. The primary questions arising from this genetic interaction is how these signaling pathways can overcome the apparent cellular stress and reduction in proliferation and induce the opposite effect, a massively increased rate of proliferation. Several lines of evidence suggest that over-proliferation in DlOE, COX7RNAi eye imaginal discs is controlled by pH changes induced by LDH that modify the activity of Notch downstream effectors. First, in COX7a-depleted cells, the metabolism is switched to aerobic glycolysis, leading to an increased production of lactate due to the activity of LDH. And, consistent with an accumulation of this metabolic acid, this study found the intracellular pH to be reduced in COX7RNAi cells. Second, DlOE, COX7RNAi-mediated over-proliferation was rescued by ATF4 knockdown and, to a lesser extent, pH buffering, showing that intracellular pH changes (downstream of ATF4 and LDH) play an important role in proliferation control. Third, LDH phenocopies COX7RNAi, indicating that most of the cooperative effects of Dl overexpression and COX7a knockdown are mediated by the ATF4 target LDH. In the same line, expression of LDH as one of the many ATF4 targets was sufficient to drive Dl-expressing cells into over-proliferation, strongly suggesting that the processes downstream of LDH-in particular, the changes in intracellular pH-lead to a modification of the Notch pathway. Finally, a cooperation of Notch and the COX7a nuclear effector ATF4 on the transcriptional level was not observed, showing that the Notch pathway is not hyper-activated, but arguing that over-proliferation is due to changes in the activity of Notch downstream effectors. The next obvious question is how changes in intracellular pH can modify the activity of signaling pathways. It is known that the intracellular pH can control the protonation of specific histidine residues in proteins acting as pH sensors, leading to changes in protein properties. Importantly, it has been shown recently in chicken embryos that intracellular pH changes induced by a Warburg-like metabolism control the acetylation of the Wnt effector &betsa;-catenin, thereby mediating Wnt signaling activation. Thus, this study envisions that pH changes induced by LDH expression lead to a (non-enzymatic) modification of Notch effectors, thereby increasing fitness and proliferation rates of eye progenitor cells (Sorge, 2020).

This is a very attractive model; however, one result was puzzling. Although LDH is sufficient to induce over-proliferation when combined with the Notch pathway, no rescue (but an increase in the severity) of the DlOE, COX7RNAi phenotype was observed when LDH was selectively depleted. This obvious discrepancy can be explained by different hypotheses. One of them is based on a recent study showing that LDHA inhibition in melanoma cell lines also failed to impact cell proliferation, survival, or tumor growth. In this context, LDHA inhibition engaged the GCN2-ATF4 signaling axis to initiate an expansive pro-survival response, including the upregulation of the glutamine transporter SLC1A5 and glutamine uptake, as well as mTORC1 activation. Another hypothesis is based on the finding that a major driver of over-proliferation is the intracellular pH. Since LDH catalyzes the conversion of pyruvate to lactate (and back), reducing LDH levels will affect the ratio of lactate to pyruvate, leading to an increase of the even stronger metabolic acid pyruvate. It has been shown that pyruvate as lactate induces a concentration-dependent intracellular acidification. Thus, it could be envisioned that an enhancement of proliferation rates beyond those observed in DlOE, COX7RNAi cells is a consequence of pyruvate accumulation in the absence of LDH, which enhances the decrease in the intracellular pH, resulting in the increase in proliferation rates (Sorge, 2020).

Translational induction of ATF4 during integrated stress response requires noncanonical initiation factors eIF2D and DENR

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA (Met-tRNAiMet) for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. This study reports the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and Density regulated protein (DENR), a non-canonical translation initiation factor, in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, these findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo (Vasudevan, 2020).

The integrated stress response (ISR) in animals and a related-general amino acid control in yeast are adaptive signaling pathways that respond to a variety of stress conditions. Dysregulation of the ISR pathway is associated with a wide variety of diseases ranging from diabetes to neurodegenerative diseases such as Alzheimer's, reflecting the importance of cellular stress adaptation in health. In addition, activation of the ISR aids cancer cell survival and metastasis by enhancing cellular adaptation to extrinsic stresses in the tumor microenvironment (Vasudevan, 2020).

ISR signaling is initiated by stress-activated kinases that phosphorylate the α-subunit of the eIF2 complex, thereby inhibiting eIF2's ability to deliver initiator methionyl-tRNA (Met-tRNAiMet) to ribosomes. Such conditions reduce general mRNA translation in cells, but they also stimulate translation of the yeast GCN4 and the metazoan ATF4 main open reading frames, which encode transcription factors that mediate a signaling response. These mRNAs have regulatory 5' leader sequences containing multiple upstream open reading frames (uORFs), which interfere with main ORF translation in unstressed cells. Upon cellular stress that prompts eIF2α phosphorylation, this 5' leader sequence now stimulates the main ORF translation (Vasudevan, 2020).

This paper reports eIF2D and DENR as previously unrecognized regulators of ATF4 translation in Drosophila and human cells. Though several studies have examined regulatory targets of DENR individually, none have uncovered the link between these translational initiation factors and ATF4-mediated ISR signaling. The current data show that in the absence of both eIF2D and DENR, ATF4 fails to be induced in Drosophila and human HAP1 cells. Furthermore, it was demonstrated that the loss of eIF2D and DENR disrupts a number of pathophysiological processes in Drosophila that also require ATF4 signaling (Vasudevan, 2020).

