InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

Programmed cell death, or apoptosis, is the regulated elimination of cells that occurs naturally during the course of development. The same process is carried out in many pathological circumstances that required cell death for the benefit of the organism. This delibrate elimination of cells occurs in a morphologically distinct manner suggesting an active, gene-directed process. Investigation of the pathways involved in apoptosis provides a fascinating exercise in unraveling complex gene interactions.

In Drosophila, the earliest normal appearance of non-pathological cell death is observed in three places in the head, in the dorsal cephalic region, within the gnathal segments, and in the clypeolabrum as the germ band begins to retract (stage 11). Thereafter, as germ band retraction [Images] proceeds (stages 12 and 13), cell death becomes widespread throughout the embryo, particularly in the ventrolateral portions and around the procephalic lobes. Cell death becomes prominent within the most posterior abdominal segments; early signs of degeneration along the ventral midline can be observed within the most anterior thoracic segments. Scattered cell deaths also begin to appear in a segmentally reiterated pattern within the lateral portions of the ventral region, and in the ventral neurogenic region. Large numbers of degenerating cells accumulate in the interstitial spaces beneath the dorsal ridge. During dorsal closure [Images], a zone of degenerating cells, organized in the shape of a ring, forms around the closing dorsal tissue (stage 14). As head involution becomes well advanced (stage 15), zones of death apparent in earlier stages subside, and scattered subepidermal death appears throughout the embryo. Eventually, prominent cell death appears throughout the CNS as the ventral nerve cord condenses (stage 16). With the exception of death in the tightly packed cell body layer of the CNS, cell death is accompanied by the engulfment of dying and dead cells by circulating phagocytic macrophages (Abrams, 1993).

As with many complex developmental processes, programmed cell death requires a large number of interacting proteins. Central to the process is the gene reaper (rpr). The intriguing aspect of reaper is its possession of a conserved sequence domain called the "death" domain, involved in the apoptosis process in many other species. What is the death domain and how does it function in apoptosis? Once the sequence of Reaper was made available in the literature, it was noted that Reaper bears homology to mammalian proteins involved in programmed cell death. The mammalian receptor Fas is involved in immune regulation. Likewise activation of the mammalian receptor Tumor necrosis factor receptor 1 (TNFR1) can lead to apoptosis. Both Fas and TNFR1 intracellular domains bear homology to Reaper (Golstein, 1995).

Two other Drosophila genes, grim and Wrinkled, also known as head involution defective (hid), are closely linked to reaper on the chomosome. Both proteins bear sequence homology to Reaper and other death domain proteins and both are involved in programmed cell death. Whereas reaper is a relatively small protein of 65 amino acids, HID is a large novel 410 amino acid protein with homology to RPR at its N-terminal region (Grether, 1995). As with rpr, ectopic expression of hid and grim is sufficient to induce apoptosis. Grim is sufficient to elicit apoptosis in at least one context, where RPR expression is not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors (Chen, 1996).

Mutants of reaper contain many extra cells and fail to hatch, but many other aspects of development appear to be quite normal. One cell type that normally undergoes programmed death in insects is the abdominal neuroblast. In the fly, approximately 25 cells are born in each abdominal neuromere, but only six cells persist to eventually produce neurons in the imaginal ganglia. In rpr mutants 20 or more cells are found in some abdominal segments. A similar increase is found in the number of cells in the larval photoreceptor organ (White, 1994).

Deletion of reaper protects embryos from apoptosis caused by x-irradiation and developmental defects. Mutation of the gene crumbs leads to widespread defects in the development of the epithelial tissues, followed by massive cell death during embryogenesis. reaper deletion blocks the massive ectopic death seen in crumbs mutant embryos (White, 1994).

Reaper and other death domain proteins initiate a cascade of protein activity that includes activation of cysteine proteases known as ICE/CED-3 like proteases. The death domain is likely to be a protein interaction domain, assemblying other proteins involved in the activation of ICE proteases. Direct evidence that proteases are centrally involved in the regulation of the cell death process has come from studies on the nematode C. elegans. A series of genes that control various elements of the programmed cell death process in this worm have been identified, two of which, ced-3 and ced-4 are required for cell death during development. Subsequently, ced-3 was found to exhibit significant homology to mammalian Ice, which converts the 33 kDa protease form of Interleukin-1ß to an active 17.5 kDa form. Ectopic expression of ice in fibroblasts results in apoptosis, suggesting that Ice is both functionally as well as structurally homologous to ced-3 (Martin, 1995 and references).

Other evidence suggests that cell death induced by Reaper occurs by a mechanism distinct from that induced by mammalian death domain proteins. Transient expression of Drosophila rpr in the lepidopteran SF-21 cell line induces apoptosis displaying nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Inhibitors of ICE-family proteases p35 and crmA, as well as members of the iap class of genes, Op-iap and D-iap2, but not bcl-2 family members (see death executioner Bcl-2 homologue), block rpr-induced apoptosis. Mutational analysis of rpr provides no support for the proposed sequence similarity of Reaper and death domain proteins. Mutations in the N-terminal region of Reaper, which displays sequence similarity to Hid and Grim, other Drosophila gene products, correlate with the initiation of apoptosis, suggesting that these residues might be functionally important. The mammalian cDNA encoding FADD (Fas-associating protein with a death domain) also induces cell death in SF-21 cells, but death progresses more slowly and with features which distinguished it from rpr-induced apoptosis. Several bcl-2 family members delay or block FADD-induced cellular death in SF-21 cells (For information about FADD and bcl-2 see the Reaper Evolutionary Homologs section). Thus, apoptosis initiated by Reaper progresses by a faster path, one which appears to differ from that of FADD-induced apoptosis. Mutational analysis or residues originally proposed to be conserved between Reaper and death domain proteins fails to support a functional significance for these proposed sequence alignments. Mutations in the residues that show the strongest conservation display no observable alteration in Reaper activity. It is concluded that cell death induced by Reaper occurs by a mechanism distinct from that induced by mammalian death domain proteins. Reaper operates through a distinct alternative cell death pathway (Vucic, 1997b).

The first Drosophila caspase identified is known as Death Caspase-1. Inhibitors of ICE/CED-3 proteases have also been identified. Existence of these inhibitors, provides indirect evidence that ICE/CED-3 proteases are involved in Drosophila programmed cell death. Drosophila inhibitors of apoptosis function to block cell death induced by Reaper or Wrinkled/Head involution defective. Two proteins, Drosophila IAP1 and Drosophila IAP2 (DIAP1/Thread and DIAP2 respectively) are homologous to a baculovirus protein IAP, a protein that can block cell death induced by stimuli other than viral infection. Baculoviruses are insect viruses that infect Bombax mori, as well as other insects. Drosophila IAP1 is allelic to the gene thread (th), which when mutated gives rise to viable flies whose aristae lack normally occurring side branches. DIAPs share common domains with baculovirus IAP as well as mammalian IAPs and human apoptosis inhibitory protein. DIAPs as well as most other members of this protein family contain RING finger motifs at the C-terminus, thought to be a protein interaction motif that forms zinc-binding sites. The proteins also contain two or three tandem repeats of about 70 amino acids, termed the BIR motif. N-terminal fragments of IAPs containing the BIRs are sufficient to prevent normally occurring and ectopically induced cell death. Mammalian IAPs inactivate interleukin-1ß converting enzyme (ICE)-like cysteine proteases known to play an important evolutionarily conserved role in bringing about cell death (Hay, 1995 and Martin, 1995 and references).

The Drosophila proapoptotic proteins (Reaper, HID, and Grim) are substrates for IAP-mediated ubiquitination. Ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, regulation of Reaper by ubiquitination has been shown to be a significant factor in determining Reaper biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance (Olson, 2003).

Drosophila Reaper can induce rapid apoptosis in vitro using an apoptotic reconstitution system derived from Xenopus eggs. To directly demonstrate Reaper-induced activation of caspases in Xenopus extracts, trace amounts of various 35S-labeled substrates of these proteases were added to the extract. The substrates used were poly(ADP) ribose polymerase (PARP), the zymogens pro-caspase 3 (Yama/CPP32), pro-caspase 1 (ICE), and pro-caspase 7 (ICE-LAP3) and baculovirus p35, which acts as a competitive inhibitor of caspases while being cleaved by them. With the sole exception of pro-caspase 1, all of these substrates are cleaved to their characteristic apoptotic fragments in extracts to which Reaper has been added. Routinely, cleavage of all these proteins precedes the initial stages of nuclear fragmentation by ~10 min. It is unclear whether the failure to cleave pro-caspase 1 reflects the fact that caspase 1 is not activated during Reaper-induced apoptosis or Is due to some incompatibility between the heterologous components in this assay (Xenopus extract, Drosophila Reaper protein and human caspase 1). However, it is striking that Reaper can engage the Xenopus apoptotic machinery, triggering endogenous protease activation (Evans, 1997).

It is known that regulated release of cytochrome c from mitochondria accompanies activation of the apoptotic program, both in mammalian cells and in the Xenopus cell-free system. Bcl-2, a potent inhibitor of apoptosis, acts, at least in part, by inhibiting cytochrome c release. The absence of cytochrome c in the reconstituted Xenopus extracts (lacking mitochondria) might account for the inability of Reaper to induce apoptosis. Therefore, bovine or equine heart cytochrome c were added to the reconstituted extracts in the presence and absence of Reaper and the appearance of apoptotic changes was examined. Between 70 and 85 min after initiation of room temperature incubation the nuclei in these extracts begin to enter apoptosis, as monitored by fluorescence microscopy, regardless of whether Reaper is present. This is in marked contrast to extracts lacking cytochrome c, which show no signs of apoptotic nuclear fragmentation, even 6 h after Reaper addition. These results were confirmed at a biochemical level by analysis of baculovirus p35 degradation. Addition of cytochrome c to extracts lacking mitochondria promotes rapid apoptotic cleavage of p35 regardless of whether or not Reaper is present. Even after careful titration a concentration of cytochrome c which can accelerate apoptosis only in the presence of reaper cannot not be found. Interestingly, addition of free cytochrome c to extracts containing mitochondria also greatly accelerates apoptosis, consistent with the idea that release of cytochrome c from mitochondria is a rate limiting step in this process. Taken together these data suggest that Reaper might induce downstream release of cytochrome c from mitochondria, thereby triggering caspase activation. To determine whether Reaper can indeed induce mitochondrial cytochrome c release, Reaper was incubated in unfractionated Xenopus egg extracts. Aliquots were assayed at various times for cytochrome c release and caspase activity. Although the exact timing of apoptotic events varies between extracts, Reaper accelerates both the release of cytochrome c from mitochondria and the consequent activation of DEVD-cleaving caspases. Bcl-2 antagonizes these effects, but high levels of Reaper can overcome the Bcl-2 block. These results demonstrate that Reaper can function in a vertebrate context, suggesting that Reaper-responsive factors are conserved elements of the apoptotic program (Evans, 1997).

Coordinated expression of cell death genes regulates neuroblast apoptosis

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. This study used loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. Expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process (Tan, 2011).

The patterns of developmental cell death are extremely complex and dynamic, and the fate of an individual cell probably represents the integration of multiple signaling pathways. Intrinsic pathways that define the identity and health of a cell, as well as extrinsic growth and survival pathways, are all likely to contribute to the life or death decision. In Drosophila, the transcriptional regulation of the RHG genes is an important output of the pathways that regulate cell death. The clustering of these genes to a single large genomic locus suggests that cell death signals can be integrated by regulatory sequences that control the coordinated expression of these genes (Tan, 2011).

This work shows that rpr, grim and skl are co-regulated to eliminate NBs during embryonic development. A genomic region important for this regulation was identified, located between rpr and grim. Identified genes or transcripts are surprisingly lacking in the 93 kb region between these genes, which is highly conserved between Drosophila species. This suggests that crucial cis-regulatory elements might be localized to this region (Tan, 2011).

The genomic region important for NB apoptosis was narrowed down to 22 kb. The number of NBs expressing grim and skl is significantly decreased in stage 14/15 embryos homozygous for a deletion of this region. The number of NBs expressing rpr is also affected, but to a smaller extent. Interestingly, there is still expression of grim, rpr and skl in NBs from stage 10 through stage 16. However, in the wild type, the number of NBs expressing all three genes increases at stage 14, presaging the loss of these cells by several hours. This increase in NBs expressing rpr, grim and skl is not seen in MM3 homozygous embryos. As overall grim, rpr and skl expression levels are not significantly altered in the mutants, the deleted regulatory sequences must control grim, rpr and skl expression in both a temporal- and tissue-specific manner, and baseline NB expression must be regulated by sequences outside of this region (Tan, 2011).

It is also interesting to note that the MM3 deletion has a more significant effect on grim and skl expression than on rpr expression. grim and skl are co-expressed in most cells of the stage 15 embryonic CNS, whereas rpr expression is less overlapping. As grim and skl lie 134 kb apart, on either side of rpr, long range enhancer interactions might regulate subsets of genes in this cell death locus. This type of long-range interaction has been implicated previously in the response to irradiation. Here again, a single regulatory region regulates multiple cell death genes at a great distance. The radiation responsive element activates hid and rpr, which are 200 kb apart, without having a major effect on the intervening gene grim. It will be important to examine higher order chromatin interactions to understand these long-range enhancer interactions (Tan, 2011).

The structure of the RHG cell death locus is conserved in other Drosophila species, but not obviously in more distantly related insect species, even though IAP inhibitory proteins are found in other species. This suggests that a coordinately regulated RHG gene cluster might be particularly important for Drosophila development (Tan, 2011).

As demonstrated by these studies, the individual roles for the cell death genes in developmental apoptosis are not well understood. Strong genetic and molecular data support a role for hid in regulating cell death in the embryonic head and in the developing eye. A role for grim in the death of microchaete glial cells has been described recently (Wu, 2010). Shown here is a specific role for grim in the elimination of the normal abdominal neuroblasts in the third instar larvae. As described in this work, rpr alone is not required for abdominal NB death in the embryo. However, this double mutant analysis demonstrates that rpr and grim must act together to eliminate these cells (Tan, 2011).

A possible role for hid in NB death cannot be eliminated, although no hid mRNA is detected in the VNC outside of the midline. A recent study suggests that rpr requires hid for efficient induction of apoptosis (Sandu, 2010). However, loss of hid does not result in increased NB survival, as would be expected if hid is required for rpr activity. Furthermore, loss of one copy of hid along with rpr and grim does not increase NB survival. Knockdown of rpr, grim and hid in NBs with an RHG miRNA also does not increase the number of surviving NBs over loss of rpr and grim. Further studies are needed to examine how hid and grim, rpr and skl differentially contribute to developmental apoptosis (Tan, 2011).

Although skl has similar pro-apoptotic activity to hid, grim and rpr, a role for skl in developmental apoptosis has not previously been demonstrated, owing to the lack of a skl mutation. This study shows that skl deletion alone does not alter NB death. In addition, it was found that deletion of one copy of skl along with grim and rpr slightly increase NB survival over deletion of grim and rpr alone. Deletion of skl and rpr, along with the NBRR in XR38/X20 also results in a larger VNC than deletion of rpr and the NBRR in XR38/H88, again suggesting a role for skl in cell death in this tissue. Additional double mutant analysis will be needed to test whether deletion of skl along with grim or rpr supports a role for skl in developmental apoptosis. Given the strongly overlapping expression of grim and skl in the embryonic VNC, it might be found that skl is redundant with grim in the CNS (Tan, 2011).

What are the developmental advantages of multiple cell death regulators? This work demonstrates that multiple RHG genes are activated within a single NB to activate apoptosis. In the salivary gland, hid and rpr, but not grim, are transcribed in the dying tissue, whereas the radiation response appears to involve hid, rpr and skl. These data suggest that the complex upstream regulation of cell death is likely to impact on different subsets of the RHG genes. If each gene is controlled by different combinations of enhancers this could provide greater flexibility in controlling the activation of cell death. In addition, a requirement for multiple cell death activators could ensure that cells are not killed inappropriately by the accidental activation of a single RHG gene (Tan, 2011).

The requirement for multiple RHG genes to kill cells might also reflect differences in the pro-apoptotic activities of these genes. For example, the RHG proteins show differential binding preferences to specific domains of the DIAP1 protein, and inhibit DIAP1 anti-apoptotic functions through different mechanisms. Additional activities, such as mitochondrial permeabilization, translational repression or interaction with other proteins such as Bruce or Scythe, also differ between RHG genes. Furthermore, post-translational regulation of particular RHG proteins might regulate their activity. This has been demonstrated for Hid; phosphorylation by MAPK downregulates the killing activity of Hid. It remains to be demonstrated whether different cell types have differential sensitivities to particular combinations of cell death genes when expressed at normal levels. This might provide yet another layer of control of the cell death process (Tan, 2011).

In sum, this work demonstrates that the regulation of NB apoptosis during development involves the coordinated temporal and spatial regulation of multiple cell death genes, controlled by a common regulatory region. By dissecting the RHG gene locus, specific genomic elements that regulate these genes in other developmentally important cell deaths are likely to be identified (Tan, 2011).

