The Interactive Fly
Zygotically transcribed genes
Cell death regulation in Drosophila: Conservation of mechanism and unique insights
Autophagy in neurodegeneration: two sides of the same coin
Genes involved in autophagic cell death
The caspase family of cysteine proteases is central to apoptotic signaling and cell execution in all animals that have been studied, including worms, flies, and vertebrates. As with many proteases, caspases are synthesized as inactive zymogens, known as procaspases, and are generally thought to be present in all cells at levels sufficient to induce apoptosis when activated. Death stimuli lead to one or more cleavages COOH-terminal to specific aspartate residues. These cleavage events separate the large and small subunits that make up the active caspase. Two sets of these subunits assemble to form the active caspase heterotetramer, which has two active sites. Frequently an NH2-terminal prodomain is also removed during caspase processing. An important point is that the sites cleaved to produce an active caspase often correspond to caspase target sites. Thus, once activated, caspases can participate in proteolytic cascades (Vernooy, 2000 and references therein).
Caspases play two roles in bringing about the death of the cell. They transduce death signals that are generated in specific cellular compartments, and they cleave a number of cellular proteins, resulting in the activation of some and the inactivation of others. These latter cleavage events are thought to lead, through a number of mechanisms, to many of the biochemical and morphological changes associated with apoptosis. Caspases that act as signal transducers (known as apical or upstream caspases) have long prodomains. These regions contain specific sequence motifs (known as death effector domains [DEDs] or caspase recruitment domains [CARDs]) that are thought to mediate procaspase recruitment into complexes in which caspase activation occurs in response to forced oligomerization. Some caspases may also become activated as a consequence of prodomain-dependent homodimerization. Once activated, long prodomain caspases are thought to cleave and activate short prodomain caspases (known as downstream or executioner caspases) that rely on cleavage by other caspases for activation. It is important to note that, in mammals and flies, mutant phenotypes suggest caspases can also play important nonapoptotic roles, and the functions of a number of caspases are still unclear (Vernooy, 2000 and references therein).
Drosophila encodes three long prodomain caspases: dcp-2/dredd, dronc (Dorstyn, 1999a), and dream, as well as four caspases with short prodomains: dcp-1, drICE (Fraser, 1997), decay (Dorstyn, 1999b), and daydream. An eighth Drosophila caspase, a head-to-head partial duplication of daydream, is likely to be nonfunctional because of numerous mutations (including premature stop codons and deletions). The Caenorhabditis elegans genome encodes three caspases, the known apoptosis inducer ced-3, and csp-1 and csp-2, all of which have long prodomains. 14 caspases have been identified in mammals, 10 of which have long prodomains (Vernooy, 2000 and references therein).
All long prodomain caspases that have been identified to date in mammals contain either CARD or DED sequences. In contrast, both Drosophila and C. elegans encode caspases that have long prodomains with unique sequences, as well as a single caspase with a CARD. The unique prodomain sequences in these caspases may promote death-inducing caspase activation in response to unknown stimuli. Alternatively, they may regulate caspase activation in contexts other than cell death. Several Drosophila and C. elegans caspases, Dronc and Csp-1a and Csp-2a, respectively, are unique in a second way as well. Caspases are described as being specific for cleavage after aspartate and typically have an active site that conforms to the consensus QAC(R/Q/G)(G/E) (catalytic cysteine is underlined). Dronc, Csp-1a, and Csp-2a have active sites that differ in the first two positions. Because the glutamine at the first position of the active site pentapeptide QACRG is part of the substrate binding pocket, it is likely that caspases with different amino acids at this position will have unique cleavage preferences. In support of this hypothesis, Dronc, which has the active site sequence PFCRG, cleaves itself after glutamate rather than aspartate, and cleaves tetrapeptide substrates after glutamate as well as aspartate (Hawkins, 2000). Cleavage specificity data for Csp-1 and Csp-2 have not been reported. Why might these caspases have altered cleavage specificity? All are long prodomain caspases, suggesting that they act to transduce signals. One possibility is simply that these proteins have unique substrates (which may or may not be death related) that require an altered cleavage specificity. The altered cleavage specificity may also have evolved to be able to efficiently cleave the sequences present between their large and small caspase subunits, which contain sequences predicted to be very poor target sites for traditional caspases. An altered cleavage specificity, in conjunction with an absence of good target sites for other caspases in the linker region, may also serve as a way of making the activation of these caspases more strictly dependent on oligomerization rather than activation by other caspases (Vernooy, 2000 and references therein).