Previously reported biochemical functions of eIF2D and DENR-MCTS-1 as noncanonical Met-tRNAiMet delivering translation initiation factors (Dmitriev, 2010; Skabkin, 2013) provide mechanistic insights regarding the translational regulation of ATF4. For mRNAs with single ORFs in their 5' leaders, the AUG codon procures the first methionine through ribosomes associated with the eIF2-GTP-Met-tRNAiMet ternary complex (Hinnebusch, 2016). However, during translation of mRNAs with multiple uORFs, such as those seen in the ATF4 5' leader, the scanning ribosome consumes the Met-tRNAiMet upon encountering the uORFs. Therefore, translation initiation at the ATF4 main ORF requires the scanning ribosomes to acquire another Met-tRNAiMet. It is posited that eIF2D and DENR (together with MCTS-1) could act as Met-tRNAiMet delivery factors for the ATF4 ORF. This is supported by previous in vitro experiments showing that eIF2D can act as a noncanonical Met-tRNAiMet carrier (Skabkin, 2010; Skabkin, 2013), and by the data showing that the RNA binding interface within the PUA domain of eIF2D is required for regulation of ATF4 via the 5' leader. This model of eIF2D and DENR-MCTS1 acting as noncanonical Met-tRNAiMet carriers would predict a decreased occupancy of ribosomes at the ATF4 main ORF in the absence of eIF2D and DENR-MCTS1. An accompanying manuscript (Bohlen, 2020) also independently reports that loss of DENR negatively affects ATF4 translation due to decreased ribosome occupancy at the ATF4 ORF, validating the hypothesis presented in this paper. These data support the model that eIF2D and DENR affect translation of ATF4, and warrant future studies to further understand the molecular mechanism of this model (Vasudevan, 2020).

eIF2D and DENR-MCTS-1 have been demonstrated to also participate in ribosome recycling after translation termination. If this were the primary function of eIF2D and DENR-MCTS-1 in translation of the ATF4 mRNA, decreased ribosome recycling in eIF2D DENR double mutants would be predicted to have the effect of enhancing ATF4 main ORF translation. In Saccharomyces cerevisae, the homologs of eIF2D, DENR, and MCTS-1 (Tma64 Tma22, and Tma20, respectively) have more prominent roles in ribosome recycling as evidenced by increased translation of downstream of ORFs and uORFs. The translation of GCN4, the S. cerevisiae equivalent of ATF4, also increases upon the loss of these recycling factors. Together, these findings suggest that the in vivo roles of eIF2D, DENR, and MCTS have diverged during evolution, with metazoan factors playing more prominent roles in translational initiation of ATF4 mRNA (Vasudevan, 2020).

In conclusion, this study reports that unconventional Met-tRNAiMet delivery factors are required for ATF4 induction during ISR signaling in Drosophila and humans. This discovery provides insights into how ATF4 translation occurs efficiently in stressed cells, where the canonical Met-tRNAiMet delivery factor, eIF2, is inhibited by eIF2α kinases. Additionally, since abnormal ISR signaling is associated with a wide range of metabolic and degenerative diseases in humans, it is anticipated these findings to have clinical impact (Vasudevan, 2020).


The defender against apoptotic cell death 1 gene is required for tissue growth and efficient N-glycosylation in Drosophila melanogaster

How organ growth is regulated in multicellular organisms is a long-standing question in developmental biology. It is known that coordination of cell apoptosis and proliferation is critical in cell number and overall organ size control, while how these processes are regulated is still under investigation. This study found that functional loss of a gene in Drosophila, named Drosophila Defender against apoptotic cell death 1 (dDad1), leads to a reduction of tissue growth due to increased apoptosis and lack of cell proliferation. The dDad1 protein, an orthologue of mammalian Dad1, was found to be crucial for protein N-glycosylation in developing tissues. Loss of dDad1 function activates JNK signaling and blocking the JNK pathway in dDad1 knock-down tissues suppresses cell apoptosis and partially restores organ size. In addition, reduction of dDad1 triggers ER stress and activates unfolded protein response (UPR) signaling, prior to the activation of JNK signaling. Furthermore, Perk-Atf4 signaling, one branch of UPR pathways, appears to play a dual role in inducing cell apoptosis and mediating compensatory cell proliferation in this dDad1 knock-down model (Zhang, 2016).

Cell apoptosis, or programmed cell death (PCD), is one of the most fundamental processes essential for normal development in multicellular organisms. Cell apoptosis is tightly controlled to eliminate excess or damaged cells during development, while dysregulation can lead to pathological diseases, such as neurodegenerative diseases and cancer. Although regulation of cell apoptosis involves a sophisticated and complex molecular cascade, the core machinery that initiates, mediates and executes programmed cell death as well as related regulatory pathways are evolutionary conserved among species. Drosophila melanogaster, with its well-annotated genome and established genetic tools, is commonly used to study mechanism of cell apoptosis and discover novel regulatory pathways and genes for regulating development and tissue homeostasis (Zhang, 2016).

The defender against apoptotic cell death 1 (Dad1) gene was first identified and considered as a negative regulator of cell apoptosis from a temperature-sensitive hamster cell line, tsBN7. With the temperature sensitive mutation in Dad1 gene, cells are normal at permissive temperature, while shifting to restrictive temperature led to programmed cell death. Homologous proteins of hamster Dad1 from other species were found to be highly conserved in sequence and function. The yeast homologue of Dad1 is called Ost2 with 40% identity compared to hamster Dad1 and ost2 mutant induces yeast cell apoptosis. The function of Dad1 has been studied in multicellular organisms as well. Mouse Dad1 mutants exhibited developmental delay, aberrant morphology and increased cell apoptosis during embryogenesis and only survived up to stage of midgestation. In Caenorhabditis elegans, overexpression of either Caenorhabditis Dad1 or human Dad1 induced by a heat-shock promoter inhibited developmental cell death during embryogenesis. While ectopic expression of Dad1 in mouse thymocytes did not increase survival of T cells under apoptotic stimuli, peripheral T cells from spleen and lymph nodes in Dad1 transgenic mice increased cell proliferation. These results indicate that Dad1 plays a critical role in regulating cell viability and apoptosis (Zhang, 2016).

Dad1 is identified as a subunit of oligosaccharyltransferase complex (OST), which acts at the first step for protein N-linked glycosylation. N-linked glycosylation is an important protein modification process, which transfers oligosaccharide to selected asparagine of target nascent proteins to ensure their proper folding and maturation in the endoplasmic reticulum (ER). In eukaryotes, OST complex has a catalytic subunit Stt3 as a core and other six non-catalytic subunits, including Ribophorin I, Ribophorin II, Ost48, Dad1/Ost2, N33/Ost3, and Ost4. There are two OST catalytic subunit genes in vertebrates and insects, referred to as Stt3A and Stt3B. Non-catalytic subunits facilitate the catalytic function of Stt3A and Stt3B. Among them, Ost48 and Dad1 are critical for assembly and stability of Stt3 as well as the OST complex, and loss of them can lead to hypoglycosylation. Ribophorin I escorts the N-glycosylation of OST complex on specific substrates, especially some membrane proteins. Because loss of any one non-catalytic subunit does not fully abolish the function of OST, it is possible that they may have redundant roles or different non-catalytic subunits assist the glycosylation of specific targets (Zhang, 2016).