REGULATION

Promoter

The genomic region containing reaper, grim, and head involution defective is required for all cell death in Drosophila embryos, including radiation-induced apoptosis. rpr is transcriptionally induced in embryos following irradiation, and an 11 kb sequence upstream of the rpr start codon is sufficient to confer radiation responsiveness on a lacZ reporter transgene. To identify the minimal radiation-responsive cis-elements upstream of rpr, the ability of smaller fragments of this 11 kb regulatory region to activate lacZ transcription was tested. Each transgenic strain was tested for radiation-induced expression of beta-galactosidase. Multiple constructs containing sequences ~5 kb upstream of the rpr start codon show a robust radiation response. These experiments identify a discrete 150 bp enhancer that responds to radiation as strongly as the larger enhancer fragments tested. Since this enhancer retains radiation-responsiveness but does not recapitulate the developmental patterns of rpr expression seen with larger enhancer fragments, the results also indicate that cis-regulatory sequences responsible for damage-induced transcription of rpr can be isolated from others that respond to developmental cues (Brodsky, 2000).

Within the 150 bp enhancer, a 20 bp sequence was identified that strongly resembles the consensus for human p53 DNA-binding sites. This 20 bp sequence is referred to as the p53 response element (p53RE) to reflect its response to Dmp53 in yeast. Like those found upstream of the human target genes mdm-2 and p21/WAF1, this putative p53 binding site upstream of rpr contains two tandemly arrayed 10mers, each of which matches the consensus motif at nine of ten positions. The two mismatches occur at the outer positions of the 20 bp element; the invariant core nucleotides of each 10mer motif match the consensus perfectly (Brodsky, 2000).

Yeast one-hybrid assays were used to see whether Drosophila p53 interacts with the p53RE. For these studies, a reporter plasmid containing the p53RE upstream of the beta-galactosidase gene was integrated into the yeast genome to produce the p53RE bait strain. Next, this p53RE bait strain was transformed with test plasmids expressing either wild-type Dmp53 or Dmp53(259H) fused to the GAL4 activation domain. These strains were assayed for beta-galactosidase activity. Reporter expression in strains with Dmp53(259H) or the empty vector control (expressing the Gal4 activation domain alone) are indistinguishable from each other. Compared to these controls, each of the four independent transformants carrying the wild-type Dmp53 plasmid shows a substantial increase in beta-galactosidase levels. Based on these results, it has been concluded that the 150 bp radiation-responsive enhancer upstream of rpr contains a 20 bp binding site for Dmp53 (Brodsky, 2000).

A test was performed to see whether the p53RE is sufficient to confer radiation-responsive transcriptional activation on a lacZ reporter construct in vivo. A transgene containing four copies of the p53RE and the minimal hsp70 promoter has showennegligible expression in untreated embryos but is substantially induced following irradiation. Therefore, the 20 bp Dmp53 binding site from the rpr locus is sufficient to mediate a transcriptional response to radiation and may define a minimal radiation responsive sequence. When analyzed in parallel to the 150-lacZ reporter, containing 150 bases surrounding the p53RE, the p53RE-lacZ reporter exhibits less robust and less uniform beta-galactosidase activity following irradiation. Reduced activity is often observed when DNA elements are tested in isolation from the normal flanking sequences and, in this instance, may reflect the influence of other factors that interact with the 150 bp enhancer sequence (Brodsky, 2000).

Disruptions of development are often associated with excess apoptosis. For example, in a crumbs (crb) mutant background, abnormal epidermal development in the embryo leads to widespread apoptosis. This apoptosis is fully suppressed by deletions for the genomic region containing rpr, hid, and grim and is preceded by a dramatic induction of rpr expression, similar to that seen in irradiated embryos. A test was performed to see whether transcriptional activation mediated by p53RE represents a specific response to radiation damage or a common integration point for multiple pathways that lead to excess apoptosis. Beta-galactosidase expression was examined in wild-type and crb embryos carrying either the p53RE-lacZ or the 2kb-lacZ reporter constructs. In stage 12/13 wild-type embryos, expression of the 2kb-lacZ transgene is normally confined to the developing gut but, in similarly aged crb embryos, expression is induced throughout the epidermis. In contrast, the p53RE-lacZ transgene exhibits only basal expression in either wild-type or crb embryos. Thus, despite widespread apoptosis in crb embryos, there is no induction of reporter expression from the p53RE. These results indicate that the p53RE specifically responds to radiation damage, not generally to all proapoptotic signals. They also indicate that irradiation and disrupted development may activate rpr expression through distinct pathways (Brodsky, 2000).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).

This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).

The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).

The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).

Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD₅₀ values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).

The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).

(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).

(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).

Transcriptional regulation

Hormones and trophic factors provide cues that control neuronal death during development. These developmental cues in some way regulate activation of apoptosis, the mechanism by which most, if not all, developmentally programmed cell deaths occur. In Drosophila, apoptosis can be induced by the expression of the genes reaper, grim, or head involution defective. Prior to the death of a set of identifiable doomed neurons, these neurons accumulate transcripts of the reaper and grim genes, but do not accumulate transcripts of the head involution defective gene. Death of these doomed neurons can be suppressed by two manipulations: either by increasing the levels of the steroid hormone 20-hydroxyecdysone (see Ecdysone receptor) or by decapitation. The impact that these two manipulations have on reaper expression has been investigated. Steroid treatment prevents the accumulation of reaper transcripts, whereas decapitation results in the accumulation of lower levels of reaper transcripts that are not sufficient to activate apoptosis. These data demonstrate that in vivo, reaper, and grim transcripts accumulate coordinately in a set of identified doomed neurons prior to the onset of apoptosis. These observations raise the possibility that products of the reaper and grim genes act in concert in postembryonic neurons to induce apoptosis. That reaper transcript accumulation is regulated by the steroid hormone titer and by the presence of the head is evidence that developmental factors control programmed cell death by regulating the expression of genes that induce apoptosis (Robinow, 1997).

Larval midgut and salivary gland histolysis are stage-specific steroid-triggered programmed cell death responses. Larval salivary glands can be maintained for many hours in organ culture, providing an ideal opportunity to study the hormonal requirements for a variety of responses to ecdysone, including glue secretion, polytene chromosome puffing and specific gene regulation. The majority of glands cultured in the presence of ecdysone for 7 hours show a strong nuclear acridine orange stain indicating that salivary gland cell death is an ecdysone-triggered response. In vivo, dying larval midgut and salivary gland cell nuclei become permeable to the vital dye acridine orange; their DNA undergoes fragmentation, indicative of apoptosis. Crawling mid-third instar larvae were injected with ecdysone. Such injected larvae pupariate within 6-8 hours, and midguts isolated from these larvae show a uniform nuclear acridine orange staining, indicative of the onset of programmed cell death. The histolysis of these tissues can be inhibited by ectopic expression of the baculovirus anti-apoptotic protein p35, implicating a role for caspases in the death response. Coordinate stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid) immediately precedes the destruction of the larval midgut and salivary gland. The diap2 anti-cell death gene is repressed in larval salivary glands at the time that rpr and hid are induced, suggesting that the death of this tissue is under both positive and negative regulation. diap2 is repressed by ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the precise stage- and tissue-specific regulation of key death effector genes (Jiang, 1997).

The fact that tailless, brancyenteron and bowl expression at the blastoderm stage are all apparently normal in caudal-deficient embryos suggests that a hindgut primordium is established in the absence of cad activity. The lack of proper blastoderm stage expression of fkh and wg, however, indicates that this hindgut primordium is not properly specified. In byn mutant embryos one of the earliest phenotypic manifestations of an abnormally specified hindgut primordium is ectopic expression of the cell death gene reaper (rpr). To ask whether the extremely reduced hindgut in cad-deficient embryos might result from a similar course of programmed cell death, expression of rpr was examined in embryos lacking cad. A striking pattern of ectopic rpr expression is observed in cad minus embryos, beginning during stage 7 and continuing into stage 8 (gastrulation), in a ring at the circumference of the amnioproctodeal plate. However, the actual loss of cells that is presumably initiated by this ectopic rpr expression does not begin until after early stage 10, because the hindgut primordium is present at this stage in cad-deficient embryos, as indicated by its expression of byn and fkh. By stage 13, the cad-deficient embryo has a very short hindgut and no detectable anal pads; in sections of stage 13 embryos there are numerous apoptotic cells in the region of the hindgut remnant (Wu, 1998).

A steroid-triggered transcriptional hierarchy controls salivary gland cell death during Drosophila metamorphosis

The steroid hormone ecdysone signals the stage-specific programmed cell death of the larval salivary glands during Drosophila metamorphosis. This response is preceded by an ecdysone-triggered switch in gene expression in which the diap2 death inhibitor is repressed and the reaper (rpr) and head involution defective (hid) death activators are induced. rpr is induced directly by the ecdysone-receptor complex through an essential response element in the rpr promoter. The Broad-Complex (BR-C) is required for both rpr and hid transcription, while E74A is required for maximal levels of hid induction. diap2 induction is dependent on FTZ-F1, while E75A and E75B are each sufficient to repress diap2. This study identifies transcriptional regulators of programmed cell death in Drosophila and provides a direct link between a steroid signal and a programmed cell death response (Jiang, 2000).

Although initial studies had indicated that rpr and hid are coordinately induced in the salivary glands approximately 12 hr after puparium formation, more recent work has shown that rpr is induced approximately 1.5 hr earlier than hid, suggesting that these death activators are regulated by distinct mechanisms. The timing of rpr induction is synchronous with the prepupal ecdysone pulse, suggesting that it may be induced as a primary response to the hormone, while the delay in hid induction suggests that it may be a secondary response to ecdysone. These two modes of regulation can be distinguished by their different sensitivity to the inhibition of protein synthesis. Salivary glands were dissected from 10 hr wild-type prepupae and cultured in insect medium supplemented with 20-hydroxyecdysone, either in the presence or absence of cycloheximide. Total RNA was extracted after 0, 2, or 4 hr of culture and analyzed by Northern blot hybridization. Both rpr and hid are induced within 2 hr of hormone treatment, consistent with the proposal that these genes are induced by ecdysone in late prepupal salivary glands. In the presence of the protein synthesis inhibitor cycloheximide, rpr transcription is both delayed and reduced, while hid expression is completely eliminated. These observations indicate that rpr is induced directly by the hormone-receptor complex, although maximal levels of rpr transcription also require the synthesis of ecdysone-induced proteins. In contrast, hid is induced solely as a secondary response to ecdysone. These observations are consistent with the timing of rpr and hid induction in staged salivary glands and provide a framework for defining the molecular mechanisms by which ecdysone regulates rpr and hid transcription, triggering salivary gland cell death (Jiang, 2000).

The BR-C is defined by three genetic functions: broad (br), reduced bristles on palpus (rbp), and l(1)2Bc. Earlier studies have shown that the rbp function of the BR-C is required for salivary gland cell death during metamorphosis. This result has been confirmed by finding that larval salivary glands are not destroyed by 22 hr after puparium formation in pupae that carry the rbp⁵ null allele. The high penetrance of this mutant phenotype suggests that rpr and hid may not be properly expressed in rbp⁵ mutant salivary glands (Jiang, 2000).

To test this hypothesis, salivary glands were dissected from staged rbp⁵ mutants, and rpr and hid expression was examined by Northern blot hybridization. Both rpr and hid transcription is significantly reduced in rbp⁵ mutant salivary glands, indicating that the failure of salivary gland cell death in this mutant can be attributed to its inability to express these death activators. Both betaFTZ-F1 and the ecdysone-inducible E93 early gene are expressed in rbp⁵ mutant salivary glands, indicating that the block in rpr and hid transcription is not simply due to developmental arrest of the mutant animals. BR-C is expressed in midprepupal salivary glands and thus would be present in the late prepupal glands used for the cycloheximide experiment described above. This explains why the reduced level of rpr transcription observed in the absence of protein synthesis is not as severe as the rbp⁵ mutant phenotype (Jiang, 2000).

Both molecular and genetic studies have indicated that the BR-C and E74 function together in common developmental pathways during the onset of metamorphosis. It was therefore asked whether, like the BR-C, E74 might contribute to the ecdysone-triggered destruction of larval salivary glands. In support of this proposal, salivary gland cell death is significantly delayed in E74^P[neo] animals. This mutation is a null allele that inactivates the E74A promoter. While salivary glands in control animals are completely destroyed by 16 hr after puparium formation, approximately 20% of E74^P[neo]Df(3L)st-81k19 animals have salivary glands at 24 hr after puparium formation. This partially penetrant cell death defect suggests that rpr and hid expression may be reduced in E74A mutant salivary glands. To test this hypothesis, salivary glands were dissected from staged E74^P[neo]/Df(3L)st-81k19 mutants, and rpr and hid expression was examined by Northern blot hybridization. Although rpr transcription is unaffected by the E74^P[neo] mutation, the levels of hid transcription are significantly reduced. This observation indicates that E74A is required for the maximal induction of hid but not rpr (Jiang, 2000).

The observation that rpr transcription is induced directly by ecdysone in cultured larval salivary glands indicates that one or more EcR/USP binding sites should be present in the rpr promoter. As a first step toward identifying these regulatory elements, the sequences required for ecdysone-inducible rpr transcription in larval salivary glands were mapped. 9.6 kb of the rpr promoter is sufficient to recapitulate certain aspects of the complex pattern of rpr expression during embryogenesis. Four P element constructs were made that carry either 9.5, 6.1, 3.9, or 1.2 kb of DNA upstream from the rpr transcription start site and 125 bp downstream from the transcription start site, with the rpr 5' untranslated region fused to a lacZ reporter gene. These constructs were introduced into the Drosophila germline by P element-mediated transformation, and the patterns of lacZ transcription in staged salivary glands were compared with those of the endogenous rpr gene by Northern blot hybridization. An increased level of rpr promoter activity is seen upon deletion of sequences between -9.5 and -6.1 kb relative to the start site of rpr transcription. The overall level of lacZ transcription is then reduced as more rpr regulatory sequences are deleted. However, 1.3 kb of the rpr promoter is sufficient to direct lacZ induction in synchrony with that of the endogenous rpr gene, indicating that this region contains the sequences required for proper temporal regulation (Jiang, 2000).

DNA fragments from the 1.3 kb rpr promoter region were generated by PCR and tested for their ability bind EcR: a 274 bp fragment extending from -195 bp to +80 bp relative to the rpr transcription start site binds EcR. Sequence analysis has shown a single imperfect palindromic EcR/USP binding site within this fragment. This rpr EcRE matches 10 out of 13 positions with the consensus EcR/USP binding site. The rpr element is not as strong of a binding site as a canonical hsp27 element. This observation is consistent with the deviations from the consensus at positions +2 and +3 in the rpr EcRE. These and other results strongly suggest that the ecdysone-receptor complex directly regulates rpr transcription through at least one binding site in the rpr promoter (Jiang, 2000).

Therefore ecdysone-regulated transcription factors encoded by betaFTZ-F1, BR-C, E74, and E75 function together to direct a burst of the diap2 death inhibitor followed by induction of the rpr and hid death activators. It is proposed that cooperation between rpr and hid allows these genes to overcome the inhibitory effect of diap2, by precisely coordinating when the salivary glands are destroyed. Evidence that the ecdysone-receptor complex directly induces rpr transcription through an essential response element in the promoter, providing a direct link between the steroid signal and a programmed cell death response. The diap2 death inhibitor is expressed briefly in the salivary glands of late prepupae, foreshadowing the imminent destruction of this tissue. This transient expression is directed by at least three ecdysone-regulated transcription factors: betaFTZ-F1, E75A, and E75B. diap2 induction is dependent on the betaFTZ-F1 orphan nuclear receptor. This is consistent with the timing of betaFTZ-F1 expression, which immediately precedes that of diap2, as well as the known role of betaFTZ-F1 as an activator of gene expression in late prepupae (Jiang, 2000).

Drosophila Immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis

Transgenic flies were examined in which immune deficiency (imd) expression was placed under the control of the ubiquitous da-GAL4 driver. It was surprising to observe 100% lethality in these flies during early larval development. The lethality was partially rescued by coexpression of the viral caspase inhibitor P35. This protein inactivates most of the executioner caspases of the death program. It was further noted that overexpression of imd by the fat body-specific driver yolk-GAL4 induced the transcription of a reaper-lacZ reporter in this tissue. Reaper is a key activator of apoptosis in Drosophila. A TUNEL analysis of transgenic flies overexpressing imd also revealed a remarkably large number of labeled nuclei in fat body cells, as compared to controls. This effect was suppressed by coexpression of the antiapoptotic protein P35. Transmission electron microscopy analysis revealed that the fat body cells exhibit the classical morphological aspects of apoptosis, that is, densification and fragmentation of the cytoplasm, membrane blebbing, and stacking of the endoplasmic reticulum (Georgel, 2001).