In mammals, three pathways have been described that lead to caspase activation. In one pathway a serine protease, granzyme B, is delivered directly into the cytoplasm of target cells from cytotoxic T cells, where it activates executioner caspases. In the other two pathways, cytoplasmic adaptor proteins link a cell death signal transducer to a long prodomain caspase through homophilic receptor-adaptor and adaptor-caspase interactions leading to caspase activation. In one pathway, initiating at the plasma membrane, caspase recruitment is initiated by the binding of ligands to receptors of the tumor necrosis factor/nerve growth factor receptor superfamily. The cytoplasmic region of these receptors contains a region known as the death domain (DD). Ligand-dependent receptor multimerization results in the recruitment of DD-containing cytoplasmic adaptors such as Fas-associated death domain (FADD) through homophilic DD interactions. FADD and related adaptors also contain a second motif known as DED, copies of which are also present in the prodomains of caspase-8 and caspase-10. Homophilic interactions between the DEDs present in receptor-bound adaptors and procaspases leads to caspase oligomerization and subsequent autoactivation. Other adaptors that include DD and CARD domains may also couple activated receptors to CARD domain-containing caspases (Vernooy, 2000 and references therein).
Database searches were used to find candidate death receptors (predicted type 1 transmembrane proteins containing intracellular DDs) in the fly genome. A number of proteins or predicted proteins with DD homology were found, including the kinase Pelle, a Drosophila netrin receptor, a protein with a number of ankyrin repeats (CG7462), and three other proteins that lack significant similarity to other proteins (CG2031, AF22205, and AF22206). However, none of these also shows DED or CARD homology. The prodomain of Dcp-2/Dredd does share weak homology with that of caspase-8, but the Dcp-2/Dredd prodomain is not itself identified in searches for Drosophila proteins. In fact, no Drosophila proteins with significant DED homology were identified in similar searches. These observations suggest several possibilities. One is that Drosophila lacks death receptor signaling pathways. A second possibility is that Drosophila has a death receptor pathway analogous to that found in mammals, but that the level of homology of these proteins with their mammalian counterparts is very low. Finally, Drosophila death receptors may incorporate a distinct set of oligomerization motifs. In the context of this possibility, it will be interesting to identify proteins that interact with the Dream and Dcp-2/Dredd prodomains (Vernooy, 2000 and references therein).
In a second major pathway of apical caspase activation in mammals, cellular stress of various sorts leads to the release of mitochondrial cytochrome c, which in conjunction with the cytosolic adapter protein Apaf-1, promotes caspase-9 activation. Apaf-1 shows large regions of homology with the C. elegans apoptosis inducer, Ced-4. In both organisms, caspase-activating adapter-caspase interactions are dependent on homophilic interactions between the two proteins, mediated at least, in part, by CARDs present at the NH2 terminus of Ced-4/Apaf-1 and in the caspase prodomain. In the case of worms, caspase activation by Ced-4 requires disruption of an association between Ced-4 and the apoptosis inhibitor and Bcl-2 family member Ced-9 by Egl-1, which is a second Bcl-2 family member that acts as an apoptosis inducer. Activation of Apaf-1 in mammals in vitro requires cytochrome c, which stably interacts with WD-40 repeats present at the COOH terminus of Apaf-1 but which are absent in Ced-4. The Apaf-1 WD-40 repeats inhibit its function, and this inhibition is relieved after cytochrome c binding in the presence of ATP/dATP, allowing the formation of a multimeric Apaf-1/cytochrome c complex. Procaspase-9 is recruited to this complex and activated through autocatalysis. Recently, several Apaf-1-like genes have been identified in vertebrates. The proteins encoded by these genes contain distinct NH2- and COOH-terminal sequences, suggesting that they may activate other caspases through different upstream signaling pathways (Vernooy, 2000 and references therein).