Dad1, as an N-glycosylation regulatory protein, plays an important role in cell survival, but up to now no detailed mechanism is known about how loss of Dad1 induces cell apoptosis. Based on the evidence from studies in different organisms, Dad1 may facilitate the OST complex to target specific proteins that directly maintain cell survival. Another possibility is that accumulation of unfolded or misfolded proteins of hypoglycosylation at ER triggers stress signaling and initiates programmed cell death. It is also possible that Dad1 affects cell viability in an OST-independent manner. For instance, Dad1 interacts with a Bcl2 family protein, Mcl1, which might lead to apoptosis inhibition (Zhang, 2016).

This study used Drosophila Dad1 (dDad1), which shows more than 70% identity with human DAD1, to study how loss of Dad1 function affects cell viability and tissue growth. Loss of Dad1 in Drosophila was found to decreased tissue growth due to induction of apoptosis and absence of cell proliferation. dDad1 is required for efficient N-glycosylation in developing tissues and the small wing phenotype induced by dDad1 knock-down can be enhanced by reducing gene dosage of the OST catalytic subunit. In addition, the c-Jun N-terminal kinase (JNK) pathway was found to mediate dDad1 knockdown-induced cell apoptosis in wing discs through mitogen-activated protein kinase kinase kinase 1 (Mekk1) and wallenda (wnd). Moreover, in dDad1 knock-down tissues, ER stress is induced and Perk-Atf4 pathway functions upstream of the JNK pathway. Intriguingly, unfolded protein response (UPR) signaling appears to play a dual role in inducing cell death and stimulating compensatory proliferation of neighboring cells for tissue homeostasis (Zhang, 2016).

This study found that loss of dDad1 in the wing interferes with N-linked protein glycosylation, which triggers ER stress and activates the downstream JNK pathway to cause apoptosis. Interestingly, although this study suggests that dDad1 regulates tissue growth through the OST complex, only one of two catalytic subunits, Stt3A (CG1518), genetically interacts with dDad1 in growth control. With the association of dDad1, Stt3A may become the major functioning complex modifying crucial proteins or more nascent proteins in developing tissues, therefore inducing severe or prolonged ER stress if dDad1 activity is absent. Different tissues or regions of tissues may have different levels of sensitivity to the loss of dDad1, which could explain why cell apoptosis was more frequently observed in wing pouch than other region of the wing disc in these experiments. Similar phenomena were also reported in other studies. For instance, Dad1 mutant mice exhibit cell death which leads to developmental defect and embryonic lethality, but the death of cells only happens in some tissues of the embryo (Zhang, 2016).

One study reports that dDad1 homozygous mutant clones in larval fact body induced cell autophagy. However, the current study did not observe cell autophagy using lyso-tracker staining in dDad1 mutant clones in larval wing discs. The different response may be due to different tissue-specific context. Therefore, apoptosis induced by loss of dDad1 in the wing is unlikely to be dependent of autophagy process (Zhang, 2016).

As an N-glycosylation regulator, dDad1 may have other target proteins essential for animal viability. In this study, although blocking Perk-Atf4 or JNK pathway suppresses apoptosis induced by loss of dDad1, the lethality of en-Gal4/UAS-dDad1 RNAi individuals cannot be rescued. Thus, cell apoptosis induced by loss of dDad1 may not be the major reason for causing lethality of the mutant organism. It would be interesting to identify potential targets of dDad1 that are essential for normal development of Drosophila (Zhang, 2016).

The Perk-Atf4 pathway may play both protective and destructive roles in growth control in loss of dDad1 model: on one hand, it activates JNK signaling to cause cell apoptosis; on the other, it may send out signals to neighboring cells to increase cell proliferation (Zhang, 2016).

In Drosophila, it is known that prolonged ER stress induces cell apoptosis, however the mechanism of ER stress activates apoptosis remains elusive. A few Drosophila ER stress models have been established and their connection with apoptosis has been validated. Nevertheless, they suggested different pathways leading to cell death and none of them is the same as what was found in the current study. In a chronic ER stress model due to Presenilin (Psn) overexpression, cell apoptosis is induced in a Perk-Atf4 dependent but JNK-independent manner. Although the study found that the JNK pathway is activated by Perk-Atf4, it induces dilp8 expression which extends developmental stage to keep tissue homeostasis, rather than stimulates cell death. In the current study, induction of cell apoptosis in the absence of dDad1 function was found to be dependent on both Perk-Atf4 and JNK pathways. The JNK pathway acts downstream of Perk-Atf4 and causes cell apoptosis. It was also found that Mekk1 and Wnd are two major JNKKK's that activate JNK pathway in response to loss-of-dDad1 induced ER stress. This is consistent with the observations from another ER stress model, autosomal dominant retinitis pigmentosa (ADRP) model. However, ADRP model suggests that activation of JNK pathway and cell apoptosis does not rely on UPR signaling. In mammalian models of ER stress, cell apoptosis is induced through activation of IRE1-XBP1 branch, followed by JNK pathway activation via TRAF2/ASK1. In the current study, it was not possible to test the role of Ire1-Xbp1 branch due to the embryonic lethality caused by expression of Ire1 RNAi or Xbp1 RNAi driven by en-Gal4, although its activation was observed. Ask1 does not seem to play a role in mediating cell apoptosis in the loss-of-dDad1 model, since knocking down of Ask1 cannot restore loss-of-dDad1 induced wing size deduction (Zhang, 2016).

Compensatory proliferation is a self-rescue mechanism to keep tissue homeostasis when some cells are under stress or apoptosis. Mechanism of compensatory proliferation was mostly studied in Drosophila. In fly wing discs, Dronc in the apoptotic cells stimulates JNK pathway, inducing expression of mitogens, Dpp and Wg, to presumably promote proliferation of neighboring cells. Interestingly, in this study, activation of Perk-Atf4 pathway appears to play a role in inducing extra cell proliferation but does not rely on activation of the JNK pathway nor cell death. It suggests that Atf4 transcription factor may induce transcription of mitogenic genes to stimulate cell proliferation, in addition to the genes that are related to stress response (Zhang, 2016).