Steroid regulation of midgut cell death during Drosophila development

Steroid hormones trigger dynamic tissue changes during animal development by activating cell proliferation, cell differentiation, and cell death. Steroid regulation of changes have been characterized in midgut structure during the onset of Drosophila metamorphosis. Following an increase in the steroid 20-hydroxyecdysone (ecdysone) at the end of larval development, future adult midgut epithelium is formed, and the larval midgut is rapidly destroyed. Mutations in the steroid-regulated genes BR-C and E93 differentially impact larval midgut cell death but do not affect the formation of adult midgut epithelia. In contrast, mutations in the ecdysone-regulated E74A and E74B genes do not appear to perturb midgut development during metamorphosis. Larval midgut cells possess vacuoles that contain cellular organelles, indicating that these cells die by autophagy. While mutations in the BR-C, E74, and E93 genes do not impact DNA degradation during this cell death, mutations in BR-C inhibit destruction of larval midgut structures, including the proventriculus and gastric caeca, and E93 mutants exhibit decreased formation of autophagic vacuoles. Dying midguts express the rpr, hid, ark, dronc, and crq cell death genes, suggesting that the core cell death machinery is involved in larval midgut cell death. The transcription of rpr, hid, and crq are altered in BR-C mutants, and E93 mutants possess altered transcription of the caspase dronc, providing a mechanism for the disruption of midgut cell death in these mutant animals. These studies indicate that ecdysone triggers a two-step hierarchy composed of steroid-induced regulatory genes and apoptosis genes that, in turn, regulate the autophagic death of midgut cells during development (Lee, 2002).

Transcription of rpr, hid, ark, dronc, and crq increases in wild-type animals following the late larval pulse of ecdysone that triggers larval midgut cell death. Since mutations in the BR-C and E93 genes prevent proper destruction of larval midguts, Northern blots were prepared from midguts of these mutants at stages preceding and during cell death. BR-C 2Bc² mutants have altered transcription of rpr, hid, and crq, but do not impact the transcription of ark and dronc. In contrast, E93 mutants possess altered transcription of dronc, but do not change the transcript levels of the other cell death genes known to be expressed in dying midguts. Although midguts die by autophagy, they transcribe core apoptosis regulators during this cell death, and mutants that prevent autophagy alter transcription of apoptosis genes (Lee, 2002).

The distributed association of future adult cells within the epithelium of larval midguts is another important difference between ecdysone-regulated midgut and salivary gland programmed cell death. The close association of larval and adult midgut cells may be one of the reasons why larval midgut exhibits a less synchronized cell death than salivary glands. Both salivary glands and midguts require the function of the E93 and BR-C genes. However, mutations in these genes appear to result in different effects in salivary glands and midguts; BR-C appears to play a more important role in midguts. While both salivary glands and midguts express the cell death genes rpr, hid, ark, dronc, and crq, the impact of mutations in BR-C and E93 are very different in the midgut than in salivary glands. BR-C affects transcription of rpr, hid, and crq, but E93 mutants only affect dronc transcription in midguts. In contrast, mutations in E93 prevent proper transcription of all of these cell death genes in dying salivary glands. Clearly, many more genes may be involved in the complicated autophagic cell death of midguts. While several similarities and differences have been identified between salivary gland and midgut death, future analyses are needed to clarify the mechanism by which the steroid ecdysone triggers midgut programmed cell death (Lee, 2002).

The Drosophila Hox gene Deformed sculpts head morphology via direct regulation of the apoptosis activator reaper

Hox proteins control morphological diversity along the anterior-posterior body axis of animals, but the cellular processes these proteins regulate directly are poorly understood. During early Drosophila development, the Hox protein Deformed (Dfd) maintains the boundary between the maxillary and mandibular head lobes by activating localized apoptosis. Dfd accomplishes this by directly activating the cell death promoting gene reaper (rpr). One other Hox gene, Abdominal-B (Abd-B), also regulates segment boundaries through the regional activation of apoptosis. Thus, one mechanism used by Drosophila Hox genes to modulate segmental morphology is to regulate programmed cell death, which literally sculpts segments into distinct shapes. This and other emerging evidence suggests that Hox proteins may often regulate the maintenance of segment boundaries (Lohmann, 2002).

Several lines of evidence -- the effects of manipulating rpr expression in embryos, in vitro DNA binding studies with Dfd protein and mutagenesis of Dfd binding sites in the rpr enhancer, the phenocopy of the Dfd mutant boundary defect with an apoptosis inhibitory gene, its rescue with an apoptosis promoting gene, and the phenotype of rpr mutants -- show that the Hox protein Dfd is a direct transcriptional activator of rpr in the anterior maxillary segment, and that rpr expression and apoptosis are necessary to maintain the maxillary/mandibular boundary. At least in part, this Dfd-dependent, anterior maxillary expression of rpr is conferred by a 674 bp enhancer (rpr 4-S3) that maps 3.1 kb upstream of the rpr transcription start. This demonstrates that a Hox protein directly regulates a cell biological effector gene that mediates a morphological subroutine for that Hox function. Therefore, rpr qualifies as a directly regulated realizator. Interestingly, although Dfd is expressed in nearly all maxillary cells, the loss of Dfd function does not influence rpr expression in the posterior maxillary segment, indicating that other activators and/or repressors of rpr are distributed in maxillary cells that influence the transcriptional activity of Dfd protein on this locus. In the tail region, Abd-B is also required for the formation of normal boundaries between the abdominal segments A6/A7 and A7/A8, and their maintenance correlates, as in the case of the maxillary/mandibular boundary, with the localized activation of rpr. Thus, at both termini of the Drosophila body, Hox control of apoptosis is used for segment boundary maintenance (Lohmann, 2002).

Hox proteins may have a wider role in the programming of segmental boundaries than is currently believed. There is strong evidence that two Drosophila homeobox genes that are used to control segment number, even-skipped and fushi-tarazu, are independently derived from genes that still possess Hox segment identity functions in most insects and other arthropods. In addition, mutants in the mouse Hoxa-2 gene have segmentation defects in the hindbrain. Although a segmental boundary is normally established between rhombomeres 1 and 2 in the Hoxa-2 mutants, it is not maintained, which is reminiscent of the defect in boundary maintenance observed in Dfd mutant embryos (Lohmann, 2002).

Surprisingly, although rpr is required for maxillary/mandibular boundary maintenance during embryogenesis, flies lacking rpr function survive to adulthood with only minor defects. One possible explanation is that other cell death activators can compensate for the absence of rpr at later stages of development, since other apoptosis genes, like hid, grim, and sickle, are expressed in overlapping patterns with rpr and share IAP binding motifs in their N-terminal protein sequence. This may also explain why the maxillary/mandibular segmentation defect is less severe in Dfd null mutants and in XR38/H99 mutants when compared to homozygous Df(3L)H99 mutants. Since rpr and hid, but not grim, are expressed in anterior maxillary cells at many developmental stages, and since the combined activities of hid and rpr dictate the probability of a cell to undergo apoptosis, it is suggested that in wild-type embryos the combination of rpr and hid are required to kill cells at the maxillary/mandibular boundary (Lohmann, 2002).

Many Drosophila genes are known to be regulated in a Hox-dependent manner, but most encode either transcriptional regulators or cell signaling molecules. These Hox effectors presumably act both independently and/or in parallel to Hox genes to indirectly influence cell type identity and morphology. For example, the Hox target gene Distal-less (Dll) is required for the development of embryonic appendages and is directly repressed in abdominal segments by the Hox proteins Ubx and Abd-A. However, at some point the Hox proteins, their downstream effectors, and other cofactors must affect cellular changes by means of the class of realizer genes (Lohmann, 2002).

There are three good candidates for realizer genes in Drosophila: connectin, centrosomin, and ß-tubulin. connectin encodes an extracellular cell surface protein with leucine-rich repeats. It mediates cell-cell adhesion in cell culture assays and acts as a homophilic cell adhesion molecule in the lateral transverse muscles. In the nervous system, connectin expression is under the control of Ubx; a small regulatory fragment that mediates portions of connectin expression has been isolated by its affinity for Ubx in coimmunoprecitation assays. In some tissues, connectin is under the direct control of Ubx protein, but it is still unclear which of the morphogenetic subfunctions of Ubx require connectin function. centrosomin is a subunit of the centrosome and is necessary for the proper development of the CNS, PNS, and midgut. During the formation of the second midgut constriction, the functions of both Ubx and centrosomin are required, and centrosomin is lost in the visceral mesoderm cells of Ubx mutants. The ß-tubulin gene encodes a major component of microtubules and contains a cis-regulatory element that is regulated by Ubx in the visceral mesoderm (Lohmann, 2002).

In Drosophila, as in vertebrates, programmed cell death is used for the sculpting of morphological structures. For example, limb formation in amniotes is accompanied by massive cell death in almost all the interdigital mesenchymal tissue located between the chondrifying digits, eliminating the cells located between the differentiating cartilages and thus sculpting the shape of the limb. Interestingly, in Hoxa13 heterozygous mutant mice, the apoptosis that normally occurs in the interdigital regions is reduced, leading to a partial fusion of digits II and III in adult mice. In Hoxa13 homozygous mutant mice, there is no interdigital apoptosis and no digit separation in 14-day-old embryos. Although it remains to be seen whether Hoxa13 and other Hox genes are direct regulators of apoptotic genes in amniotes and other animals, one intriguing possibility is that Hox-dependent regulation of apoptosis is a more general mechanism used to generate and maintain metameric pattern during animal development (Lohmann, 2002).

Binary cell death decision regulated by unequal partitioning of Numb at mitosis

An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. A Drosophila sensory organ lineage is described that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. This study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner (Orgogozo, 2002).

The vmd1a neuron is located within a cluster of five multidendritic (md) neurons in the ventral region of abdominal segments A1-A7. The vmd1a neuron can be distinguished from the other ventral md neurons (vmd1-4) using the B6-2-25 enhancer-trap marker. The origin of this vmd1a neuron is not known. vmd1-4 neurons are generated by the four vp1-4 external sensory (es) organ primary precursor (pI) cells. Each vp1-4 pI cell follows a lineage called the md-es lineage. This lineage is composed of four successive asymmetric cell divisions that generate five distinct cells, the four cells of the es organ at the position where the pI cell has formed and one md neuron that will then migrate to the ventral md cluster. In the md-es lineage, the membrane-associated protein Numb is segregated into one of the two daughter cells at each cell division. Numb establishes a difference in cell fate by antagonizing Notch in the Numb-receiving cell. Because no es organ is found in the vicinity of the vmd1a neuron, this neuron is probably not generated by a md-es lineage (Orgogozo, 2002).

rpr and grim, but not hid, are expressed specifically in the pIIa and pIIIb cells of the vmd1a lineage. By contrast, these genes are not expressed in cells of the vp1-4 lineages. In embryos in which a pIIb cell divides at the vp1 position in at least one abdominal segment, most segments contain a vmd1a pIIa-pIIb pair with one cell expressing rpr or grim. This cell is the pIIa cell fated to die. In some other segments, neither of these two cells accumulates rpr (25%) or grim (8%). Since the development of segments is not perfectly synchronous, it is assumed that this represents a situation preceding the onset of rpr and grim expression in the pIIa cell. In the remaining segments, a single Cut-positive cell is detected indicating that the pIIa cell has died. In those segments, expression of rpr and grim is never detected in the remaining pIIb cell (Orgogozo, 2002).

During the pIIb division, Numb was shown to segregate into the dorsal pIIb daughter cell. This cell is not fated to die and differentiates as a vmd1a neuron. By contrast, it could not be directly determined which one of the two pI daughter cells inherits Numb. Indeed, since the orientation of the vmd1a pI cell division is random, the pIIa and pIIb cells could not be identified from their relative positions. Nevertheless the vmd1a pIIa and pIIIb cells appear to generate ectopic shaft/socket and neuron/sheath cell pairs when cell death is prevented. In the md-es lineage, these cell pairs are the progeny of the cells that do not inherit Numb. This suggests that both the vmd1a pIIIb cell and the pIIa cell do not inherit Numb. Thus, Numb appears to segregate in the cells that do not die in the vmd1a lineage (Orgogozo, 2002).

The role of Numb was tested in regulating rpr and grim expression as well as cell death in the vmd1a lineage. In numb mutant embryos in which a secondary precursor cell divides at the vp1 position in at least one abdominal segment, it was observed that the two Cut-positive vmd1a pI daughter cells accumulate rpr or grim transcripts (54% of the segments for rpr, 52% for grim). In other segments a single Cut-positive pI daughter cell was found accumulating rpr or grim. In these segments one pI daughter cell has already died and the other one is undergoing apoptosis. These two phenotypes are not seen in wild-type embryos. Thus, in the absence of numb, both pI daughter cells undergo programmed cell death. Consistently, no Cut-positive cell is observed at the vmd1a position in numb mutant embryos in most segments. It is concluded that numb is required to inhibit the expression of rpr and grim and to prevent cell death in the pIIb cell (Orgogozo, 2002).

To test whether numb is sufficient to prevent cell death, the progeny of the vmd1a pI cell was analyzed in arm-Gal4 UAS-numb embryos that express high levels of Numb. In wild-type embryos in which a vp1 pIIIb cell is dividing in at least one segment, one or two Cut-positive cells are observed at the vmd1a position. In contrast, four Cut-positive cells are observed in 50% of the segments in arm-Gal4 UAS-numb embryos at the same stage. In 8 out of the 9 segments with four cells, two cells accumulating high levels of Pros and two cells accumulating low levels of Pros are seen, suggesting that these cells are two vmd1a neurons and two pIIIb cells. These data indicate that the pIIa cell death is inhibited and that the pIIa cell is transformed into a pIIb-like cell (Orgogozo, 2002).

Numb is known to function by antagonizing Notch activity. This therefore suggests that Notch promotes cell death in the vmd1a lineage and that Numb blocks this activity of Notch. Unfortunately, the strong effect of Notch loss-of-function alleles on the selection of the vmd1a pI cell means that it was not possible to test directly whether Notch is required for cell death in the vmd1a lineage. Therefore the conditional Notch^ts1 allele was used. However, when Notch^ts1 embryos are shifted to a restrictive temperature (31°C) soon after the specification of the vmd1a pI cell (i.e., at 13-14.5 hours after egg laying at 19°C), no significant reduction was seen in the number of rpr- or grim-expressing pIIa cells. A stronger Notch^ts1/Notch^55e11 combination causes the appearance of additional vmd1a pI cells even at the permissive temperature (19°C). It is therefore not possible to determine whether an increase in the number of rpr- or grim-negative cells results from a lack of Notch-dependent apoptosis or from an excess of vmd1a pI cells due to reduced Notch signaling during lateral inhibition (Orgogozo, 2002).

Therefore a test was performed to see whether an activated form of Notch, Nintra, can promote the death of the pIIb cell when expressed around the time of the vmd1a pI cell division. In 6% of the segments from embryos in which at least one segment shows a dividing vp1 pIIb cell, rpr or grim transcripts accumulate in both vmd1a pI daughter cells. In other segments, a single Cut-positive cell remains at the vmd1a position and accumulates rpr or grim. These expression patterns are not seen in heat-shocked control embryos. Importantly, these observations are similar to those made in numb mutant embryos. Thus, both loss of numb activity and ectopic Notch signaling lead to transcriptional activation of pro-apoptotic genes in the pIIb cell. Finally, a similar effect of Nintra on rpr and grim expression is seen in the vmd1a pIIb daughter cells when Nintra expression was induced at a later stage, i.e., when the vmd1a pIIb cell is dividing. Together, these results indicate that Notch signaling is sufficient to promote cell death in the vmd1a lineage (Orgogozo, 2002).

In summary, the lineage generating the vmd1a neuron has been described. This lineage is composed of two asymmetric divisions following which one daughter cell undergoes apoptosis. These two binary cell death decisions are regulated by the unequal segregation of Numb at mitosis. Therefore, the data provide the first experimental evidence that alternative cell death decision can be regulated by the unequal segregation of a cell fate determinant. The conserved role of Numb and Notch in neuronal specification in flies and vertebrates suggests that Numb-mediated inhibition of Notch may play a similar role in regulating cell death decisions in vertebrates (Orgogozo, 2002).

Segment-specific prevention of pioneer neuron apoptosis by cell-autonomous, postmitotic Hox gene activity

In vertebrates, neurons often undergo apoptosis after differentiating and extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. The Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role in guiding follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously by repressing the RHG-motif cell death activators reaper and grim. These results identify the first clear case of a cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox positional information to differences in neuronal architecture along the anteroposterior axis by the selective elimination of mature neurons (Miguel-Aliaga, 2004).

How does Abd-B prevent the function of RHG-motif genes? It is likely that Abd-B prevents pioneer neuron apoptosis by repressing the transcription of, at least, rpr and grim. This idea is supported by four facts: (1) the H99 deletion is epistatic to (functions downstream of) Abd-B; (2) Abd-B is a transcription factor; (3) rpr^GAL4 is activated posteriorly in Abd-Bm mutants; (4) when misexpressed postmitotically, Abd-B can fully rescue both types of pioneer neurons. Given that loss of rpr is critical for anterior dMP2 survival, whereas loss of grim is critical for anterior MP1s, Abd-B must prevent the expression of at least these two cell death activators (Miguel-Aliaga, 2004).