The Drosophila genome has one Ced-4/Apaf-1 homolog, variously known as dapaf-1(Kanuka, 1999), dark (Rodriguez, 1999), or hac-1 (Zhou, 1999). Here, this gene is referred to as apaf-1-related killer (ark), its designation in the FlyBase. This gene encodes two splice forms. The long form most closely resembles Apaf-1, in that it contains a series of COOH-terminal WD-40 repeats that presumably mediate regulation by cytochrome c. The short form most closely resembles CED-4, which lacks these repeats, and would thus be predicted to be constitutively active. Genetic evidence indicates that Ark is important for cell death induction in the fly (as well as other processes such as specification of photoreceptor number), and biochemical data point toward interactions between Ark, cytochrome c, and Drosophila caspases. Mitochondrial cytochrome c is at least shifted in localization (Varkey, 1999), and perhaps released into the cytoplasm during apoptosis (Kanuka, 1999). Thus, the weight of evidence suggests that in Drosophila , as in vertebrates, cytochrome c functions to transduce apoptotic signals through Apaf-1 (Vernooy, 2000 and references therein).
Since proteolysis is irreversible, and caspases have the potential to engage in amplifying cascades of proteolysis, caspase activation and activity must be carefully regulated in cells that normally live. The only known cellular caspase inhibitors are members of the inhibitor of apoptosis (IAP) family. Genetic and biochemical evidence from Drosophila argues that IAP-dependent inhibition of caspase activity is essential for cell survival, and that one mechanism for cell death activation involves inhibition of IAP function (Wang, 1999; Goyal, 2000; Lisi, 2000; Vernooy, 2000 and references therein).
IAPs were first identified as baculovirus-encoded cell death inhibitors. These proteins contain several NH2-terminal repeats of an ~70-amino acid motif known as a baculovirus IAP repeat (BIR) as well as a COOH-terminal RING finger domain. RING fingers have since been found in proteins that function in a number of different contexts. For a number of proteins this domain confers E3 ubiquitin protein ligase activity. A number of cellular proteins that share homology with the viral IAPs, based on the presence of one or more BIR repeats (referred to as BIR repeat-containing proteins, or BIRPs) have now been identified in organisms ranging from yeast to humans. The Drosophila genome encodes four BIRPs, including DIAP1, the product of thread locus, Inhibitor of apoptosis 2, deterin, a homolog of Survivin (Jones, 2000), and Bruce, a homolog of BRUCE. A number of the cellular BIRPs, including XIAP, cIAP-1, cIAP-2, NAIP, and Survivin in mammals, and DIAP1, DIAP2, and Deterin in Drosophila, have been tested and shown to act as cell death inhibitors. Notable exceptions are BIRPs from C. elegans and yeast, which regulate cell division. Thus, whereas all IAPs contain BIR repeats by definition, not all proteins with BIRs are IAPs. Many of the death-inhibiting BIRPs, including XIAP, cIAP-1, cIAP-2, Survivin, and DIAP1, have been shown to directly inhibit caspase activation or activity. However, IAPs have been found to associate with a number of different proteins, and may have multiple mechanisms of action. This is particularly suggested in the case of those proteins that contain domains associated with ubiquitin conjugation (Vernooy, 2000 and references therein).