N-glycosylation of Wg was affected in dDad1 mutant cells. However, it turned out that N-glycosylation of Wg is unimportant for its secretion and signaling in fly tissues. Regardless, it is unlikely that Wg was responsible for mediating extra cell proliferation in this loss-of-dDad1 model based on the following observations. Firstly, compensatory cell proliferation can still occur in the absence of wg and dpp in tissues undergoing massive cell apoptosis. Moreover, this study found that proliferation of extra cells can still occur when JNK signaling was blocked, while the JNK pathway is presumably responsible for Wg induction. Therefore, further studies are needed to address how Perk-Atf4 signaling acts to induce compensatory cell proliferation to maintain tissue homeostasis (Zhang, 2016).

Some studies suggest that Dad1 may have a direct effect on cell death suppression. Overexpression of hDAD1 or ceDAD1 has been shown to be sufficient to inhibit developmental cell apoptosis in C. elegans. Overexpression of DAD1 in mouse thymocyte did not increase survival of T cells under apoptosis stimuli, however peripheral T cells from spleen and lymph node in Dad1 transgenic mice have increased cell proliferation. This work examined the phenotypes of dDad1 transgenic flies in different tissues. Drosophila pupa eye is a good model to study developmental apoptosis because wild-type flies have fixed number of interommatidial cells in mid-pupal eye discs due to the elimination of excess cells by apoptosis. This study found that overexpression of dDad1 in the eye did not cause the accumulation of extra interommatidial cells in mid-pupal eye discs, which indicates that dDad1 is not sufficient to block normal apoptosis during eye development. In addition, this study overexpressed apoptosis-inducing genes in Drosophila wing and eye to test if dDad1 is able to suppress induced apoptosis. The cell apoptosis in the wing induced by overexpression of UAS-eiger failed to be suppressed by dDad1 overexpression. However, overexpression of dDad1 could moderately rescue cell death induced by GMR-hid in adult eyes. These observations indicate that Drosophila dDad1 inhibits induced cell apoptosis moderately and conditionally, but exhibits no effect on normal developmental apoptosis. Therefore, these observations suggest that dDad1 may not function as an active defender of cell apoptosis in Drosophila (Zhang, 2016).

Targets of Activity

Transcriptional regulation of neuropeptide and peptide hormone expression by the Drosophila dimmed and cryptocephal genes

The regulation of neuropeptide and peptide hormone gene expression is essential for the development and function of neuroendocrine cells in integrated physiological networks. In insects, a decline in circulating ecdysteroids triggers the activation of a neuroendocrine system to stimulate ecdysis, the behaviors used to shed the old cuticle at the culmination of each molt. Two evolutionarily conserved transcription factor genes, the basic helix-loop-helix (bHLH) gene dimmed (dimm) and the basic-leucine zipper (bZIP) gene cryptocephal (crc), control expression of diverse neuropeptides and peptide hormones in Drosophila. Central nervous system expression of three neuropeptide genes (Dromyosuppressin, FMRFamide-related and Leucokinin) is activated by dimm. Expression of Ecdysis triggering hormone (ETH) in the endocrine Inka cells requires crc; homozygous crc mutant larvae display markedly reduced ETH levels and corresponding defects in ecdysis. crc activates ETH expression though a 382 bp enhancer, which completely recapitulates the ETH expression pattern. The enhancer contains two evolutionarily conserved regions, and both are imperfect matches to recognition elements for activating transcription factor-4 (ATF-4), the vertebrate ortholog of the CRC protein and an important intermediate in cellular responses to endoplasmic reticulum stress. These regions also contain a putative ecdysteroid response element and a predicted binding site for the products of the E74 ecdysone response gene. These results suggest that convergence between ATF-related signaling and an important intracellular steroid response pathway may contribute to the neuroendocrine regulation of insect molting (Gauthier, 2006).

DIMM has been proposed as a direct regulator of neuroendocrine gene expression in most neuropeptidergic cells. Quantitative RTPCR results, supplemented by in situ hybridization, show that DIMM upregulates the levels of mRNAs derived from at least three neuropeptide genes, Fmrf, Lk and Dms. These findings provide strong support for DIMM as a key regulator of multiple neuroendocrine genes. The LIM-homeodomain gene apterous (ap) also controls Fmrf and Lk gene expression. ap acts cell-autonomously to stimulate dimm expression, but the AP and DIMM proteins can also physically interact, and they may function together in regulating Fmrf. Several other factors, including the transcriptional co-factors encoded by dachshund and eyes absent, the zinc-finger gene squeeze, and the retrograde bone morphogenetic protein (BMP) pathway, act in combinatorial fashion with dimm and ap to control Fmrf expression. However, other neuropeptidergic cells appear to use only portions of this code. For example, ap and dimm appear to contribute to the expression of Lk in Fmrf-negative cells (the segmental cells A1-A7 and possibly the brain lobe cells Br1). Even within the population of Lk cells, loss of dimm results in very different effects in different neurons, with a reduction in Lk transcript levels in cells A1-A7, and an increase (or no change) in Lk levels in the Br1 and the subesophageal SE neurons. How do these relatively widely expressed factors interact with other regulatory proteins to produce cell type-specific patterns of neuropeptide gene expression? It will be of interest to determine which other elements of the combinatorial pro-Fmrf code are used to control Lk and Dms expression, and to identify additional factors that interact with dimm to control expression of these neuropeptide genes (Gauthier, 2006).

Does dimm control neuropeptide levels through an additional indirect mechanism? No changes were detected in levels of three neuropeptide biosynthetic enzyme mRNAs, Phm, Fur1 and amon, in the qRTPCR analysis. This is in contrast to earlier immunocytochemical studies, in which a marked reduction was observed in the protein products of these genes in dimm mutant CNS. In some cases, these differences may reflect the spatial insensitivity of the qRTPCR methods, as was confirmed by in situ hybridization analysis of Lk expression. Phm, in particular, may belong in this category. Although levels of PHM and DIMM expression are strongly correlated, PHM is also highly expressed in many other tissues that do not express dimm. Any dimm-dependent change in Phm expression may have been obscured by the much larger pool of dimm-independent Phm mRNA in whole-animal qRTPCR analysis (Gauthier, 2006).