In the developing vertebrate neural tube, a number of studies have shown that Hox genes are critical for AP organization and for proper neuronal specification. Although their action may be largely confined to progenitor cells, recent studies have revealed that Hox genes can also act to control the identity of early postmitotic neurons. In the light of the current findings, it will be of interest to determine if selective, Hox-dependent elimination of mature neurons gives rise to differences in motor neuron numbers along the AP axis of the vertebrate spinal cord. Increased apoptosis of postmitotic motor neurons has been observed in mouse mutants lacking Hoxc-8, one of the vertebrate homologues of abd-A. This may be the result of the aberrant connectivity pattern of Hoxc-8-deficient motor neurons, which would restrict their access to target-derived neurotrophic factors. However, this increase in cell death is also consistent with the possibility that Hoxc-8 normally acts to prevent apoptosis of postmitotic neurons in its expression domain (Miguel-Aliaga, 2004).

The results contrast with the previous finding that Abd-B appears to activate rpr transcription to regulate segment boundary formation in the posterior region of early Drosophila embryos. Decreased apoptosis has also been observed in mouse mutants lacking Hoxb13, one of the vertebrate homologues of Abd-B. It has previously been shown that the target functions of Hox genes are highly dependent on cellular context, and the regulation of apoptosis appears to be no exception. This context dependence may not be unique to the Abd-B gene. abd-A has been previously reported to activate apoptosis in post-embryonic neuroblasts during normal development. When Antp and Ubx were misexpressed in these neuroblasts, they too were able to trigger apoptosis. In contrast, none of these genes acted in a pro-apoptotic manner in the current study. It is, therefore, conceivable that the pro-apoptotic function of Hox genes is confined to progenitors, at least in the nervous system. Alternatively, or additionally, availability of certain cofactors may determine whether a Hox gene activates or represses transcription of pro-apoptotic genes in a specific cell (Miguel-Aliaga, 2004).

In addition to their dependence on cellular context, specific Hox proteins may control pro-apoptotic genes differently. Abd-B and its vertebrate homologues share several properties that distinguish them from other Hox proteins, such as the absence of a hexapeptide motif and a preference for a different DNA core sequence. Together, these differences may confer unique transcriptional properties on proteins of the Abd-B family, and may explain why Abd-B is the only Hox protein capable of fully rescuing anterior pioneer neurons. The finding that Abd-B is the only Hox gene that was unable to rescue the embryonic brain phenotypes of Drosophila mutants for the Hox gene labial is consistent with this idea (Miguel-Aliaga, 2004).

Is the cellular control of Hox gene expression functionally relevant? The results show that while Hox genes are broadly expressed within their domains, they are largely absent from certain cell populations; at stage 16, few glial cells express Hox genes in the VNC. Since many Drosophila neuroblasts give rise to both neurons and glia, it is possible that Hox gene expression is actively suppressed by factors promoting glial fate. Alternatively, an initial wave of Hox expression in progenitors could be followed by a second, neuron-specific re-activation of Hox expression. In any case, it will be of interest to identify the molecular mechanism by which Hox gene expression is confined to specific populations of postmitotic cells in the nervous system (Miguel-Aliaga, 2004).

While cellular context may determine whether a Hox gene acts in a pro- or anti-apoptotic manner, apoptosis of specific cells within a Hox expression domain may also be achieved by differential Hox gene expression. For example, while Abd-A is broadly expressed in abdominal segments during larval stages, it is absent from post-embryonic neuroblasts. However, at the last larval instar, a neuroblast-specific pulse of abd-A results in the activation of the cell death program in these cells. Similarly, and given the novel role for Hox proteins in the apoptosis and differentiation of postmitotic neurons, the expression of Hox genes in specific postmitotic neurons is likely to be of functional significance. Together, these findings are not consistent with the view that Hox genes solely function as 'segment identity' factors specifying global properties of the segments in which they are active. Instead, they lend functional support to the proposal that Hox genes are required for a number of decisions taken at the cellular level (Miguel-Aliaga, 2004).

The combined activity of RHG-motif genes is critical to the initiation of all cell death in the Drosophila embryo. These genes act in an additive manner. However, not all cell death activators are simultaneously expressed in every cell fated to die, and their specific expression patterns do not always overlap. Therefore, it is likely that they are differentially regulated by specific developmental signals. While Abd-B acts to repress rpr and grim function in posterior pioneer neurons, the developmental stimulus activating their expression in these neurons throughout the cord is currently unknown. Three developmental signals are known to regulate the function of RHG-motif genes in the Drosophila nervous system. The insect hormone ecdysone appears to be important for blocking cell death of certain peptidergic neurons during metamorphosis. However, the ecdysone-receptor complex has also been shown to promote cell death by activating rpr transcription in other tissues during Drosophila metamorphosis. While an embryonic ecdysone pulse occurs around the time when pioneer neurons die, preliminary experiments have failed to lend any support to an ecdysone-dependent activation of apoptosis in these neurons. The EGF-receptor/Ras/MAPK pathway has been shown to phosphorylate Hid protein, thereby preventing apoptosis of midline glial cells. However, neither Rpr nor Grim appear to be regulated in this fashion, and this model would not address the specific transcriptional activation of these genes in pioneer neurons. Lastly, Notch signaling has been described as resulting in both activation and inhibition of apoptosis. In Drosophila, recent studies have revealed that Notch can act cell-autonomously to induce apoptosis during final mitotic divisions both in the central and peripheral nervous systems. Although this Notch-induced developmental apoptosis is prevented in H99 mutant embryos, the molecular mechanisms by which activated Notch signaling results in the activation of IAP inhibitors are still unknown. Nevertheless, Notch signaling is unlikely to be relevant to dMP2 death, since it is not active in dMP2 neurons. It is, therefore, likely that an as yet unidentified factor is responsible for the activation of the apoptotic machinery in pioneer neurons. This factor could be Odd-skipped, given its specific expression in dMP2 and MP1 neurons. Because of the early role of odd in embryonic patterning, its possible postmitotic function in these neurons cannot be addressed using the currently available odd mutants (Miguel-Aliaga, 2004).

Developmental apoptosis in invertebrate embryos typically occurs shortly after cells are generated. In Drosophila, this has often precluded the identification of dying cells until apoptosis has been genetically prevented. Consequently, progress in identification of the mechanisms controlling apoptosis has been relatively slow, and little is known about the upstream pathways that initiate cell death in specific tissues or lineages. Furthermore, in the Drosophila VNC, studies have shown that apoptotic corpses are engulfed by glia, transported to the dorsal surface of the VNC and transferred to macrophages for final destruction. The molecular genetic mechanisms underlying this intriguing series of events are only just beginning to be unraveled. The identification of a late apoptotic event in two of the best-studied and least complex lineages in the Drosophila CNS, as well as the characterization of the dMP2-GAL4 line, should contribute to the elucidation of the mechanisms involved in both the developmental initiation and execution of apoptosis (Miguel-Aliaga, 2004).

Apical deficiency triggers JNK-dependent apoptosis in the embryonic epidermis of Drosophila

Epithelial homeostasis and the avoidance of diseases such as cancer require the elimination of defective cells by apoptosis. This study investigated how loss of apical determinants triggers apoptosis in the embryonic epidermis of Drosophila. Transcriptional profiling and in situ hybridisation show that JNK signalling is upregulated in mutants lacking Crumbs or other apical determinants. This leads to transcriptional activation of the pro-apoptotic gene reaper and to apoptosis. Suppression of JNK signalling by overexpression of Puckered, a feedback inhibitor of the pathway, prevents reaper upregulation and apoptosis. Moreover, removal of endogenous Puckered leads to ectopic reaper expression. Importantly, disruption of the basolateral domain in the embryonic epidermis does not trigger JNK signalling or apoptosis. It is suggested that apical, not basolateral, integrity could be intrinsically required for the survival of epithelial cells. In apically deficient embryos, JNK signalling is activated throughout the epidermis. Yet, in the dorsal region, reaper expression is not activated and cells survive. One characteristic of these surviving cells is that they retain discernible adherens junctions despite the apical deficit. It is suggested that junctional integrity could restrain the pro-apoptotic influence of JNK signalling (Kolahgar, 2011).

This study has shown that apical, but not basolateral, disruption leads to apoptosis in a developing epithelium. It was also showed that JNK signalling is a key intermediate in the signal transduction mechanism that triggers apoptosis in response to the loss of apical determinants. Apical disruption leads to activation of JNK signalling, which in turn activates transcription of the pro-apoptotic gene rpr. Moreover, rpr expression is not activated in apically disrupted embryos that are prevented from activating JNK signalling. Interestingly, in bicoid-deficient embryos and other segmentation mutants, cell fate misspecification requires activation of a different pro-apoptotic gene, hid. Therefore, distinct quality control pathways might exist to ensure that different forms of defective cells are removed from developing epithelia. JNK signalling has been shown to mediate apoptosis in a variety of other situations, including after DNA damage. However, JNK signalling does not necessarily cause apoptosis. Indeed, this pathway modulates many other cell activities, such as proliferation, differentiation and morphogenesis. What conditions determine whether JNK triggers apoptosis or not is an important issue. Another obvious question raised by the current findings concerns the nature of the mechanism that triggers JNK signalling following the loss of apical determinants (Kolahgar, 2011).

This study has shown that, in the embryonic epidermis, JNK signalling is activated by the loss of apical, not basolateral, determinants. In fact, reduction of lgl activity prevents JNK activation in crb mutant embryos. Similarly, Scrib knockdown prevents JNK activity in the mouse mammary epithelium, suggesting that the loss of the basolateral domain could have a general anti-JNK (and perhaps anti-apoptotic) activity. Although JNK activation has been documented in tissues that lack a basolateral determinant, it is suggested that this might be an indirect consequence of cell competition, which triggers JNK signalling, or of the specific experimental conditions (partial reduction of Puc activity). Overall, the results suggest the existence of an apical domain-dependent activity that modulates JNK signalling in the embryonic epidermis of Drosophila. This activity could be similar to that postulated to be at work in cultured MDCK cells, but is likely to be distinct from the process that leads to apoptosis in response to mosaic disruption of the basolateral domain in imaginal discs (Kolahgar, 2011).

The mechanism underlying the activation of JNK signalling by loss of apical determinants remains unknown. For example, it is uncertain at this point whether there is an apically localised activity that directly modulates JNK signalling or whether a more indirect route is at work (paths 1 and 2 respectively; see Signalling upstream and downstream of JNK). Since apical organisation is required for the establishment of adherens junctions, it is conceivable that the effect of apical disruption on JNK signalling is mediated by junctional disruption. This possibility is compatible with the absence of ectopic JNK activation in basolateral mutants, in which E-cadherin remains localised to patches at the cell surfac. However, one would have to invoke that slight junctional disruption is sufficient to trigger JNK signalling, as this pathway is upregulated in the dorsal region of crb mutants, where the extent of junctional disruption is relatively mild. It has not been possible to discriminate between paths 1 and 2, partly because of the current difficulty in eliminating adherens junctions from early Drosophila embryos. Future work will require novel means of interfering specifically with adherens junctions. Considering the lack of involvement of Egr, it will also be necessary to identify the upstream components of JNK signalling that respond to epithelial disruption (Kolahgar, 2011).

Overexpression of Puc, a feedback inhibitor of JNK signalling, prevents apoptosis in the ventral epidermis of crb embryos. This is clear evidence that JNK signalling is required for apical deficit to trigger apoptosis. However, it is well established that JNK signalling does not necessarily lead to apoptosis. This is particularly well illustrated by the situation at the dorsal edge of wild-type embryos, where JNK is highly active without triggering apoptosis. Moreover, in crb mutants, a 6- to 10-cell-wide band of dorsal tissue is refractory to the pro-apoptotic influence of JNK signalling. Therefore, additional conditions must be met for JNK signalling to activate rpr expression and trigger apoptosis. This study has identified two situations when refractory cells succumb to the pressure of JNK signalling (see Signalling upstream and downstream of JNK). One involves the reduction of Puc and the other the removal of zygotic E-cadherin activity. The first situation suggests that endogenous Puc can limit the ability of JNK to activate rpr expression and trigger apoptosis. The important role of Puc in preventing cell death is also highlighted by the extensive apoptosis seen in embryos lacking both maternal and zygotic Puc activity. Puc could act solely by limiting the extent of JNK signalling, thus preventing the very high level of signalling required for rpr expression. Alternatively, or in addition, Puc could have an activity that specifically prevents certain genes, such as rpr, from being spuriously activated. In any case, it is likely that the regulatory relationships between JNK, Puc and apoptosis are influenced by the cellular context (e.g., the state of adherens junctions (Kolahgar, 2011).

JNK signalling triggers rpr expression (and apoptosis) more readily if adherens junctions are weakened or disrupted. Therefore, junctional integrity could also protect epithelial cells from the pro-apoptotic effects of JNK signalling. Dorsoventral differences in junctional integrity and remodelling have been noted in the embryonic epidermis of Drosophila and these might explain why these two regions respond differently to the loss of crb. The results suggest that residual junctional integrity in the dorsal epidermis prevents JNK signalling from activating rpr expression. It is conceivable that a protective signal emanates from adherens junctions. Alternatively, junctional disruption could interfere with the ability of Puc to rein in the effect of JNK signalling on rpr expression. Although differential junctional dynamics between the dorsal and ventral epidermis could determine the propensity to undergo apoptosis, the possibility cannot be excluded that other dorsoventral determinants are at work too (Kolahgar, 2011).

This study has shown that loss of apical polarity leads to apoptosis by activating JNK signalling and causing junctional disruption. It is expected that this response, which is readily detectable in the crb mutant condition, might reflect a process that ensures the removal of abnormal and damaged cells during epithelial homeostasis. It is hoped that understanding the machinery that links epithelial disruption to JNK signalling and the transcription of pro-apoptotic genes will suggest means of reactivating this pathway in pathological situations (Kolahgar, 2011).

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, binding regions were identified for the Hox TF Deformed (Dfd) in the Drosophila genome. This analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. It was also found that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, these results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo (Sorge, 2012).

In order to quantitatively identify genomic regions bound by the Hox TF Dfd in Drosophila, two complementing approaches were employed: ChIP-seq, which has been successfully applied previously to identify stage- and tissue-specific enhancer activities, and computational detection of clusters of TF binding sequences, which allows the identification of cis-regulatory modules irrespective of temporal and spatial context. To generate genome-wide maps of Dfd binding in vivo, ChIP was performed using stage 10-12 Drosophila embryos and a Dfd-specific antibod. Stage-independent in silico Dfd-specific Hox response elements (HREs) were identified by searching for clusters of conserved Dfd binding motifs, as defined by a position weight matrix (PWM), in the non-coding regions of the genomes of 12 distinct Drosophila species. By applying both approaches, 4526 genomic regions containing clusters of Dfd binding sites and 1079 Dfd ChIP-seq enrichment peaks were identified, including two out of the three well-characterized Dfd-HREs, namely rpr-4S3 and Dfd-EAE. To study the regulatory capacity of novel in silico and ChIP-seq detected HREs, cell culture-based enhancer assays were performed for 11 randomly selected HREs, and it was found that reporter expression driven by the identified genomic regions was in all cases dependent on Dfd binding. In vivo activity was tested of 21 arbitrarily selected enhancers in transgenic reporter lines, revealing that 7 out of 11 ChIP-identified and 5 out of 10 in silico-predicted Dfd-HREs recapitulate the spatio-temporal expression of adjacent genes). Most importantly, it was possible to demonstrate Dfd-dependent regulation of both transgenic reporter expression and endogenous gene expression, suggesting that they are bona fide direct Dfd target genes. Thus, the identified Dfd-HREs represent a data set of biologically relevant regulatory regions and an excellent resource to unravel sequence features within Hox responsive enhancers that might be essential for the highly selective Hox target gene regulation (Sorge, 2012).