Mitochondria are necessary for cellular energy production, and, thus, are essential for cell survival. In vertebrates (and probably also in Drosophila ) the mitochondria are an important site of integration for cell death and survival signals. The decision to release cytochrome c constitutes one proapoptotic output of this calculation. A second proapoptotic protein released from mitochondria is apoptosis-inducing factor (AIF), which in mammals translocates from the mitochondria to the nucleus upon receipt of a death signal and causes large-scale fragmentation of the DNA. Drosophila , but not C. elegans, encodes a clear AIF homolog (CG7263) (Vernooy, 2000 and references therein).
In some cells undergoing apoptosis, caspase inhibitors are unable to prevent cell death. One cause of this caspase-independent death is thought to be due to mitochondrial damage that occurs upstream of caspase activation. The Bcl-2 family of proteins constitutes a major family of cell death regulators, and many of their pro- and anti-apoptotic functions in vertebrates can be traced to their effects on mitochondrial function. Currently 19 distinct vertebrate Bcl-2 family members have been identified that share up to four Bcl-2 homology domains (BH1-4). Some also have a hydrophobic COOH terminus that targets them to membranes. An important aspect of Bcl-2 family member function is that pro- and anti-apoptotic proteins can heterodimerize (though this is not always required for function), and a large body of evidence argues that they titrate each other's function. However, exactly how these proteins regulate cell death is still unclear. Drosophila encodes two clear Bcl-2 family members. The first is known variously as debcl, drob-1, dBorg-1, or dbok. The second gene is known as buffy (Colussi, 2000) or dBorg-2 (Brachmann, 2000). Both proteins have BH1, BH2, and BH3 domains. Weak BH4 domain homology may also be present. They show the greatest overall homology to the mammalian proapoptotic protein Bok/Mtd, and have proapoptotic function. Genes encoding candidate prosurvival Bcl-2 proteins are not apparent in the fly genome. One possibility is that prosurvival Bcl-2 proteins do not exist. Alternatively, prosurvival members may exist, but have such low homology that it was not possible to identify them. Finally, prosurvival Bcl-2 function may be obtained from posttranslational conversion of one or both of these proteins into an antiapoptotic form (Brachmann, 2000; Vernooy, 2000 and references therein).
A common feature of apoptotic cell death is nuclear condensation and extensive DNA degradation. Apoptotic DNA degradation involves at least several steps. In vertebrates, the initial degradation of DNA is triggered by the caspase-dependent activation of a 40-kD nuclease known as CPAN/CAD/DFF. This protein is synthesized in a form that is complexed to a specific chaperone/inhibitor known as DFF45/ICAD. Caspase cleavage of DFF45/ICAD by caspase-3, releases CPAN/DFF40/CAD, which moves to the nucleus and cleaves DNA. Both DFF45/ICAD and CPAN/DFF40/CAD, as well as several other vertebrate proteins, contain a motif known as a CIDE domain. Experimental observations suggest that CIDE-CIDE interactions are important for regulation of CPAN/DFF40/CAD activity. Degradation of DNA after cell death also occurs in Drosophila and C. elegans. The fly genome encodes functional homologs of caspase-activated DNase (CAD) and CAD inhibitor (ICAD), as well as several other predicted proteins that have CIDE domains (Inohara, 1998; Inohara, 1999; Yokoyama, 2000). CAD-like DNases or other proteins with CIDE domains have not been identified in the C. elegans genome. However, DNA fragmentation occurs cell autonomously in a CED-3-dependent manner in dying cells, suggesting that a CAD-like activity is present. In a second step in apoptotic DNA degradation, which involves the participation of cells that engulf the dying cell, DNA is further processed by an acidic endonuclease. In mammals, this activity is probably an acid lysosomal DNase, either DNase II or a DNase II-like enzyme, and in C. elegans it is the product of the nuc-1 gene. Drosophila also encodes a DNase II-like protein (CG7780), and it seems likely that this form of DNA degradation occurs in flies as well (Vernooy, 2000 and references therein).