DIMM may regulate levels of other neuroendocrine proteins through a route that does not involve interactions between DIMM and cis-regulatory elements in the respective genes. Evidence was obtained in support of this hypothesis in an earlier analysis of an ectopically expressed neuropeptide in dimm mutant cells; levels of ectopic PDF protein were strongly reduced while dimm had no effect on levels of the cognate Pdf mRNA. This study showed that larvae homozygous for a specific loss-of-function mutation in dimm displayed reduced levels of endogenous ETH-like protein(s), but not ETH mRNA, in the endocrine Inka cells, a site of dimm gene expression. This may occur simply through a dimm-dependent change in levels of one secreted protein, such as PHM, that may disrupt the formation of multi-protein aggregates required for neuropeptide sorting into secretory granules. Alternatively, recent studies on the mouse ortholog of dimm, Mist1, suggest that dimm may play a more direct role in the management of secretory granule budding from the trans-Golgi network. In Mist1 knockout mice (Mist1KO), pancreatic exocrine cells display reduced intracellular organization. Moreover, the Mist1KO phenotype is partially phenocopied in animals mutant for the Rab3D gene, a small GTPase involved in secretory granule trafficking. Further studies on the regulation of ETH, PHM and Rab3-like proteins, and on the biochemical interactions among them, may shed light on the cellular mechanisms underlying the indirect actions of DIMM (Gauthier, 2006).

Mutations in the crc gene result in pleiotropic defects in ecdysone-regulated events during molting and metamorphosis. Many of the morphological defects are associated with a failure of the insect to properly complete ecdysis, a stereotyped set of behaviors required for shedding of the old cuticle at the culmination of each molt. Several neuropeptides and peptide hormones, including ETH, play critical roles in organizing and triggering ecdysis behavior (Gauthier, 2006).

This study provides four independent lines of evidence that demonstrate a crucial role for crc in the upregulation of ETH mRNA levels: (1) a marked reduction by qRTPCR is observed in levels of ETH transcripts [but not in mRNAs encoding CCAP or EH, two other neuropeptides involved in the neuropeptide hierarchy controlling ecdysis in crc mutant larvae; (2) in situ hybridization revealed a strong reduction in ETH mRNA levels in the endocrine Inka cells in crc mutant larvae; (3) the intensity of anti-PETH immunoreactivity was markedly reduced in crc1/crc1 homozygotes; (4) EGFP fluorescence driven by an ETH-EGFP reporter gene was reduced in crc mutant larvae. Therefore, CRC is a strong activator of ETH gene expression, and loss of CRC results in a corresponding reduction in levels of the ETH protein (Gauthier, 2006).

Despite the molecular identification of the crc locus, almost six decades after the original description of the first crc allele, the causes of the molting and metamorphosis defects in crc mutants remained unclear. The current results suggest a simple model to explain the crc mutant phenotype. Strong hypomorphic or null mutations in crc and ETH both severely disrupt ecdysis. These defects include weak, irregular and slower ecdysis contractions and a failure to shed old cuticular structures, leading to retention of two and sometimes three sets of mouthparts into the next larval stage. These similarities in molting defects, taken together with the observation that crc is required for normal expression of ETH mRNA and ETH protein, point to the loss of ETH signaling as the likely proximate cause of the ecdysis defects observed in crc mutants (Gauthier, 2006).

Despite the specific effects of crc on ETH transcription in the Inka cells, crc is widely expressed, suggesting a cellular housekeeping function. The vertebrate ATF-4 protein is also ubiquitously expressed. In addition, the upregulation of ATF-4 constitutes a milestone of many cellular stress response pathways including oxidative stress, amino acid deprivation, and hypoxia. In the tobacco hornworm, Manduca sexta, levels of ETH fluctuate during the molts and are regulated by circulating ecdysteroids. It is hypothesized that CRC contributes to the regulation of ETH gene expression during this period, perhaps in response to signals from the tracheae (Gauthier, 2006).

Peaks in circulating levels of the ecdysteroid hormone, 20-hydroxyecdysone (20HE), initiate and coordinate each molt. A subsequent decline in 20HE levels is required for ecdysis, and the activation of these behaviors involves a hierarchical cascade of peptide hormone and neuropeptide signals that is triggered by ETH. Is CRC required in order to maintain ETH expression, or is CRC involved in regulating transcription of the ETH gene during the molts? While it is not known whether ETH mRNA levels fluctuate during Drosophila post-embryonic development, the regulation of ETH levels by ecdysteroids in molting Manduca sexta, and the analysis of the conserved region sequences CR1 and CR2 (located 91-171 bp upstream of the ETH translational start site), provides tantalizing clues to possible coordinate regulation of ETH gene expression by CRC and ecdysone response genes. There is substantial overlap between the predicted CRC binding site in CR1 and a putative ecdysteroid response element (EcRE). In addition, a potential binding site in CR2 for products of the E74 early ecdysone-inducible gene. E74 expression is induced directly by 20HE, and it encodes transcription factors that regulate other ecdysone response genes. Mutations that specifically disrupt E74B, which likely binds the same consensus as E74A, display defects associated with pupal ecdysis that closely phenocopy crc. In future, studies will focus on whether ETH expression is regulated by elements in both CR1 and CR2 in an ecdysteroid-dependent manner, and whether CRC, E74B and other factors in the ecdysone-response pathway interact competitively or cooperatively at these sites (Gauthier, 2006).

4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP (Thor) is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. This study shows that GCN2 and its downstream transcription factor, ATF4 (Cryptocephal), mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging (Kang, 2016).

Previous studies had established the importance of 4E-BP transcription by FOXO in several distinct biological contexts, including the regulation of cell number, metabolism, response to oxidative stress, and cardiac function. Alternative transcriptional regulatory mechanisms for 4E-BP and their biological significance have remained poorly characterized. This study shows evidence that another pathway, mediated by GCN2 and ATF4, mediates the induction of 4E-BP transcription in response to the restriction of amino acids in the diet and during the development of specific tissues. The specific data presented in this study include examination of 4E-BP protein through Western blot from starved larval extracts and examination of transcripts through quantitative PCR in cultured S2 cells, larvae, and adult tissues. A new 4E-BP intron reporter, which responds to ATF4 activation, is widely expressed in Drosophila, indicating that ATF4 is a major mediator of 4E-BP induction during normal development as well as in response to dietary restriction of amino acids (Kang, 2016).