Transcriptional regulation in many cases relies on the assembly of regulatory protein complexes mediated by closely spaced TF binding sites within a cis-regulatory module and previous studies have shown that Hox proteins employ this mechanism to control target gene activity in small subsets of cells. The novel HREs were systematically scanned for TF binding motifs appearing in close proximity to Dfd binding sites. Using a statistical test for pair-wise distance distributions, w11 overrepresented DNA motifs for known TFs were found adjoining to Dfd binding sites with 5 of the motifs occurring in both the ChIP-seq and in silico-identified Dfd-HREs. When the expression patterns of six of these transcriptional regulators known to bind to the 11 motifs that were identified were examined, colocalization with Dfd was found in different sub-populations of cells in all cases. Colocalization was already known for two TFs, whose binding sites were coupled to Dfd motifs, including Extradenticle (Exd) , which is known to cooperatively bind with Hox proteins to DNA and thereby increase Hox DNA-binding selectivity. It was next asked whether the short-distance arrangements in Dfd-HREs are of biological relevance and translated into the regulation of similar classes of target genes. To this end, the overrepresentation was statistically tested of expression and biological terms of genes associated with HREs harbouring specific combinations of Dfd and close-by motifs. This analysis revealed that only those Dfd-HREs with short distance intervals between the Dfd and adjacent motifs were coupled to similar gene classes, while random distance intervals did not show any correlation. Strikingly, genes associated with specific short-distance HREs had similar expression and functional annotations as the TFs interacting with the Hox adjoining motifs, suggesting that time and place of Hox action is dictated by spatio-temporally restricted co-regulators. Support for this hypothesis stems from the observation that one of the close-distance partners, Optix, regulates similar processes as Dfd, since Dfd and Optix mutants displayed comparable morphological defects in the head region, such as the absence of mouth hooks, a maxillary segment-derived structure known to be specified by Dfd. In addition, one of the genes associated with a Dfd-Optix HRE, the known Dfd target gene reaper (rpr), is expressed in the ventral epidermis primordium as predicted by its HRE architecture, and regulated by Dfd and Optix in ventral-maxillary cells, which also express these factors. A cell-culture assay using the well-established Dfd responsive module responsible for rpr expression in a few anterior-maxillary cells, the rpr-4S3 Dfd-HRE, with wild-type or mutated Dfd binding sites or reduction of Dfd levels by RNAi confirmed the requirement for simultaneous activity of Dfd and Optix on the rpr-4S3 Dfd-HRE for strong reporter gene induction. Optix binding to the rpr-4S3 Dfd-HRE was additionally confirmed by electrophoretic mobility shift assay (EMSA) experiments. Furthermore, transgenic reporter expression induced by the rpr-4S3 Dfd-HRE was lost in Optix mutant embryos or when the Optix binding sites were mutated. These results demonstrate that Optix, one of the newly identified factors, is a Dfd co-regulator required for proper regulation of the important Hox target gene rpr (Sorge, 2012).

Whether The precise spacing between Hox and adjacent binding sites plays a role for enhancer activity was explored. The rpr-4S3 Dfd HRE, which induces gene expression in a few anterior-maxillary cells, has previously been shown to be under the control of Dfd and Glial cells missing (Gcm), a Dfd co-regulator also identified in this study. Dfd and Gcm as well as Optix binding sites within the rpr-4S3 HRE are directly adjacent to each other, thus a 5- and 10-bp spacer was introduced to interfere with potential interactions of the proteins on the enhancer. In all cases, reporter gene expression was strongly reduced or completely abolished, showing that the close-distance arrangements between Dfd and Gcm as well as Dfd and Optix are required for the in vivo activity of the rpr-4S3 enhancer (Sorge, 2012).

While the results regarding the close-distance arrangement of Dfd and Gcm binding sites suggested the formation of a Dfd-Gcm protein complex, like in the case of Dfd and Exd, only independent binding of the two proteins to the rpr-4S3 enhancer was observed in EMSA experiments , supporting the idea of Hox proteins collaborating with other TFs on target HREs in the absence of physical contact. It has been shown before that Hox proteins together with other TFs that bind in the immediate vicinity recruit non-DNA binding cofactors to HREs. To test if such factors could interact with Dfd and the newly identified short distance binding TFs, the modENCODE data set was scanned and it was found that dCBP/Nej, a member of the CBP/p300 family of transcriptional co-activators bearing acetyltransferase activity, binds to the rpr-4S3 enhancer in vivo. As nej has been previously reported to genetically interact with Dfd, its function was examined in Dfd/Gcm-mediated transcriptional activation. Both factors, Dfd and Gcm, are required for transcriptional activation, since expression of Gcm in Drosophila D.Mel-2 cells, which have basal levels of Dfd activity, resulted in strong induction of reporter gene expression, while abolishing Dfd binding to the rpr-4S3 HRE by mutating all Dfd binding sites or by reducing Dfd protein levels in D.Mel-2 cells using RNAi, strongly reduced reporter gene expression in the presence of Gcm. Strikingly, Dfd- and Gcm-mediated reporter gene expression was strongly reduced in nej dsRNA-treated cells, whereas inhibition of protein deacetylation by Trichostatin A (TSA0) restored reporter gene expression. Consistently, rpr expression was abolished in nej mutant embryos. These results demonstrate that dCBP/Nej-mediated protein acetylation/histone modification is important for the combined activity of Dfd and Gcm on the rpr-4S3 HRE. While it was not possible to demonstrate that nej physically interacts with Dfd protein using various assays, EMSA experiments show that nej interacts with Gcm. Furthermore, acetylation of transiently transfected Gcm was detected in cultured Drosophila cells. Acetylation of Gcm is dependent on Nej, as it was reduced upon RNAi-mediated downregulation of nej. These results are consistent with published work demonstrating that in human cells CBP interacts with Gcma, resulting in its acetylation and stimulation of its transcriptional activity. Since about 10% of all Dfd and nej in vivo genomic binding events during embryonic stages 10-12 overlap, the functional interaction of Dfd and nej observed at the rpr locus does not seem an exception. This finding suggests that the interaction of co-activators (and co-repressors) with Hox proteins and close distance binding TFs on enhancer modules could be a commonly used mechanism to achieve highly specific spatio-temporal control of target gene activity. In this scenario, Hox proteins would control downstream genes by direct transcriptional and/or epigenetic regulation depending on HRE composition and thus cofactor identity and recruitment (Sorge, 2012)

Despite very similar DNA binding behaviour in vitro, Hox proteins regulate distinct morphological features along the anterior-posterior body axis in animal systems. To elucidate the mechanistic basis for the differences in their regulatory properties, Dfd-HREs identified in this study were compared to genomic regions bound by the Hox TF Ultrabithorax (Ubx) at identical developmental stages, as identified by the modENCODE consortium. Searching for overrepresented DNA motifs in both enriched ChIP regions, it was found that Dfd and Ubx bind to identical DNA sequences in vivo, reminiscent to in vitro systems. However, individual binding motifs seem to play only a minor role for Hox binding site selection in vivo, since this analysis revealed that Dfd and Ubx exclusively interact with non-overlapping genomic regions in embryonic stages 9-12. Consequently, Dfd- and Ubx-HREs were found to be associated with distinct classes of genes, revealing that genes with roles in the epidermis are primarily under the control of Dfd at the analysed embryonic stages while genes with mesoderm-related functions are predominantly regulated by Ubx. Consistently, it was found that the expression of tartan (trn), one of the genes associated with a Dfd-HRE, is regulated exclusively by Dfd, but not by Ubx, in epidermal cells, while parcas (pcs), one of the genes linked to a Ubx-HRE is under the selective control of Ubx in mesodermal cells. Furthermore, only Ubx-HREs were found to substantially overlap with cis-regulatory elements stage specifically bound by the mesoderm-specifying TFs Myocyte enhancer factor 2 (Mef2), Twist and Tinman. In contrast, the common ability of both Dfd and Ubx to regulate genes involved in nervous system development was underlined by comparable representations of binding motifs for the neuronal-specifying TFs Asense, Deadpan and Snail in Dfd- and Ubx-HREs (Sorge, 2012).

Strikingly, the basic design principles of Dfd- and Ubx-HREs were found to be similar: like in Dfd-HREs, six binding motifs for known TFs were located adjacent to Ubx binding sites and colocalization studies showed that they are expressed in subsets of Ubx-positive cells. Again, Ubx binding sites and motifs for potential co-regulators occurred most frequently in specific short intervals and only those Ubx-HREs with the preferred distance were associated with specific gene classes. This analysis also revealed that four of the six short-distance motifs were specific for Ubx-HREs, which is consistent with the data showing that Hox proteins interact with different and spatially restricted co-regulators to control target gene expression in selected cells. Importantly, in the cases of the close-distance motifs detected in both HREs, namely the binding sites for the TFs Ladybird early (Lbe) and Cut (Ct), the associated target genes were also expressed in non-overlapping tissues. This raised the question of how different Hox proteins can act on distinct target genes, even when their target HREs exhibit similar binding site compositions including short-distance arrangements. Since Lbe is active in both mesodermal and epidermal cells, one Dfd-Lbe and one Ubx-Lbe HRE was exemplarily analysed, and binding of Lbe protein was confirmed to both HREs by EMSAs. As predicted by the presence of Lbe binding sequences. Complex formation between the Hox protein and Lbe was observed in the case of Ubx and Lbe while Dfd and Lbe interact independently with the Dfd-Lbe HRE, indicating that the two Hox proteins employ different mechanisms for binding to the selected HREs. Lbe interaction with the Dfd-Lbe and Ubx-Lbe HREs is essential for in vivo activity, since in both cases ectopic reporter gene expression was observed when Lbe binding sites were mutated. Even more important, reporter gene expression was specifically changed only in segments in which either Dfd or Ubx is active, meaning in the case of the Dfd-Lbe HRE in maxillary cells and in the case of the Ubx-Lbe HRE in abdominal segments A1-A7. Taken together, these results demonstrate that the combined activity of Lbe and the Hox proteins Dfd or Ubx on selected HREs is critical for the precise spatiotemporal and segment-specific control of HRE activity. It was next asked whether additional (DNA- and non-DNA-binding) factors contribute to the predicted cell type-specific expression of the Dfd-Lbe and Ubx-Lbe HREs. Using the Drosophila Interactions Database (DroID; Murali, 2011) and published genome-wide DNA binding studies a search was carried out for unique Dfd-lbe and Ubx-lbe interactors. It was discovered that almost 20% of all Ubx-Lbe HREs but none of the Dfd-Lbe HREs were found to interact with the mesoderm-specifying factor Mef2 in vivo, while H3K9me3 histone marks, which are mediated by one of the unique Dfd-lbe interactors, Enhancer of zeste E(z), are enriched only within Dfd-Lbe HREs. Interestingly, E(z) modifies chromatin also by trimethylating H3K27 residues, a histone mark highly enriched at the genomic region spanning the ChIP-detected Dfd-Lbe HRE. Consistent with the repressive function of this histone modification, loss of Lbe binding to the Dfd-Lbe HRE results in ectopic reporter gene expression, suggesting that Lbe (and Dfd) recruits E(z) to the Dfd-Lbe HRE for cell type-specific target gene repression (Sorge, 2012).

Taken together, these results demonstrate that Hox proteins interact with different regulatory proteins on HREs, which allows them to differentially regulate their target genes despite their similar DNA binding properties. The fact that these interactions occur only in a few cells for a short period of time is very likely one of the major reasons why the identification of factors conferring regulatory precision and specificity to Hox function has met with little success so far (Sorge, 2012).

This study, has identified crucial features of HREs, which are essential for cell type-specific regulation of Hox target genes in vivo. In addition to motif composition the exact spatial arrangement of TF binding elements is critical to translate Dfd function into transcriptional regulation in vivo. These architectural features of Dfd-HREs alone accurately predict target gene function and expression patterns. Furthermore, it was found that epigenetic regulators bind to HREs on a genome-wide scale, suggesting that they generally collaborate with Hox proteins to achieve stable target gene regulation. This is in line with recent findings showing that chromatin modifications at enhancers strongly correlate with functional enhancer activity and tissue specificity. By comparing HREs regulated by Dfd and Ubx, two different Hox proteins with different embryonic regulatory specificities, this study shows that while similar design principles apply, specificity is encoded by distinct sets of co-occurring DNA motifs. Due to the highly dynamic regulatory output of Hox TFs in space and time, cell type-specific approaches are required in future to elucidate all relevant aspects of Hox-chromatin and Hox-cofactor interactions (Sorge, 2012).

A p53 enhancer region regulates target genes through chromatin conformations in cis and in trans

How a p53 enhancer transmits regulatory information was examined in vivo. Using genetic ablation together with digital chromosome conformation capture and fluorescent in situ hybridization, this study found that a Drosophila p53 enhancer region (referred to as the p53 response element [p53RE]) physically contacts targets in cis and across the centromere to control stress-responsive transcription at these sites. Furthermore, when placed at ectopic genomic positions, fragments spanning this element re-established chromatin contacts and partially restore target gene regulation to mutants lacking the native p53RE. Therefore, a defined p53 enhancer region is sufficient for long-range chromatin interactions that enable multigenic regulation (Link, 2013).

This study present in vivo functional evidence that a single enhancer region can specify regulation of multiple targets in cis and in trans. Using tailored deletions, it was found that a p53 regulatory element controlled stimulus-dependent induction of multiple genes, with effects on targets that range from 4 kb to 330 kb throughout the Drosophila Reaper region. In these studies, the p53RE also regulated xrp1, a genetically unlinked target residing across the centromere. Furthermore, when transplanted to ectopic locations, contacts with target sites were re-established and regulation of some target genes was restored. Together, these functional studies offer compelling evidence that an enhancer transmits regulatory activity in trans through direct physical contact (Link, 2013).

In principle, long-range regulation of xrp1 by the native p53RE could involve local induction of an activator that subsequently induces distant genes, but this type of expression cascade would not explain the data. First, no correlation exists between the timing of RIPD gene induction and proximity to the p53RE. Second, cis targets in the Reaper interval encode products with no known function in the nucleus or in transcription. Third, conventional expression cascades would not account for the restoration of regulation and contacts by a transgenic rescue fragment. Therefore, the idea is favored that long-range regulation by the p53RE involves chromosomal architectures that link this enhancer to target genes regardless of whether they are in cis or in trans (Link, 2013).

Using either 3C or direct visualization, suggestive chromatin links between enhancers and distant genomic sites in trans have been reported. Few have been genetically tested, and, where functionally studied, detectable effects were not seen. the current finding that productive looping contacts can be assembled from a foreign site suggests that determinants of long-range chromatin interactions are modular and probably specified through sequence motifs, secondary structures, and epigenetic features that occur in vivo. It is further noted that the presence of contacts is not sufficient for target induction. For example, despite loops between the native p53RE and sites near grim or contacts between the ectopic p53RE and sites near rpr and skl, transcriptional induction was not seen. Therefore, elements that map outside of the rescue fragment or constraints imposed by flanking chromatin may also be important (Link, 2013).

Given that p53 enhancers in both flies and humans share a common sequence motif, mechanisms by which these response elements form long-range interactions in trans may be conserved. It will be interesting to see whether other enhancer regions share this property. Likewise, it will be important to determine whether these contacts are mediated through complexes involving proteins such as Cohesin, Mediator, Ldb1, Polycomb, or CTCF. If broadly generalized, the precedent established here could offer a framework that helps explain genetic disease alleles mapping to noncoding sequences (Link, 2013).

The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development

A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or 'true' joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. This study shows that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. A dedicated dys cis-regulatory module was identified that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis (Cordoba, 2014: PubMed).

Targets of Activity

To discover whether expression of apoptosis activators reaper, grim and hid triggers the accumulation of Death related ced-3/Nedd2-like protein (DREDD) mRNA, the three apoptosis activators were ectopically expressed in mesoderm, and the expression of DREDD mRNA examined. Expression of the apoptosis activators triggers excessive apoptosis in mesoderm. During stage 13 and beyond, DREDD mRNA is not widely expressed in the developing musculature in wild-type flies. However, when misexpression of each of the death activators is directed to these tissues, prominent levels of ectopic DREDD mRNA are detected. Expression of grim in the ectoderm also results in DREDD mRNA accumulation. DREDD mRNA accumulation has also been examined in embryos homozygous for crumbs (crb). In crb mutants, reaper is ectopically expressed in the disorganized epidermis. As anticipated, ectopic accumulation of DREDD mRNA is found scattered throughout the ectoderm in crb embryos, coincident with widespread patterns of rpr expression. Perhaps the most compelling evidence for a direct role for Dredd in apoptosis comes from an examination of accumulation of DREDD mRNA in embryos carrying a homozygous deletion of the entire reaper region (mutated for rpr, hid, and grim). No apoptosis occurs in these deletion mutants. The selective accumulation of DREDD mRNA fails to occur in these mutants. This is the first report of a molecular activity that is completely blocked by the absence of H99-associated signaling (Chen, 1998).

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. An overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoke apoptogenic cytochrome c display and, surprisingly, caspase activity is necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation is triggered by mitochondria from apoptotic cells but identical preparations from healthy cells are inactive. These observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases (Varkey, 1999).

Two cytochrome c genes, DC4 and DC3, have been described in Drosophila studies at the level of protein and at the level of RNA that suggest that DC4 (which shows >86% identity with its rat counterpart) is either the predominant or only form of actively expressed product. An existing panel of mAbs, directed against mammalian versions of cytochrome c, was screened in a search for possible probes for in situ analyses of the fly counterpart. Two mAbs, 6H2 and 2G8, recognize Drosophila cytochrome c. Both antibodies detected a doublet that comigrates with mammalian cytochrome c at ~13 kD. While mAb 2G8 preferentially precipitates the upper band, mAb 6H2 has about equal affinity for both forms of cytochrome c. No obvious correlation between the relative abundance of the two cytochrome c bands and apoptosis is observed. These bands clearly represent distinct cytochrome c species. Although the biochemical nature of these different forms is unresolved, mAb 2G8 preferentially recognizes the higher molecular weight form of the doublet. This product occurs in relatively small amounts that are not overtly affected by the extent of apoptosis in the cultures (Varkey, 1999).