Two other mammalian proteins that promote nuclear apoptotic events are AIF and acinus. AIF translocates from the mitochondria to cause chromatin condensation and large-scale DNA fragmentation. Acinus, a DNA-condensing factor with no nuclease activity, localizes to the nucleus, and is activated during apoptosis by combined caspase and serine protease cleavage. Drosophila, but not C. elegans, encodes clear homologs of both these proteins Acinus and AIF) (Vernooy, 2000 and references therein).
One of the reasons for working with a model system such as the fly is the hope of finding a different perspective that will afford unique insight into a conserved, but complex process such as apoptosis. Drosophila has arguably been in this position for some time. An early genetic screen identified a genomic region at 75C that contained genes required for essentially all normally occurring cell deaths during Drosophila embryogenesis. Three genes within this region, reaper, head involution defective, and grim, mediate this proapoptotic requirement. A large body of evidence argues that they act to integrate and transduce many different cell death signals that, ultimately, lead to the activation of caspase-dependent cell death. Rpr, Hid, and Grim have only very limited homology with each other (a short stretch of roughly 14 amino acids near their NH2 termini), and sequence homologs have not been identified in other organisms. However, recent observations argue that the mechanisms of action defined by these genes are likely to be conserved: (1) each of these proteins induces apoptosis in mammalian cells, strongly suggesting that some aspect of their function is evolutionarily conserved; (2) despite their very low level of homology with each other, they each interact with several different conserved death regulators. This suggests that putative mammalian homologs may also be quite divergent in sequence. For example, they each bind the Drosophila caspase inhibitor DIAP1 through interactions that require their NH2 termini, and genetic and biochemical data argue that one way they promote apoptosis is by inhibiting DIAP1's ability to prevent death-inducing caspase activity. Since IAPs and caspases also function to regulate death in vertebrates, it seems reasonable that Rpr, Hid, and Grim orthologs exist that perform a similar death-promoting function. A mammalian protein called Smac/DIABLO, which appears to play such a role has recently been described (Du, 2000). Rpr, Hid, and Grim also bind a Xenopus protein, Scythe, in an interaction that does not require their NH2 termini. In the case of at least Rpr this interaction leads to release of a Scythe-bound proapoptotic factor that promotes cytochrome c release. Drosophila encodes a Scythe homolog (CG7546), suggesting that a similar pathway may exist in flies as well (Vernooy, 2000 and references therein).
Autophagy is a bulk lysosomal degradation process important in development, differentiation and cellular homeostasis in multiple organs. Interestingly, neuronal survival is highly dependent on autophagy due to its post-mitotic nature, polarized morphology and active protein trafficking. A growing body of evidence now suggests that alteration or dysfunction of autophagy causes accumulation of abnormal proteins and/or damaged organelles, thereby leading to neurodegenerative disease. Although autophagy generally prevents neuronal cell death, it plays a protective or detrimental role in neurodegenerative disease depending on the environment. This review describes the two sides of autophagy, the ability to protect or impair cell survival depending on the physiological and pathological environment (Lee, 2009. Full text of article).
Programmed cell death (PCD), important in normal animal physiology and disease, can be divided into at least two morphological subtypes, including type I, or apoptosis, and type II, or autophagic cell death. This study reports the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo. During Drosophila metamorphosis, the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade. To identify and analyze the genes expressed, wild-type patterns of gene expression were examined in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE). 1244 transcripts, including genes involved in autophagy, defense response, cytoskeleton remodeling, noncaspase proteolysis, and apoptosis, were expressed differentially prior to salivary gland death. Expression was detected of the steroid hormone 20-hydroxyecdysone (ecdysone)-induced primary response genes E74, E75, and E93 and the cell death genes ark, dronc, crq, rpr, and iap2. Mutant expression analysis has indicated that several of these genes are regulated by E93, a gene required for salivary gland cell death. These analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis, and that there are important differences (Gorsky, 2003).