The results also show that Drosophila gcn2 mutants have a shorter lifespan than wild-type controls when reared in food with low yeast content. These results are similar to what had been observed with mutants of C. elegans gcn2 and yeast GCN4, an ATF4 equivalent gene in that organism. The Drosophila gcn2 mutant phenotype is also similar to the reported phenotype of 4E-BP mutant flies. However, this study did not examine through double-mutant analysis whether the two genes have a strictly linear genetic relationship in regulating lifespan. Based on current understanding, the two genes do not have a strictly linear relationship: GCN2-ATF4 has other transcriptional targets that also contribute to their phenotypes, and ATF4-independent regulatory inputs into 4E-BP exist, such as those mediated by FOXO and TOR. Thus, it is speculated that the similar reported phenotypes of gcn2 and 4E-BP mutants on lifespan may be due to a broad effect of 4E-BP on other GCN2-ATF4 target gene expression, as 4E-BP's target, eIF-4E, is thought to be involved in the expression of most eukaryotic genes (Kang, 2016).

Emerging evidence indicates that 4E-BP is not indiscriminate in the inhibition of general translation. For example, ribosome profiling studies in mammalian cultured cells have found that 4E-BP1's effect on translation is highly selective, with some transcripts being highly sensitive to 4E-BP1 and others indifferent. Accordingly, it appears that 4E-BP activation would have cells shift their overall protein synthesis profile. The data in this study are consistent with that view. Specifically, it was found that BiP and other stress-responsive transcripts score positive in the Internal Ribosomal Entry Site (IRES) assay and are resistant to suppression by 4E-BP. It is noted that mammalian BiP also reportedly has an IRES element in its 5' UTR. The finding that 4E-BP is a target of the UPR helps make sense of such an observation; IRES would help transcripts evade suppression by 4E-BP, whose expression level is high in stressed cells, allowing BiP to be expressed and help resolve stress. As 4E-BP activation results in a specific biological phenotype of enhanced stress resistance and lifespan extension, it appears that the proteome shift brought on by 4E-BP favors stress-responsive gene expression (Kang, 2016).

In Drosophila, 4E-BP is widely understood as a transcriptional target of FOXO. However, the role of FOXO in mediating the effects of dietary restriction of amino acids has been disputed. The experiments presented in this paper show that the loss of foxo does not impair 4E-BP transcription, at least under conditions of amino acid restriction. Notably, a foxo mutant allele was used that is different from those used in the earlier studies on 4E-BP. Although the earlier studies had used foxo21/25 alleles with premature stop codons, recent studies indicate that full-length FOXO protein is still expressed in the foxo25 mutants. Thus, it is possible that these alleles have neomorphic properties that may have led to results different from the current work. On the other hand, the foxo mutant allele used in this study has been validated to be a null allele. The negative result with the foxo mutant is mostly related to the amino acid deprivation response and does not contradict FOXO's known role in the induction of 4E-BP in other contexts (Kang, 2016).

In regards to the cellular response to amino acid deprivation, much focus had been placed on the TOR signaling pathway. It is interesting that the other amino acid-response pathway mediated by GCN2 leads to the transcriptional regulation of this TOR phosphorylation substrate. The current observation suggests that the two amino acid-responsive pathways work cooperatively (Kang, 2016).

Protein Interactions

Cryptocephal, the Drosophila melanogaster ATF4, is a specific coactivator for ecdysone receptor isoform B2

The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here Cryptocephal (CRC), the Drosophila homolog of ATF4, is shown to be an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. It is proposed that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators (Gauthier, 2012).

The experiments suggest that the 17-residue B2-specific N-terminus binds to the bZIP region of CRC, that an ionic interaction between EcR-B2-E9 and CRC-R361 plays some role in the binding, and that the interaction of the two proteins plays a crucial role in those tissues where EcR-B2 is essential. These tissues include the endocrine Inka cells, which display ecdysone-dependent upregulation of ETH transcripts and which require EcR-B2 and CRC for full ETH expression. Taken together, these findings implicate CRC as an isoform-specific transcriptional activator for EcR-B2 (Gauthier, 2012).

In diverse systems, bZIP proteins interact with dyadic or palindromic promoter sequences as homodimers or heterodimers with other bZIP partners. Dimerization involves regularly spaced hydrophobic amino acids that form a coiled-coil between two leucine zipper domains. Other bZIP transcription factors are known to interact with nuclear receptors, modulating the activities of either AF1 or AF2, but in the cases reported previously, bZIP proteins bind either to the DNA-binding domain or to the hinge domain of the nuclear receptor. By contrast, CRC (through its bZIP domain) appears to bind directly to the EcR-B2 AF1 region, and its interaction is specific to one EcR isoform (Gauthier, 2012).

ATF4, the mammalian homolog of CRC, plays a central role in stress responses. The role of CRC in ecdysone signaling suggests the possibility of interesting and unexpected connections between stress responses and the control of developmental timing and metamorphosis (Gauthier, 2012).

The ETH promoter contains sequences matching the consensus half-sites for binding of ATF4 and EcR to DNA. These half-sites are separated by 4 nucleotides, and they are located within a highly conserved sequence (comparing D. melanogaster to several other Drosophila species) that is 138-171 nucleotides upstream of the ETH transcriptional start site. Since bZIP proteins may bind first sequentially as monomers and then dimerize while bound to DNA, these observations suggest a model in which CRC participates in the stabilization of EcR-B2 binding to the ETH promoter. This interaction provides a basis for understanding some of the differences in transcriptional properties that are displayed by the EcR isoforms and perhaps other nuclear receptors with distinct AF1-coactivators (Gauthier, 2012).


in situ hybridization was performed to determine the expression pattern of crc mRNAs. In wild-type, wandering stage third instar larvae, specific hybridization was seen in several tissues. The imaginal discs and CNS displayed the strongest signals. There was strong, relatively uniform staining in the T1-T3 leg discs and detectable, though weaker, staining in the wing discs, labial discs, and in large cells associated with the anterior spiracles. Within the CNS, the strongest expression was observed in or near the optic lobe proliferation zones. The rest of the brain and ventral nerve cord showed strong, uniform hybridization, although less hybridization was observed in the posterior abdominal neuromeres. Specific hybridization was seen in patches of small cells located throughout the midgut (Hewes, 2000).