Between stages 11 and 13 of Drosophila oogenesis, programmed cell death eliminates nurse cells which nourish the developing egg. The apoptotic nature of nurse cell death is indicated by two distinct markers: acridine orange staining and TUNEL labeling. These readily identifiable cells offer a unique opportunity to examine pre-apoptotic events before their eventual demise. To directly demonstrate the involvement of cytochrome c during apoptosis, Drosophila ovaries were stained with the anti-cytochrome c mAbs described above and egg chambers at all stages of development were analyzed. Only nurse cells at stage 10B exhibit pronounced cytochrome c immunoreactivity distributed as characteristically punctate labeling of the cytoplasm in a pattern consistent with localization to mitochondria. To determine the chronology of cytochrome c display, relative to other apoptotic changes, the onset of mAb binding was compared with other degenerative changes known to occur in these cells. Though nurse cells at stage 10B show pronounced exposure of the epitope for mAb 2G8, no signs of apoptosis are apparent in either the cytoplasm or the nuclei of these cells. By stages 12-13 (at least 0.5 to 4 h later) the nuclei of the dying nurse cells adopt characteristic apoptotic features as evidenced by the TUNEL assay and acridine orange staining. These results demonstrate that cytochrome c display precedes overt signs of apoptosis in intact organs (Varkey, 1999).

Previous studies on Drosophila SL2 cells have shown that conditional expression of rpr or grim triggers apoptosis in cultured cells and in transgenic animals. Transiently transfected SL2 cells were induced for rpr or grim and, at various time intervals after induction, the preparations were examined for cytochrome c immunoreactivity with mAb 2G8. Apoptotic cultures exhibit profound staining with the antibody. To test the possibility that cytochrome c might be released into the cytosol during apoptosis, healthy SL2 cells and apoptotic rpr- or grim-expressing cells were fractionated, and assayed for cytochrome c in the mitochondrial and cytosolic fractions. Surprisingly, these cells showed no difference in cytochrome c distribution and no evidence was found for the transit of cytochrome c to the cytosol as a correlate to apoptosis. Biochemical data indicating retention of cytochrome c in mitochondria during apoptosis is consistent with cytological studies. These observations indicate that appreciable efflux of cytochrome c from mitochondria does not occur during apoptosis in Drosophila cells (Varkey, 1999).

Mitochondria isolated from apoptotic cells trigger caspase activation in vitro. Caspase activation was measured in L2 cell cytosol that had been coincubated with mitochondria isolated from parental L2 cells or from pre-apoptotic cells (induced either for rpr or grim). Caspase activation was detected, as measured by signature cleavage of a bovine substrate, PARP. Cleavage of PARP in this assay is indistinguishable from the signature activity reported in many mammalian systems and is readily detected in the cytosol of pre-apoptotic cells but not in cytosol from parental L2. These observations emphasize the importance of one or more mitochondrial factors in the activation of caspase function triggered by rpr or grim (Varkey, 1999).

The Drosophila death activators, rpr and grim, activate one or more caspases to elicit apoptosis. To study the temporal relation of cytochrome c display with respect to caspase activity, SL2 cells were cotransfected with rpr and p35 plasmids. Six hours after induction, cells induced for rpr alone show pronounced labeling with mAb 2G8 whereas cells expressing rpr together with p35 are prevented from apoptosis and do not bind the mAb. These observations suggest that apoptogenic cytochrome c display requires caspase activity, a presumption that is further substantiated when rpr-expressing cells are treated with the peptide caspase inhibitors zDEVD-fmk and zVAD-fmk. As seen for p35-blocked cells, these inhibitors similarly prevent mAb 2G8 labeling and subsequent apoptosis. Parallel results are observed in grim-expressing cells (Varkey, 1999).

These data demonstrate that caspase activity is required for apoptogenic cytochrome c display. To determine if caspase function is sufficient to trigger this change, apoptosis was induced in SL2 cells by conditional expression of an activated version of the Drosophila caspase, dcp-1. If deleted for its prodomain, this caspase provokes considerable apoptosis in mammalian cells and SL2 cells. When labeled with mAb 2G8, cells transfected and induced for dcp-1 expression exhibit profound punctate cytochrome c staining with features indistinguishable from those associated with expression of the death activators (Varkey, 1999).

Two potential explanations reconcile the in vivo observations reported here on apoptogenic cytochrome c with reports from mammalian cell-free systems that cytochrome c can trigger caspase activation. One possibility is that the order and/or nature of cytochrome c apoptotic function is not conserved between mammals and insects and thus, relative to caspase action, cytochrome c is upstream in the former case and downstream in the latter case. This scenario, however, seems unlikely given the widespread conservation of apoptotic components, the fact that display of fly cytochrome c in the animal significantly precedes all signs of programmed cell death, and reports from mammalian systems that upstream caspases can trigger cytochrome c release. Therefore, a more likely interpretation of the results reported here is that cytochrome c propagates apoptotic physiology by functioning together with caspases in a feed-forward amplification loop. In this scenario, altered cytochrome c and caspase activity exert positive and reciprocal feedback on one another, similar to observations recently reported for caspase 8. Thus, agents that restrain caspase action (p35) are also predicted to suppress pro-apoptotic display of cytochrome c, which behaves as an amplifier of caspase function. This interpretation is also consistent with recent studies on Fas signaling in type II cells, where molecular ordering studies found that activation of an initiator caspase (caspase 8/Flice) occurs upstream of changes associated with cytochrome c (Varkey, 1999).

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis (Yoo, 2002).

Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis of apoptotic substrates. IAPs occlude the active sites of caspases to which they are bound and can function as ubiquitin ligases. IAPs are also reported to ubiquitinate themselves and caspases. Several proteins induce apoptosis, at least in part, by binding and inhibiting IAPs. Among these are the Drosophila melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal IAP-binding motif. Rpr not only inhibits IAP function, but also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation required both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibits protein translation. Since the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis (Holley, 2002).

Domain structure of Reaper and Reaper's killing activity

The Drosophila reaper, head involution defective, and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. The Gal4/UAS targeted gene expression system has been used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. In both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with reaper or hid to induce higher levels of midline cell death than observed for any of the genes individually. Finally the function was analyzed of a truncated Reaper-C protein that lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts in part to modulate Reaper activity (Wing, 2001a).

Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions

Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. This study characterized the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. Reaper concentrates are found on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper is able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation (Freel, 2008).

Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development: Differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle

MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Injection of 2′O-methyl antisense oligoribonucleotides (2'OM-ORNs) into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. These findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes (Leaman, 2005).

miR-2/13 and miR-6 depletion results in catastrophic apoptosis: Embryos injected with miR-2/13 and miR-6 antisense 2′OM-ORNs fail to differentiate normal internal and external structures. At the end of embryogenesis, the embryos fall apart on touch, and no cuticle is recovered. To determine the onset of these problems, blastoderm embryos were examined, and it was found that cellularization and early pattern formation along the anteroposterior axis occur normally for both miRNAs, indicating that early fating and morphogenesis are intact. Interestingly, in miR-6, but not miR-2/13 depleted embryos, pole cell formation at the posterior end is disrupted (Leaman, 2005).

One possible cause of the catastrophic defects observed in miR-2/13 and miR-6 depleted embryos is excessive and widespread apoptosis. In both miR-2/13 and miR-6 antisense injected embryos, the number of apoptotic cells is greatly increased compared to wild-type by stage 13. Notably, the overall morphology of miR-6 depleted embryos is much more affected than that of miR-2/13 depleted embryos. miR-6 depleted embryos are generally smaller in size and have fewer and abnormally large (para-) segments, suggesting greater excess or earlier onset of apoptosis (Leaman, 2005).

To determine the specificity of the effects of miR-6 and miR-2/13 antisense injections, genomic rescue experiments were carried out. Embryos ubiquitously overexpressing mir-6 or mir-2 (Actin-Gal4;UAS-mir6-3/2b-2) show normal cell-death patterns. When injected with miR-6 or miR-2/13 antisense, they show significant rescue of miR-6 antisense by mir-6, with respect to both cell death and morphology, and of miR-2/13 antisense by mir-2. Interestingly, crossrescue of miR-6 antisense by mir-2 overexpression and of miR-2/13 antisense by mir-6 is weak (Leaman, 2005).

The miRNA sequence family miR-6 and miR-2/13 belong to has two additional members, miR-11 and miR-308. Depletion of miR-11 results in a moderate and of miR-308 in a mild increase in apoptosis in midembryogenesis. Thus, for all members of the miR-2 family, antisense-induced depletion results in excess embryonic cell death, but with marked differences in phenotypic strength. This differential could be due to differences in expression level or to sequence divergence and thus differential interaction with target mRNAs (Leaman, 2005).

The miR-2 family regulates cell survival by translational repression of proapoptotic factors: In Drosophila, three pathways are known to control caspase activity. The main control is thought to come from the proapoptotic factors Hid, Grim, and Reaper (Rpr), which are transcriptionally activated in response to a range of natural and toxic conditions; they promote caspase activation through inhibition of the caspase inhibitor Diap1. The three factors appear to act independently, with each being sufficient to drive apoptosis. When miR-2/13 and miR-6 antisense 2′OM-ORNs are injected into embryos deficient for the hid, grim, and rpr genes (H99 deficiency), they are unable to trigger apoptosis, indicating that these miRNAs act through hid, grim, and/or rpr (Leaman, 2005).

To determine whether the regulation of the three proapoptotic factors occurs at the transcriptional or at the posttranscriptional level, their RNA expression was examined in miR-2/13 and miR-6 depleted embryos using in situ hybridization and quantitative PCR. No significant increase was found in the expression level or broadening of the pattern compared to control embryos for any of three transcripts, either at embryonic stage 13 or 1 hr earlier at embryonic stage 12. By contrast, the protein expression of Hid is dramatically increased in miR-6 depleted embryos and modestly in miR-2/13 depleted embryos. These results strongly argue against a transcriptional and in favor of a posttranscriptional regulation of the proapoptotic factors by miR-2/13 and miR-6 (Leaman, 2005).

To test this directly, two existing translation control assays were adapted to the embryonic paradigm. In the first assay, full-length 3′UTRs are fused to a ubiquitously transcribed sensor (tub-GFP); transgenic embryos are injected with sense or antisense 2′OM-ORNs, and GFP fluorescence is measured. The 3′UTRs of hid, grim, rpr, and sickle (skl, a structurally related but less potent proapoptotic factor display marked differences in sensor expression, with rpr showing no expression, hid and skl low uniform expression, and grim strong and spatially modulated expression, indicating that these proapoptotic factors experience quite different levels of translation control. To gauge the efficacy of the assay, hid GFP sensor embryos were injected with bantam antisense 2′OM-ORNs, and mild but statistically significant derepression of GFP expression was found as compared to control, consistent with the weak cell-death phenotype of bantam depleted embryos. Antisense injection of miR-2 family members reveals strong derepression of the hid GFP sensor by miR-6 antisense, but not by miR-2/13, 11, or 308 antisense. Conversely, the grim GFP sensor shows significant derepression as a result of miR-2/13, 11, and 308, but not miR-6 depletion. Finally, the skl GFP sensor shows significant derepression for all four family members (Leaman, 2005).

To assess effects on rpr, a second, more sensitive assay was developed that employs transient expression of a dual-luciferase vector in injected embryos. For initial comparison with the GFP assay, a hid luciferase sensor was tested against the entire miR-2 family and the same profile was found. The rpr luciferase sensor shows strong derepression in miR-6 and 2/13, moderate derepression in miR-11, and no significant effect in miR-308 depleted embryos. Thus, the 3′UTRs of all four proapoptotic factors are subject to translational repression by the miR-2 family, but each miRNA displays a distinct interaction profile. The interaction preferences correlate well with the observed differences in phenotype: miR-6 has the most severe death phenotype and is the only family member to regulate hid, the factor with the broadest expression and the strongest proapoptotic effect. mir-2/13 and miR-11 have the same overall profile, but they differ in the strength of their interaction with rpr and show a corresponding differential in phenotypic strength. Finally, miR-308, which has the mildest death phenotype, interacts only with the weakly proapoptotic skl and with grim (Leaman, 2005).

The differences in target interaction profile between the miR-2 family members are pronounced and do not merely reproduce differences in the strength or onset of miRNA expression. This suggests that differential pairing outside the 5′ core sequence shared by all members has an important role in target selection. Computational predictions indicate that miR-2 family binding sites are present in the 3′UTRs of all four proapoptotic factors: rpr and grim have one, hid and skl two predicted sites. All six miRNA target sites lie in sequence blocks that are conserved between the six sequenced Drosophilid species, spanning an evolutionary distance of 40 Myr. Interestingly, for all sites, absolute conservation extends well beyond the bases complementary to the 5′ core of the miRNA and includes adjacent stretches suitable for pairing with the 3′ end. All but one of the sites show Watson-Crick pairing with miRNA positions 2-7 and variable pairing at the 3′ end. One of the hid sites (hid468) has a mismatch in the core but shows strong pairing with miR-6 at the 3′ end. The rules for 3′ pairing between miRNAs and their targets are not yet well understood, but it is clear that the miR-2 family members differ considerably in their ability to form 3′ matches with the six target sites. Further experimentation will be required to better understand how the observed differences in regulatory effect relate to differences in sequence pairing (Leaman, 2005).

DEVELOPMENTAL BIOLOGY

Protein Interactions and parallel pathways

Bruce can suppress Reaper dependent cell death

Mammalian Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain. Bruce upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Using gain-of-function and deletion alleles, it has been demonstrated that Drosophila Bruce can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The Bruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. Drosophila Bruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1/Thread BIR2, be intact. Bruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that Bruce can regulate cell death at a novel point (Vernooy, 2002).

In Drosophila, the products of the reaper (rpr), head involution defective (hid), and grim genes are essential activators of caspase-dependent cell death. A genetic screen was carried out for suppressors of Rpr-, Hid-, and Grim-dependent cell death to identify regulators of their activity. Approximately 7000 new insertion lines of the GMREP P element transposon were generated. GMREP contains an engineered eye-specific enhancer sequence (GMR). This sequence is sufficient to drive the expression of linked genes in and posterior to the morphogenetic furrow during eye development. Thus, insertion of GMREP within a region can lead to the eye-specific expression of nearby genes. Each insertion line was crossed to flies that had small eyes due to the eye-specific expression of Rpr (GMR-Rpr flies), Hid (GMR-Hid flies), or Grim (GMR-Grim flies), and the progeny were scored for enhancement or suppression. A number of suppressors were identified. Five lines (GMREP-86A-1–5) mapped to the 86A region, and each strongly suppressed cell death induced by eye-specific expression of Rpr or Grim but not Hid. These lines mapped within a 6-kb interval. A number of other lines were obtained with P-element insertions located in the nearby region. Four of these, EP(3)0359, EP(3)0739, l(3)j8B6, and l(3)06142, mapped within six base pairs of the GMREP-86A-3–5 insertion sites. None of these, nor a fifth nearby line, l(3)06439, acted as a suppressor of GMR-Rpr-, GMR-Grim-, or GMR-Hid-dependent cell death. These results argue that the cell death suppression seen with the GMREP-86A lines was not due to a transposon-induced loss of function, but rather to the GMREP-dependent expression of a nearby gene. All of the GMREP-86A insertions were located 5' to a gene encoding the Drosophila homolog, Bruce, of murine Bruce (also known as Apollon in humans, suggesting this as an obvious candidate. The results of tissue in situ hybridizations with a Drosophila Bruce probe and immunocytochemistry with a Bruce-specific antibody support this possibility. Bruce transcript and protein are expressed at uniform low levels in wild-type eye discs. However, in the GMREP86A lines, they are expressed at high levels in and posterior to the morphogenetic furrow of the eye disc, which is where the GMR element drives expression (Vernooy, 2002).

To demonstrate that Bruce is responsible for the GMREP-86A-dependent suppression of Rpr- and Grim-dependent cell death, levels of the Bruce transcript were specifically downregulated in the eyes of flies carrying a GMR-Rpr transgene as well as a GMREP-86A element. Analysis focussed on one line, GMREP-86A-1, since all five lines behaved similarly with respect to cell death suppression and Bruce overexpression. Flies were generated that carried a Bruce RNA interference (RNAi) construct driven under GMR control (GMR-Bruce-RNAi flies). The eyes of GMR-Bruce-RNAi flies were normal. These animals were crossed to flies in which GMR-Rpr-dependent cell death was suppressed by the presence of the GMREP-86A-1 transposon and progeny from this cross were identified that carried all three transgenes, GMR-Bruce-RNAi, GMR-Rpr, and GMREP-86A-1. It was reasoned that if ectopic expression of Bruce in the eye, driven by the GMREP-86A-1 insertion, was responsible for the suppression of Rpr-dependent cell death, then expression of Bruce-RNAi should downregulate levels of the Bruce sense transcript. This should lead to an attenuation of the GMR-EP-86A-1-dependent suppression of Rpr-dependent cell death, causing a decrease in eye size. Such an attenuation was in fact observed. These observations, in conjunction with those obtained from studies with Bruce deletion mutants, argue that Bruce can suppress Rpr- and Grim-dependent cell death (Vernooy, 2002).

cDNAs encompasing the Bruce coding region were sequenced. This allowed an accurate map to be assembled of the Bruce exon-intron structure, which differs in some respects from that of the BDGP predicted gene. Overall, Bruce is 30% identical to murine Bruce. However, the Bruce N-terminal BIR domain and the C-terminal E2 domain show much higher degrees of homology, 83% and 86% identity, respectively. C. elegans homologs of Bruce were not apparent. Mutations in the Bruce gene were generated by carrying out imprecise excision of a P element, EP3731, located 3' to the Bruce transcript. Two deletions were generated that extended only in one direction, into the 3' end of the Bruce coding region. E12 deleted a relatively small region of the C terminus that includes the E2 domain, while E16 deleted approximately the C-terminal half of the Bruce coding region. Both lines were homozygous viable but male sterile. The possibility that E12 and E16 represent neomorphic mutations in Bruce cannot be excluded. However, the hypothesis that they represent hypomorphs or null mutations is favored, since they had the opposite phenotype of the GMREP-86A Bruce expression lines when in combination with GMR-Rpr, acting as enhancers rather than suppressors of Rpr-dependent cell death in the eye. E12 and E16 also enhanced GMR-Grim, but this effect is much more modest. E12 and E16 have no clear effect on cell death due to expression of Hid (Vernooy, 2002).