Multiple ecdysone-induced genes were detected. Abundantly expressed were members of the L71, or Eig71E, late gene family. The function of the L71 genes has not been established, but they are reported to be induced in late third instar larvae. Their abundance at 16 hr APF and decline by 23 hr APF is consistent with a role during the early larval ecdysone pulse. Eip63F-1, a calcium binding EF-hand family member, and Eip71CD (or Eip28), a protein-methionine-S-oxide reductase, both peak in gene expression at 20 hr APF, similar to the profile observed for E74 and E75. While Eip63F-1 has been implicated in calcium-dependent salivary gland glue secretion during earlier stages of salivary gland development, a role for Eip63F-1 or Eip71CD in salivary glands at the prepupal-pupal stage transition has not been described. Similarly, a role for Hormone-receptor-like in 78 (Hr78) at this stage has not been characterized (Gorsky, 2003).
The findings indicate that transcriptional regulators other than the known ecdysone-induced factors may be involved in autophagic cell death regulation. Transcription factors with an expression profile similar to E74 and E75 (i.e., upregulated at 20 hr APF) include bunched (bun), a RNA polymerase II, and EP2237, a transcriptional activator implicated in sensory organ development. Also upregulated was Drosophila maf-S, a gene similar to a v-maf musculoaponeurotic fibrosarcoma oncogene family member in humans . Another upregulated transcription factor, CG3350, has no previous associated function (Gorsky, 2003).
Expression of genes implicated in multiple different signal transduction pathways was detected, emphasizing the likely complex interplay of signaling pathways in autophagic cell death. One gene highly induced was A kinase anchoring protein 200 (akap200). In general, Akaps function in cyclic AMP-dependent protein kinase (PKA) signal transduction, targeting bound PKA to docking sites in organelles or the cytoskeleton. Redistribution of the cytoskeleton is a feature of autophagic cell death, and it is possible that Akap200 plays a role in cytoskeleton remodeling. Genetic studies in Drosophila have also implicated akap200 as a negative regulator of Ras pathway signaling, and thus it may regulate PCD via this pathway. Another gene significantly upregulated was Darkener of apricot (Doa), a dual specificity LAMMER kinase that is involved in the differentiation of a wide variety of cell types. These findings indicate that Doa, in addition to several other differentially expressed kinases and phosphatases identified, may also be involved in regulating autophagic cell death (Gorsky, 2003).
Detection of members of the Drosophila defense response pathways (i.e., Toll pathway and imd/TNFα-like pathway) suggests that these pathways or some of their components may play a role in developmentally regulated autophagic cell death. In mammals, TNFα signaling can lead to NFκB activation or to apoptosis and has been linked to a possible autophagic type of cell death in T-lymphoblastic leukemic cells. In Drosophila, the TNFα-like pathway functions in both apoptosis and the immune response, and these results indicate that it may also be involved in autophagic cell death (Gorsky, 2003).
Multiple genes involved in apoptotic cell death are also expressed during autophagic cell death, supporting the notion that these two processes can utilize common pathways or pathway components. In addition to the previously identified cell death genes expressed in the salivary gland, additional genes associated, in other tissues, with apoptotic cell death were identifed. Besides dronc, a second caspase, dcp-1, is upregulated transcriptionally in predeath stage salivary glands. In addition to the CD36-related scavenger receptor crq, upregulation was detected of three other CD36-related scavenger receptor genes whose function has not yet been characterized. The expression of additional cell death-related genes, death executioner Bcl-2 homolog (debcl or dborg-1), buffy/dborg-2, iap-1, dredd, and sickle, was detected in salivary glands and showed low level changes or no changes in expression levels. It is possible that these genes play a role in salivary gland death but are regulated primarily at the protein level. Given the overlap of genes involved in autophagic and apoptotic cell, it is reasonable to expect that some of the novel autophagic cell death-associated genes identified in this study may also be associated with apoptotic cell death (Gorsky, 2003).