The effects of several crc alleles on the pattern of crc in situ hybridization was examined in the CNS of feeding third instar larvae. There was strong hybridization in the CNS of +/Rev8 larvae. By contrast, no signal was detected in larvae bearing a complete deletion of the crc locus (Rev8/Rev4), nor was there signal in R6/Rev8 and R1/Rev8 larvae. Both R1 and R6 delete portions of crc-a, but R1 and R6 may have differential effects on crc-b/c. Thus, under the hybridization and detection conditions used for this experiment, it appears that the crc-a isoform accounts for most if not all of the visible signal, while crc-b and crc-c were below detection. Finally, the pattern of hybridization in crc1/Rev8 larvae was the same as the pattern observed in the control, +/Rev8 larvae. Thus, crc expression in the CNS appeared to be normal in crc1 mutants, consistent with the interpretation that a defect at the protein level (Q171R) likely accounts for the crc1 mutant phenotype (Hewes, 2000).


crc alleles were generated using imprecise P-element excision and male recombination. Six partial or complete deletions of crc were generated. crcRev8 (Rev8) is a complete null; it removed all of the crc exons and several exons from gene Y. The remaining five deletions (R1, R2, R6, E85, and E98) are all partial disruptions of crc. At least two of these alleles, R2 and E98, also disrupt gene Y. E85 appears to be a specific CRC-D mutant, since it affects only exon 4b. By contrast, R1, R2, and E98 disrupt both CRC-A and CRC-D. R6, which deleted the exons encoding the bZIP domain, disrupts CRC-A and CRC-B. Because exons 1-4 remain intact in R6, this allele may not disrupt CRC-D and two small 5' RNAs (crc-a and crc-f). R1, R2, R6, and E98 each retained some or all of c929, the P element used for the mutant screens. In E85, c929 appears to have been excised completely. An additional recombinant line, R20, contained a precise excision of c929 (Hewes, 2000).

Complementation analysis revealed at least three lethal groups in 39C2-4, two of which (the 5' group and 3' group) were associated with deletions of crc exons. The 5' group includes R1, R2, E85, and E98, all deletions of 5' crc exons, as well as crc929 (929). The 3' group includes crc1 and R6, a deletion of the 3' crc exons. A third lethal complementation group was associated with disruptions of gene Y (Hewes, 2000).

With the exception of crc1, all of the crc mutant alleles share the same parental chromosome, 929. Precise excision of the c929 P element (R20) fully restored the viability of animals bearing this chromosome in trans over Rev8, a lethal deletion of the entire crc locus, and over Rev4, a larger deletion of 39C. Thus, the parental 929 chromosome displayed no lethality in 39C2-4 independent of the P-element insertion (Hewes, 2000).

The crc 5' complementation group is associated with isoform-specific disruptions of the crc gene. For example, 929 is semilethal in trans over TW161, Rev4, and Rev8 (all of which completely delete crc) but not over TW1, which leaves intact the entire crc gene as well as ~15 kb of DNA upstream of the putative crc-a transcriptional start site. Since the 929 P element is inserted in an intron of crc upstream of the putative crc-b/c transcriptional start site, the lethality caused by 929 may reflect a specific disruption of the crc-a mRNA isoform. Similar results were obtained with R1, which deletes all of the 5' exons of crc (leaving the exons encoding crc-b and crc-c intact). Both R1 and 929 display similar lethality (with variable penetrance) in crosses to the deficiencies TW161, Rev4, and Rev8. R1 is semilethal in homozygotes, whereas 929 homozygotes are fully viable. Thus, R1 appears to be a slightly more severe allele. This difference may stem from the fact that R1 disrupts the crc-d-f mRNAs in addition to crc-a. Consistent with this hypothesis, E85, a smaller deletion that disrupts an exon specific to crc-d, displayed significant lethality in trans with TW161 and Rev4. The E85 chromosome also appears to bear a lethal mutation at a second, distant site: E85 homozygotes displayed greater lethality than E85 hemizygotes, and in contrast with the larger R1 deletion, E85 displayed some lethality in trans with TW1. Finally, there are two stronger lethal alleles, E98 and R2, and the degree of lethality associated with these alleles is correlated with the distal extent of these deletions (Hewes, 2000).

The crc 3' complementation group is associated with disruptions of both of the major crc mRNA isoforms, crc-a and crc-b. R6 deletes all of the 3' crc exons shared by these two mRNAs. crc1 and R6 both were lethal in trans with deletions of the crc locus, and crc1 and R6 fail to complement each other. By contrast, crc1 was fully viable over deletions that extend distally from the c929 P-element insertion site. Thus, the wild-type function(s) of the crc gene must include contributions by transcription units located proximal to the c929 insertion, such as crc-b and crc-c. Up to 2% adult escapers were observed among hemizygous crc1 progeny. Hemizygous R6 adult escapers were never observed. Thus, although both crc1 and R6 are very strong hypomorphs, it is concluded that R6 is a more severe allele. R6 is not a complete crc amorph, since it complements E85 (Hewes, 2000).

Crosses revealed two largely distinct phenotypes, each generally associated with only one of the crc complementation groups. This result further indicates that the 5' and 3' groups represent distinct genetic functions. For the 3' group (crc1 and R6), there were several lethal phases during larval, pupal, and adult development. Both crc1 and R6 hemizygotes displayed 15-50% of their lethality after pupariation. At each stage, the R6 allele displayed a more severe phenotype than crc1 (Hewes, 2000).

The molts between successive larval stages are disrupted in crc1 mutants, and this phenotype is accompanied by significant lethality (Chadfield, 1985). A comparable phenotype was observed in the R6 mutants, and the presence of supernumerary mouthparts was strongly correlated with larval lethality. In addition to the larval molting defects, R6 hemizygotes showed delayed and defective pupariation. By contrast, crc1 hemizygotes pupariated normally (cf., Hadorn, 1943), consistent with the weaker hypomorphic phenotype of crc1. Although ~5% of the hemizygous R6 puparia were indistinguishable from wild type, the rest were aberrant to varying degrees. These defects included a failure to evert the anterior spiracles and a retention of a larval shape, which was thinned, elongated, and sometimes curved to one side. In the most severe cases, the abdominal gas bubble, which normally forms ~6 hr after pupariation, did not appear, although the larval mouthparts were later expelled (Hewes, 2000).

crc1 and R6 mutant pupae display a range of defects associated with pupation and subsequent development, as previously described for the crc1 allele (Hadorn, 1943; Fristrom, 1965; Chadfield, 1985). The pupal phenotypes of these two alleles were similar. The mutants often failed to expel or translocate the abdominal gas bubble. Head eversion failed or was incomplete, and the leg and wing discs did not completely elongate. In addition, segmentation and differentiation of the abdomen usually failed, although in some cases the anterior abdominal segments differentiated. Other aspects of adult development proceeded normally, resulting in the appearance of mature eye pigments and darkened macrochaetes and differentiation of the wings and legs (Hewes, 2000).