These results argue that endogenous Bruce levels, at least in the eye, are sufficient to act as a brake on Rpr-, and to some extent, Grim-dependent cell death. How does Bruce suppress apoptosis? A number of observations argue that Rpr- and Grim-dependent killing proceeds through distinct mechanisms and/or is regulated differently from those activities that is due to Hid. These differences are manifest at multiple points. At the level of DIAP1, point mutations of DIAP1 have effects on Rpr- and Grim-dependent cell death that are the opposite of those due to Hid. In addition, in a Drosophila extract, Hid, but not Rpr and Grim, promotes DIAP1 polyubiquitination. In contrast, in a different set of assays, Rpr and Grim, but not Hid, act as general inhibitors of protein translation. Finally, Rpr and Grim, but not Hid, show strong synergism with the effector caspase DCP-1 in terms of their ability to induce cell death in the eye. Each of these points defines a possible target for Bruce antiapoptotic action (Vernooy, 2002 and references therein).

Because Bruce strongly suppresses cell death induced by Rpr and Grim but not by Hid, one obvious possibility was that Bruce promotes Rpr and Grim ubiquitination and degradation. This hypothesis was tested by generating mutant versions of Grim and Rpr that lacked all lysines, the amino acid to which ubiquitin is added. These genes were introduced into flies under GMR control. GMR-Rpr-lys^- and GMR-Grim-lys^- flies have small eyes, indicating that these mutant proteins are effective cell death inducers. GMREP-86A-1-dependent Bruce expression suppresses this death very effectively, indicating that Bruce cannot be promoting ubiquitin-dependent degradation of Rpr or Grim. Interestingly, however, Bruce expression does not suppress cell death induced by expression of versions of Rpr (GMR-RprC) or Grim (GMR-GrimC) lacking their N termini, which are required for their IAP-caspase-disrupting interactions with the DIAP1 BIR2. This result is important because it argues that Bruce does not act to regulate this relatively uncharacterized death pathway (Vernooy, 2002).

The N-terminal Bruce BIR lacks a number of residues thought to be important for binding of Rpr, Hid, and Grim to DIAP1 BIR2. Thus, it seems unlikely that GMR-driven expression of Bruce inhibits cell death by simply titrating Rpr and Grim away from interactions with DIAP1 BIR2 as a result of similar interactions with the Bruce BIR. Nonetheless, the high degree of conservation between Bruce and mammalian Bruce in the BIR suggests that it is functionally important. To explore this role further, a fragment of Bruce that contained residues 1–531, including the BIR domain, was expressed under GMR control. Flies carrying this construct, GMR-Bruce-BIR flies, had normal appearing eyes, and in crosses to flies expressing GMR-Rpr, -Hid, or -Grim, GMR-Bruce-BIR did not enhance or suppress these eye phenotypes. These results do not rule out a role for the Bruce BIR in suppressing Rpr- and Grim-dependent cell death. However, they do suggest that the BIR alone is unlikely to mediate this inhibition (Vernooy, 2002).

Bruce overexpression in the eye also does not suppress cell death resulting from GMR-driven expression of the caspase Dronc, which is required for many apoptotic cell deaths in the fly, including those induced by expression of Rpr, Grim, and Hid. Dronc most resembles mammalian caspase-9, and its activation is likely to involve interactions with the Drosophila Apaf-1 homolog Ark. Thus, this result strongly suggests that Bruce does not block Ark-dependent Dronc activation or Dronc activity. This result is also suggested by the observation that decreasing Ark or Dronc in the eye strongly suppressed Hid-dependent cell death, which Bruce does not. A similar lack of cell death suppression is seen in the progeny of crosses between GMR-Bruce flies and flies expressing a second long prodomain caspase, Strica, whose mechanism of activation and normal functions are unknown. Finally, GMREP-86A-1 also fails to suppress the cell death due to GMR-dependent expression of the Drosophila proapoptotic Bcl-2 family member known variously as Debcl, Drob-1, dBorg-1, or Dbok (Vernooy, 2002).

Thus, the Bruce gene is found in mammals and flies, but not in the worm C. elegans. In humans, it is upregulated in some cell lines derived from gliomas and an ovarian carcinoma, and the results of antisense inhibition of Bruce suggest that it contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. The Drosophila homolog of Bruce, can potently inhibit cell death induced by Rpr and Grim but not Hid. In addition, flies with C-terminal deletions that removed the Bruce ubiquitin conjugation domain, or much larger regions of the coding region, acted as dominant enhancers of Rpr- and Grim-dependent, but not Hid-dependent, cell death. Together, these observations clearly demonstrate that Bruce can function as a cell death suppressor. The results with the deletion mutants suggest, but do not prove, that Bruce's death-inhibiting activity requires its function as a ubiquitin-conjugating enzyme. Based on the general conservation of cell death regulatory mechanisms, these results argue that mammalian Bruce is likely to facilitate oncogenesis by directly promoting cell survival in the face of specific death signals. One mechanism by which Rpr, Grim, and Hid promote apoptosis is by binding to DIAP1, thereby blocking its ability to inhibit caspase activity. It will be interesting to determine if mammalian Bruce also inhibits cell death induced by the expression of specific IAP binding proteins (Vernooy, 2002).

How does Bruce inhibit cell death? It does not promote the ubiquitination and degradation of Rpr and Grim directly. However, the possibility cannot be ruled out that Bruce somehow sequesters these proteins from their proapoptotic targets. The fact that it does not inhibit cell death due to Hid or Dronc expression argues that it is unlikely to be acting on core apoptotic regulators such as Ark, Dronc, or DIAP1, which are important for Hid-, Rpr-, and Grim-dependent cell death. An attractive hypothesis is that Bruce, perhaps in conjunction with apoptosis-inhibiting ubiquitin-protein ligases such as DIAP1 or DIAP2, promotes the ubiquitination and degradation of a component specific to Rpr- and Grim-dependent death signaling pathways. What might such a target be? Little is known about how Rpr- and Grim-dependent death signals differ from those due to Hid. However, one possibility is suggested by the recent observation that Rpr and Grim, but not Hid, can inhibit global protein translation. This creates an imbalance between levels of short-lived IAPs and the caspases they inhibit, thereby sensitizing cells to other death signals. Perhaps Bruce targets a protein(s) required for this activity (Vernooy, 2002).

Finally, Bruce is a very large protein, and thus its coding region might be expected to be subject to a relatively high frequency of mutation. Truncation of Bruce through the introduction of a stop codon or a frame shift is thus likely to be a relatively common form of Bruce mutation. The results of the deletion analysis show that C-terminal Bruce truncations act to enhance cell death in response to several different signals. Given this, it will be interesting to determine if human Bruce mutations are associated with a predisposition to pathologies that involve an inappropriate increase in cell death (Vernooy, 2002).

Grim

A new activator of apoptosis, grim, maps between two previously identified cell death genes in this region reaper (rpr) and head involution defective (hid). Expression of Grim RNA coincides with the onset of programmed cell death at all stages of embryonic development, whereas ectopic induction of grim triggers extensive apoptosis in both transgenic animals and in cell culture. Cell killing by Grim was blocked by coexpression of p35, a viral product that inactivates ICE-like proteases, and does not require the functions of rpr or hid. The predicted Grim protein shares an amino-terminal motif in common with RPR. However, Grim is sufficient to elicit apoptosis in at least one context, where RPR was not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors (Chen, 1996).

Reaper (Rpr), Hid, and Grim activate apoptosis in cells programmed to die during Drosophila development. Transient overexpression of Rpr in the lepidopteran SF-21 cell line induces apoptosis. Members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit Rpr-induced apoptosis and physically interact with Rpr through IAP family members' BIR (baculovirus IAP repeat) motifs. Transient overexpression of HID and GRIM also induces apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs block HID- and GRIM-induced apoptosis and also physically interact with them through the BIR motifs of the IAPs. The region of sequence similarity shared by Rpr, Hid, and Grim (the N-terminal 14 amino acids of each protein) is required for the induction of apoptosis by Hid and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of Hid and Grim are sufficient to induce apoptosis and confer IAP binding activity. However, Grim is more complex than HID since the C-terminal 124 amino acids of Grim retain apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within Grim. Coexpression of IAPs with Hid stabilizes Hid levels and results in the accumulation of Hid in punctate perinuclear locations that coincide with IAP localization. The physical interaction of IAPs with Rpr, Hid, and Grim provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins (Vucic, 1998).

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. grim expression is shown to induce apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. The conserved N-terminal domain is not required for either the initiation or the execution of apoptosis by Grim. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. Grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Three clustered aspartate residues near the grim C-terminus (positions 126, 128 and 129) could then be used by a caspase as a target sequence. The downshift predicted by digestion at these sites is between 1 and 1.3 kDa; however, the observed size for the short form is ~4 kDa less than that observed for the intact protein. Nevertheless, C-terminal deletions of the protein provoke downshifts greater than expected, which are in the range of the observed downshift for the proteolysed form of the protein. These results show that besides activating caspases, Grim itself can be processed as a consequence of caspase proteolytic activity. Even though inhibitors of apoptosis OpIAP and DEVD.cho do not block Grim-induced apoptosis, they are both functionally active in repressing endogenous caspases in either treated or transfected cells, since they prevented Grim cleavage. Therefore, it is likely that caspases insensitive to OpIAP and/or DEVD.cho are sufficient to achieve Grim-induced apoptosis (Clavería, 1998).

Grim-induced death is antagonized by bcl-2 in a dose-dependent manner; neither Fas signaling nor p53 are required for Grim pro-apoptotic activity. The sensitivity of the grim-induced death to bcl-2 levels suggests that Grim acts by activating a mitochondrial pathway. To determine the site of Grim action, its subcellular localization was explored in transfected cells prior to the time they show morphological symptoms of apoptosis. Pre-apoptotic fibroblasts simultaneously show Grim in two different cytoplasmic localizations: diffuse cytosolic and a punctate pattern. However, while Grim-transfected cells show an almost exclusive cytosolic localization, cells treated with cell-permeable broad specificity caspase inhibitor zVAD.fmk show predominantly the punctate pattern. The same pattern is observed in Grim-expressing p35-rescued cells. Co-localization of Grim protein with a mitochondrial-specific antibody shows that the punctate pattern corresponds to mitochondria. It is concluded that Grim localizes initially in the cytoplasm but accumulates progressively in the mitochondria, correlating with apoptosis progression. It is possible that Grim translocation to mitochondria is the event that triggers the apoptotic pathway. In that case, the increased mitochondrial localization of grim in zVAD-treated cells would be the result of the apoptosis blockade by zVAD at a point downstream of Grim incorporation in the mitochondria. These results show that Drosophila Grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases (Clavería, 1998).

It is possible that mitochondrial components of the mammalian cellular apoptotic machinery recognize the Drosophila Grim protein and respond by activating the apoptotic programme, as they would after the proper endogenous stimulus. In good agreement with this view, Grim action is counteracted by the overexpression of the anti-apoptotic factor bcl-2, which generally inhibits all mitochondrial death pathways. bcl-2 inhibits Grim-induced death in a dose-dependent manner, suggesting that they mutually antagonize to either promote or inhibit caspase activation. A bcl-2-like molecule has not yet been isolated in Drosophila, but mammalian and nematode members of this family have been shown to rescue ced-3- and Reaper-induced apoptosis in cultured fly cells. One possibility is that Grim promotes cytochrome c release from the mitochondria, as has been shown for Reaper in an in vitro amphibian system. However, Reaper does not induce apoptosis in mouse fibroblasts, arguing for a mechanism of action or regulation different from that of Grim. The activity of caspases insensitive to OpIAP, DEVD.cho and YVAD.cho is sufficient to execute the apoptosis induced by Grim. Caspase 9, which has been shown to transduce apoptotic mitochondrial signals, may be one of those activated by mitochondrial Grim. Nevertheless, group II caspases are activated as well, since Grim cleavage is inhibited by caspase inhibitors specific for this group and it cannot be excluded that activation of group II caspases may also serve to execute Grim-induced apoptosis. Inhibitor of apoptosis proteins (IAPs), the natural inhibitors of group II caspases, have been shown to inhibit apoptosis by directly binding to Grim, Reaper and Hid in a lepidopteran cell line. In contrast, neither OpIAP nor DEVD block Grim-induced death. These results, nevertheless, show that group II caspases are activated, suggesting that some of the Grim-induced caspase activation cascades are similar between mouse fibroblasts and cultured lepidopteran cells, whereas others may differ. It is possible that the particular caspase activation cascade triggered by grim depends more on the cell type in which it is expressed rather than on any intrinsic specificity of its action (Claveria, 1998).

The Drosophila reaper, head involution defective (hid), and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. In this study the Gal4/UAS targeted gene expression system was used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. In both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with either reaper or hid to induce higher levels of midline cell death than those observed for any of the genes individually. Analysis was made of the function of a truncated Reaper-C protein, which lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts in part to modulate Reaper activity (Wing, 1998).

The prototype baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) expresses p35, a potent anti cell-death gene that promotes the propagation of the virus by blocking host cell apoptosis. Infection of insect Sf-21 cells with AcMNPV lacking p35 induces apoptosis. This pro-apoptotic property of the p35 null virus was used to screen for genes encoding inhibitors of apoptosis that rescue cells infected with the p35 defective virus. Tn-IAP1, a novel member of the IAP family of cell death inhibitors, is described. Tn-IAP1 blocks cell death induced by p35 null AcMNPV, actinomycin D, and Drosophila cell-death inducers HID and GRIM. Given the conserved nature of the cell death pathway, this genetic screen can be used for rapid identification of novel inhibitors of apoptosis from diverse sources (Seshagiri, 1999).

Grim encodes a protein required for programmed cell death in Drosophila. The Grim N-terminus induces apoptosis by disrupting IAP blockage of caspases; however, N-terminally-deleted Grim retains pro apoptotic activity. This study describes GH3, a 15 amino acid internal Grim domain absolutely required for its proapoptotic activity and sufficient to induce cell death when fused to heterologous carrier proteins. A GH3 homology region is present in the Drosophila proapoptotic proteins Reaper and Sickle. The GH3 domain and the homologous regions in Reaper and Sickle are predicted to be structured as amphipathic alpha-helixes. During apoptosis induction, Grim colocalizes with mitochondria and cytochrome c in a GH3-dependent but N-terminal- and caspase activity-independent manner. When Grim is overexpressed in vivo, both the N-terminal and the GH3 domains are equally necessary, and cooperate for apoptosis induction. The N-terminal and GH3 Grim domains thus activate independent apoptotic pathways that synergize to efficiently induce programmed cell death (Clavería, 2002).

Secondary structure prediction of the Grim protein has identified three regions with a very high probability of conforming to an a-helical structure. These regions have been termed GH1, GH2 and GH3, for Grim Helix 1, 2 and 3. The GH3 domain shows similarity to a region in Reaper and Sickle, both also predicted to conform as an a-helix. The homology region spans the 15 amino acids predicted to conform as an a-helix in Grim, with two 5 amino acid regions of high similarity flanking a 5 amino acid central region with lesser homology. Representation in a helical wheel projection of GH3 residues and of those in the Reaper and Sickle homology regions reveals the amphipathic nature of the predicted a-helices (Clavería, 2002).

Tests were performed to see whether GH3 is important for Grim proapoptotic function by assaying the cell killing ability of several Grim mutants altered in the GH3 domain in Drosophila SL2 cells. Wild-type (WT) Grim induces cell death when overexpressed in this assay. In contrast, a Grim mutant form with a 13 amino acid deletion that removes the GH3 residues most reliably predicted to form an a-helix only marginally induces apoptosis. A 5'-shifted 11 amino acid deletion, such that the 3' part of GH3 was respected, was less effective in eliminating the proapoptotic activity than the complete GH3 deletion. An internal deletion removing four amino acids commonly deleted in the two larger deletions (Delta89-92) also resulted in strong impairment of Grim killing ability, but to a lesser extent than the complete GH3 deletion. The relevance was tested of L89, a conserved residue within positions 89-92, whose hydrophobicity could be relevant for the amphipathic nature of the GH3 domain. A non-conservative replacement of L89 by glutamic acid (L89E) impaired GH3 killing ability nearly to the same level as the Delta89-92 mutant. Semi-conservative replacement of L89 by alanine (L89A) had a mild effect on Grim proapoptotic function. These results show that the GH3 domain is required for Grim proapoptotic function and that L89 is a functionally relevant residue in the domain (Clavería, 2002).