The results suggest that genes associated with the process of autophagy (i.e., bulk cellular degradation) can be regulated transcriptionally and this regulation is likely integral to the mechanism of autophagic cell death. Known genes involved in autophagy have been defined largely by genetic screens in yeast and include at least 16 autophagy-defective (apg) genes and 6 autophagy (aut) genes, with overlap between the two groups. Putative Drosophila orthologs of at least ten of the apg/aut genes were identified and evidence of expression was found for at least nine of these. Strikingly, CG6194 was induced prior to cell death and is one of two Drosophila genes similar to apg4/aut2, a yeast gene encoding a novel cysteine endoprotease required for autophagy. CG6194 encodes a functional homolog of APG4/AUT2 and interacts genetically with several members of the Notch signaling pathway. Results of real-time RT-PCR analyses have indicated upregulated expression of other apg/aut-like genes including CG1643 (apg5-like), CG10861 (apg12-like), and CG5429 (apg6-like). In addition to apg/aut-like genes, evidence was found for upregulated expression of Drosophila rab-7, one of several rab gene family members implicated in autophagy in yeast and humans (Gorsky, 2003).
The terminal phase of autophagy involves autolysosome formation by fusion of the autophagosome with a lysosome and subsequent degradation of sequestered cellular components. Lysosomal components with upregulated transcripts in predeath stage salivary glands include lysozyme, β-galactosidase, and cathepsins B, D, E, F, and L. Multiple components involved in autophagy are conserved in Drosophila and likely play a role in ecdysone-induced autophagic cell death in the salivary glands (Gorsky, 2003).
To identify the genes with differential expression that are most likely associated with the autophagic cell death process, E93 mutant analyses was carried out. E93 expression appears to specifically foreshadow steroid-induced cell death, and E93 mutant salivary glands display morphological features indicative of a block in the early stages of autophagic cell death. Further, the ecdysone-induced genes BR-C, E74, and E75 and the cell death genes rpr, hid, crq, and dronc are all transcribed at reduced levels in E93 mutant salivary glands. E93 encodes a novel nuclear protein that binds to multiple sites on larval salivary gland polytene chromosomes. The map position of crq correlates with an E93 binding site and it may thus be regulated directly by E93. To identify other genes that may be regulated transcriptionally by E93 in salivary gland death, all differentially expressed genes were screened for those with a map position corresponding to E93 binding sites. Forty-three upregulated genes were identified and forty-one downregulated genes corresponding to 39 of the 65 known E93 binding sites. To test further whether these genes may be regulated directly by E93, transcription profiles were analyzed in E93 mutant salivary glands. Since previous studies indicated a role for E93 as a positive regulator of cell death gene expression, genes upregulated significantly at 23 hr APF were tested. Of 18 confirmed upregulated genes tested, all but one (Sox14) exhibited a reduction in the fold-difference in expression in the E93 mutant background compared to control genes. These results indicate that these 17 genes are regulated by E93, indirectly or directly, and that their expression is thus likely associated specifically with autophagic cell death (Gorsky, 2003).
This study represents the first comprehensive analysis of genes associated with autophagic cell death in vivo. Autophagic cell death is shown to be associated with the induction of genes that participate in protein synthesis, transcription, multiple signal transduction pathways, and two ubiquitin-like pathways required for autophagy. Multiple genes involved in apoptotic cell death also appear to be regulated in autophagic cell death, supporting the view that these two processes can utilize common pathways or pathway components. Further, many genes were implicated for the first time in cell death and represent candidate markers and/or mediators of autophagic cell death and, possibly, apoptotic cell death. In addition to similarities, likely differences were revealed between these two morphological forms of cell death. In particular, genes similar to those involved in autophagy (i.e., bulk cellular degradation) are upregulated in dying salivary gland cells, and these may prove to be useful molecular markers for the autophagic cell death process (Gorsky, 2003).
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