Adult, hemizygous crc1 females displayed markedly decreased fecundity. In addition, 5-50% (depending upon the genetic background) of the hemizygous crc1 adults of both sexes failed to expand their wings and fully tan the adult cuticle. Other hemizygous crc1 adults displayed more subtle defects involving the wings, legs, scutellum, scutellar bristles, halteres, and dorsal thorax. Many of these defects could be explained by incomplete tanning of the adult cuticle after eclosion (Hewes, 2000).

The lethal phase for the 5' group of crc alleles (R1, R2, E85, E98, and 929) was primarily after pupariation, since the number of dead pupae observed on the sides of the vials was approximately equal to the total amount of lethality. Larvae were never observedwith multiple mouthparts, and the puparia were normal in size and shape. In addition, gas bubble translocation, expulsion of the larval tracheae and mouthparts, and head eversion were all completed successfully. The 5' group of alleles displayed defects in leg and wing disc elongation that were similar to those observed for the 3' group, but they also caused novel defects in adult development ('head/abdomen-collapsed' phenotype. In contrast to crc1, the distal portions of the everted leg discs often darkened abnormally and did not differentiate further. After pupation, the abdomen shrank markedly and withdrew to a dorsal position. Subsequently, the head collapsed partially or completely into the thoracic cavity. Despite these events, many pupae developed eye pigmentation and other signs of adult differentiation (Hewes, 2000).

Although most of the mutations within the 5' group fully complemented the 3' group, R6 was an exception. R6 uncovered crc1 (3' group) as well as R1, R2, E98, and 929 (5' group). In addition, R6 mutants displayed a cryptocephal phenotype when crossed to crc1 and the head/abdomen-collapsed phenotype when crossed to alleles from the 5' group. When placed in trans with R6, the 929, R1, R2, and E98 alleles each displayed a similar degree of lethality (independent of deletion size), indicating that each of these crc 5' alleles displayed comparable defects in the function of the crc gene (Hewes, 2000).

Interestingly, the 5' group alleles and R6 (but not crc1) also displayed the head/abdomen-collapsed phenotype when heterozygous over either CyO, y+ or a second balancer, In(2LR)SLM. This dominant effect was associated with variable, but significant pupal lethality. Because the CyO, y+ and In(2LR)SLM chromosomes were created independently, it appears unlikely that these chromosomes share dominant enhancers of the head/abdomen-collapsed phenotype. Rather, this result suggests that the 5' group alleles and R6 are crc haploinsufficient in some genetic backgrounds (Hewes, 2000).

Because deletions distal to c929 complemented crc1, the genomic regions containing exons 5 and 6 and exons 7 and 8 were sequenced from crc1/crc1 larvae. Nine differences were identified between the crc1 and wild-type sequences. Of these, six corresponded to wild-type polymorphisms, and two were conservative substitutions. The remaining substitution (GATGCACAGCCAAAA; the underlined residue is G in crc1) results in a nonconservative change from glutamine to arginine (Q171R). Because Q171R was the only nonconservative substitution in the crc1 coding sequence, it is speculated that it is the cause of the associated phenotypic defects (Hewes, 2000).

To confirm the molecular identification of the crc gene, the lethality of mutant crc alleles was rescued using germ-line transformants expressing a crc cDNA. On the basis of the complementation analysis, it is predicted that ectopic expression of the CRC-B protein isoform (encoded by the crc-c mRNA) would rescue crc1 lethality. To test this hypothesis, multiple independent germ-line transformants were made with crc-c under the control of a GAL4 upstream activating sequence (UAS-crc). In six of seven lines, UAS-crc rescued 8%-21% of the lethality in crc1/Rev8 heterozygotes. The rescue was constitutive (without heat shock), presumably reflecting basal expression of UAS-crc. Heat-shock-induced expression of UAS-crc under the control of an hs-GAL4 driver caused substantial lethality in an otherwise wild-type background; thus the hs-GAL4 driver only lowered the degree of rescue seen. The degree of rescue also was influenced by the parental genotype; for insertion 9-2, ~40% rescue was obtained when both parental stocks were balanced with CyO, y+ (Hewes, 2000).

Attempts were made rescue of 5' group functions. 929 was chosed as a representative 5' group allele for two reasons. First, the complementation and molecular analyses indicated that 929 displayed lethality due to disruption of crc-a without any confounding disruption of gene Y. Second, c929 is a GAL4 enhancer trap P element, which allowed heterogeneous expression of the UAS-crc transgene in 929 mutants. The c929 GAL4 reporter gene is expressed in larvae in peptidergic central nervous system (CNS) neurons, intrinsic cells of the ring gland, salivary gland, fat body, patches of the epidermis, the PM peritracheal cells, and a few other scattered locations. By contrast, there is very restricted c929 reporter gene expression in the imaginal discs and no detectable expression in the skeletal muscles and abdominal histoblasts. Several independent UAS-crc insertions partially or completely rescued the lethality observed in 929 hemizygotes. Thus, there were two separable crc functions, and both were rescued by transgenic expression of CRC-B. Moreover, given the inclusion of numerous neurosecretory neurons in the pattern of c929 reporter gene expression, it is speculated that crc may function in close association with ecdysone biosynthesis/secretion (Hewes, 2000).


See CrebB-17A for information on CREB/ATF superfamily bZIP domain protein evolutionary homologs

See PubMed articles for information about vertebrate ATF4.


Search PubMed for articles about Drosophila Cryptocephal

Bartsch, D., et al. (1995). Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change. Cell 83(6): 979-92. Medline abstract: 8521521

Bohlen, J., Harbrecht, L., Blanco, S., Clemm von Hohenberg, K., Fenzl, K., Kramer, G., Bukau, B. and Teleman, A. A. (2020). DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4. Nat Commun 11(1): 4676. PubMed ID: 32938922

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Biological Overview

date revised: 4 April 2022

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