Since N-terminally deleted Grim can still bind IAPs, GH3 domain function could be related to Grim's ability to bind IAPs and inhibit their protective function. Deletion of the Grim N-terminal domain was found to lead to a slight reduction in its ability to bind DIAP2, however, deletion of the GH3 domain, either alone or in combination with the N-terminal deletion, does not impair Grim's ability to bind DIAP2. These results suggest that GH3 activity is unrelated to the IAP inhibitory Grim activity (Clavería, 2002).

To determine whether the GH3 domain could function as a proapoptotic motif itself, a Grim fragment containing the GH3 domain was fused to green fluorescent protein (GFP) as a carrier protein (GH3-GFP) and the ability of this fusion protein to induce apoptosis in Drosophila SL2 cells was tested. GH3-GFP induces cell death with the same efficiency as does the complete Grim-GFP fusion protein. The cell death observed is specific to GH3 domain activity, since it is largely abolished by an L-to-E mutation in the residue equivalent to Grim L89. In all cases, cell death was rescued by coexpression with the baculoviral caspase inhibitor p35. The GH3 domain is therefore sufficient to trigger a specific proapoptotic route in SL2 cells (Clavería, 2002).

Immunocytochemistry was used to identify Grim subcellular localization in Drosophila SL2 cells. In phases previous to any obvious apoptotic phenotype, Grim generally shows a diffuse distribution in the cytoplasm, but also displays rings of stronger Grim staining. Grim rings are mitochondria-associated, as indicated by the presence of a mitochondrial matrix marker, mainly inside the rings, but also in colocalization with strong Grim staining. Grim colocalization with cytochrome c is similar to that observed with the mitochondrial matrix marker but, in addition, Grim and cytochrome c show extensive colocalization in larger dots. These larger cytochrome c dots are not observed in untransfected cells: they increase in size and abundance as apoptosis progresses, and do not colocalize with a mitochondrial marker. No substantial cytochrome c release to cytosol was found. Instead, Grim promotes redistribution of cytochrome c signal in large dots in colocalization with Grim itself (Clavería, 2002).

Grim subcellular localization is dependent on the presence of an intact GH3 domain. GH3-deficient Grim shows no obvious organization in rings associated with mitochondria and does not colocalize with either the mitochondrial marker or cytochrome c. In contrast, deletion of Grim 2-14 amino acids does not alter subcellular distribution of the Grim protein, nor the changes induced in cytochrome c display (Clavería, 2002).

The relevance of the GH3 and N-terminal Grim domains was examined by overexpressing the Grim mutants in transgenic flies using the Gal4-UAS system. To drive Grim expression, the GMR-Gal4 line, which targets expression to the eye disc, and the MS1096-Gal4 line, which directs expression to the wing imaginal disc, were used. Overexpression of WT Grim with either driver causes total or partial lethality in all transgenic lines. These results suggested that leaky expression from both promoters in vital tissues produces sufficient cell death to block development. Surviving adult flies overexpressing WT Grim display a considerable reduction in eye size with the GMR driver, and wing agenesis plus notum reduction and elimination of macro- and microchaetae with the MS1096 driver (Clavería, 2002).

In contrast to these results, overexpression of a GH3-deleted Grim (Delta86-98) results in very low lethality, as well as rescue of the eye, notum and wing phenotypes. Elimination of amino acids 89-92 results in a lesser impairment of Grim killing ability, showing that the 3' part of the GH3 domain is important for proapoptotic function. In correlation with these results, the Delta89-92 mutant rescues the eye phenotype induced by WT Grim, but only partially rescues the more sensitive wing and notum phenotypes. Elimination of amino acids 83-93, which extends the deletion N-terminal to the putative helical domain, does not increase the rescue observed with the 89-92 deletion, suggesting that residues 5' of leucine 89 might be less important for proapoptotic function. The relevance of leucine 89 was again shown by the significant rescue of viability and of eye, notum and wing phenotypes in flies overexpressing the non-conservative L89E substitution. In contrast, the semi-conservative substitution L89A results in mild, but significant, impairment of Grim death induction and targeted tissue deletion (Clavería, 2002).

In accordance with the results observed in cultured cells, mutations of the GH3 domain impairs Grim proapoptotic activity in transgenic flies. Deletion of the N-terminal domain, in contrast to the results observed in cultured cells, results in highly significant elimination of Grim proapoptotic function in vivo. Both viability and appearance of the tissues targeted by the GMR and MS1096 drivers were rescued by the 2-14 deletion to a level similar to that observed for the GH3 deletion. Simultaneous deletion of the N-terminus and amino acids 89-92 of the GH3 domain results in even lower lethality and fewer alterations in the targeted tissues than those induced by each deletion in isolation (Clavería, 2002).

To determine whether the N-terminal and GH3 Grim domains can function independently of each other, tests were performed to see whether the simultaneous expression of independent 2-14- and GH3-deleted Grim proteins could induce apoptosis. Flies were generated carrying independent transgenes for 2-14 and 86-98 Grim deletion mutants driven by MS1096 expression. Whereas males carrying either protein alone showed little or no lethality and no alterations in wing development, double transgenic males simultaneously expressing Delta2-14 and Delta86-98 Grim mutants display severe lethality and reduced wings. Females did not display lethality in any situation, but frequently showed reduced wings in the double transgenics, although not in single transgenics. Functions of both the N-terminal and the GH3 domains are therefore essential for Grim activity in vivo and they independently activate specific death mechanisms that synergize to trigger apoptosis (Clavería, 2002).

Several lines of evidence point to the mitochondrial-cytochrome c pathway as the target of GH3 action. Grim associates with mitochondria in colocalization with cytochrome c and this activity resides in the GH3 domain. In vertebrate cells, Grim targets the mitochondria and induces cytochrome c release in a GH3-dependent and N-terminal-, caspase- and IAP-independent manner, suggesting functional conservation of the pathway. Interestingly, during apoptosis induction by Grim and Reaper in flies, changes in cytochrome c display are observed, rather than its free release to the cytosol as in vertebrates. Grim-expressing cells specifically show large cytoplasmic deposits of cytochrome c at sites where Grim itself is present, but other mitochondrial markers are not. The changes observed in the distribution of cytochrome c may result from its relocation from mitochondria to hypothetical specialized cytoplasmic structures involved in apoptosis induction. Alternatively, the apoptosome might be formed in the vicinity of the mitochondria, and cytochrome c deposits may constitute the remnants of damaged mitochondria, which have lost some of their constitutive components, but retain cytochrome c and Grim. The relevance of the cytochrome c proapoptotic pathway in Drosophila PCD is supported as well by the observation that elimination of Dark, a Drosophila homolog of Apaf-1 that mediates cytochrome c-primed apoptosis, impairs Reaper, Hid and Grim killing in flies. Even though no cytochrome c free release appears to take place in Drosophila cells, it is possible that a mechanism homologous to that of vertebrate cells is activated, but from different subcellular compartments (Clavería, 2002).

The involvement of the GH3 domain in a mitochondrial pathway and its predicted structure, an amphipathic a-helix, resemble the characteristics of the widespread proapoptotic BH3 domain. These similarities could be interpreted as functional homology between the two pathways; however, no association has been detected between Grim and either mammalian (Bcl-2 and Bcl-x_L) or insect (Debcl) Bcl-2-family members, as would be expected for a BH3-containing protein. Rather than representing homologous proapoptotic pathways, BH3 and GH3 domains may have converged during evolution to a similar proapoptotic mechanism. Since BH3-containing proteins coexist in Drosophila with GH3-containing proteins, the two pathways may operate in alternative routes, or even cooperate in apoptosis induction, not only in Drosophila, but perhaps also in other species (Clavería, 2002).

Two independent pathways may thus be triggered by Grim; an IAP inhibitory pathway activated by the N-terminal domain, and a mitochondrial-cytochrome c route activated by the GH3 domain. Either pathway could be alternatively or simultaneously promoted by Grim, and the relevance of each may depend on cellular context. The presence of a GH3 homology region in Reaper and Sickle suggests functional conservation of this domain in at least these other two Drosophila proapoptotic proteins. In this context, it is important to consider that Reaper promotes cytochrome c release in a cell-free Xenopus egg extract and does not require the N-terminal domain for this function (Clavería, 2002).

Although Reaper, Hid, Sickle and Grim induce specific apoptotic pathways in vertebrate cells, and in the fly participate in highly conserved routes, such as the p53 and Ras-MAPK pathways, no homolog for these proteins has been yet identified in any other organism. The vertebrate Smac/Diablo protein may, however, represent a functional homolog of the IAP inhibitory pathway. Smac/Diablo can bind to and block the protective effect of IAPs. However, it is unlikely that Smac/Diablo represent homologs of the mitochondrial-cytochrome c pathway. Database searches have failed to identify any protein with sequence similarity to the GH3 domain but, given the restricted sequence conservation among, for example, BH3 family members, this does not exclude conservation of this pathway. Whether vertebrate proapoptotic proteins exist that represent direct or functional homologs of the GH3 proapoptotic activity thus remains to be determined (Clavería, 2002).

Thread and IAPs

Induction of apoptosis in Drosophila requires the activity of three closely linked genes: reaper, hid and grim. The proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (Diap1, also known as Thread) protein. In a genetic modifier screen, both loss-of-function and gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function mutations in diap1 strongly suppress reaper-, hid- and grim-induced apoptosis. Sequence analysis of these diap1 alleles reveals that they are caused by single amino acid changes in the baculovirus IAP repeat domains of Diap1, a domain implicated in binding Reaper, Hid and Grim. Significantly, the corresponding mutant Diap1 proteins display greatly reduced binding of Reaper, Hid and Grim, indicating that Reaper, Hid and Grim kill by forming a complex with Diap1. Collectively, these data provide strong support for the idea that Reaper, Hid and Grim kill by inhibiting DIAP1's ability to antagonize caspase function (Goyal, 2000).

It is thought that the previously proposed function of IAPs upstream of reaper, hid and grim is simply an artifact of unphysiologically high levels of protein expression in heterologous systems. When IAP expression constructs are introduced into cultured cells under the control of strong promoters and at high copy numbers, the levels of proteins expressed far exceed those of the endogenous cellular IAP proteins. Under these unphysiological conditions, cellular IAPs can display properties that do not reflect their normal mechanism of action. In particular, the current results demonstrate that mutant proteins that completely lack anti-apoptotic activity in vivo can still inhibit cell death in vitro as long as they can bind to Reaper, Hid and Grim. Conversely, gain-of-function diap1 alleles that display reduced binding to Reaper, Hid and Grim have strongly increased anti-apoptotic function in vivo, but show reduced protection in heterologous cell transfection assays. These results clearly reveal the limitations of overexpression studies in cultured cells for determining the normal mechanism of action of these proteins in the cell death pathway (Goyal, 2000).

Dronc (Nedd2-like caspase) was isolated through its interaction with the effector caspase drICE (Ice). Ectopic expression of Dronc induces cell death in Schizosaccharomyces pombe, mammalian fibroblasts and the developing Drosophila eye. The caspase inhibitor p35 fails to rescue Dronc-induced cell death in vivo and is not cleaved by Dronc in vitro, making Dronc the first identified p35-resistant caspase. The Dronc pro-domain interacts with Drosophila inhibitor of apoptosis protein 1 (Diap1: known as Thread), and co-expression of DIAP1 in the developing Drosophila eye completely reverts the eye ablation phenotype induced by pro-Dronc expression. In contrast, Diap1 fails to rescue eye ablation induced by Dronc lacking the pro-domain, indicating that interaction of Diap1 with the pro-domain of Dronc is required for suppression of Dronc-mediated cell death. Heterozygosity at the Diap1 locus enhances the pro-Dronc eye phenotype, consistent with a role for endogenous Diap1 in suppression of Dronc activation. Both heterozygosity at the Dronc locus and expression of dominant-negative Dronc mutants suppress the eye phenotype caused by Reaper (Rpr) and Head involution defective (Hid), consistent with the idea that Dronc functions in the Rpr and Hid pathway (Meier, 2000).

The finding that Diap1 directly binds to and inhibits cell death caused by ectopic expression of Dronc, as well as by Rpr, Grim and Hid, underscores the key role played by Diap1 in the regulation of apoptosis in D. melanogaster and raises the possibility that Rpr, Hid or Grim may exert some, or all, of their pro-apoptotic action through displacement of Diap1 from the pro-domain of Dronc, thereby allowing activation of the caspase and consequent cell death. This idea is strongly supported by the successful isolation of Diap1 mutants that display greatly reduced binding for Rpr, Hid and Grim and significantly suppress Rpr, Hid and Grim cell killing. According to this model, IAPs function as 'guardians' of the apoptotic machinery: they act to suppress the chance of spontaneous activation of the intrinsic cell death machinery by neutralizing pro-apoptotic caspases, thereby establishing a buffered threshold that must be either exceeded or neutralized in order to initiate the destruction of a cell (Meier, 2000).

The proapoptotic genes reaper (rpr), grim, and head involution defective (hid) are required for virtually all embryonic apoptosis. The proteins encoded by these genes share a short region of homology at their amino termini. The Drosophila IAP homolog Thread/Diap1 (Th/Diap1) negatively regulates apoptosis during development. It has been proposed that Rpr, Grim, and Hid induce apoptosis by binding and inactivating TH/Diap1. The region of homology between the three proapoptotic proteins has been proposed to bind to the conserved BIR2 domain of TH/Diap1. An analysis of loss-of-function and gain-of-function alleles of th indicates that additional domains of Th/Diap1 are necessary to allow th to inhibit death induced by Rpr, Grim, and Hid. In addition, analysis of loss-of-function mutations demonstrates that th is necessary to block apoptosis very early in embryonic development. This may reflect a requirement to block maternally provided Rpr and Hid, or it may indicate another function of the Th/Diap1 protein (Lisi, 2000).

Several mechanisms of action have been suggested for the antiapoptotic properties of the IAP family of proteins. Among these are the binding of the Drosophila IAPs to the proapoptotic proteins Rpr, Grim, and Hid. This interaction has been demonstrated in overexpression systems, and has been proposed to involve the homologous amino-terminal 14 amino acid sequences of the apoptosis initiators with the second BIR domain of the IAPs. The data presented here suggest that this is an oversimplification. Another mechanism that has been proposed for IAP antiapoptotic activity is the direct binding and inhibition of caspases. Th/Diap1 binds to the Drosophila caspases drICE and DCP-1 and to inhibit their ability to induce apoptosis. Here again, this binding activity appears to rest within BIR2 (Lisi, 2000).

These physical interactions support a simple model of IAP action. In this model, IAPs act within viable cells to inhibit caspase function. The action of Rpr, Hid, and Grim interferes with the ability of IAPs to inhibit caspases, thus inducing apoptosis. On the basis of the model, the LOF mutations identified in this study would be predicted to interfere with the ability of the Th/Diap1 protein to inhibit caspase function. This is likely to be true for th^109.07, which lacks most of the protein, as well as for th⁵ and th⁴, which affect conserved residues in BIR2. BIR2 is sufficient to inhibit apoptosis induced by the active form of the Drosophila caspase drICE. The th⁹ mutation in BIR1 suggests that this BIR is also important for the full function in caspase inhibition. Alternatively, this change in BIR1 might have long-range effects on BIR2 structure or on protein stability (Lisi, 2000).

It is interesting to note that th⁷, which acts as a very strong LOF mutation and seems to show some dominant-negative properties, has only the BIR1 attached to the spacer and ring domains. Thus, despite the extensive homologies between the two BIR domains of the protein, a single BIR is not sufficient for Th/Diap1 function, at least in the presence of an attached ring domain. BIR2 of Th/Diap1 and Op-IAP, as well as the single BIR of survivin, are able to inhibit apoptosis (Lisi, 2000).

Again, on the basis of the model above, the GOF mutations identified would be predicted to bind to caspases, but not to the inducers. The th^SL mutation maps to a weakly conserved residue in BIR1 and does not result in increased th protein levels. This suggests that BIR1 is important for Rpr and Grim binding, but not for Hid binding, as Hid activity is unaffected in this mutation. Even in the context of overexpression in the eyes of transgenic flies, this mutant IAP retains some specificity for Rpr and Grim killing. This implies that the simple model of BIR2 binding to the conserved NH₂-terminal sequences of Rpr, Grim, and Hid is not accurate, and that other residues in the protein are differentially important for Rpr and Grim, as opposed to Hid binding (Lisi, 2000).

The importance of regions outside of BIR2 for Diap1 activity is supported by the analysis of the GOF1 class of mutations, th^6B and th^81.03. Both of these mutations suppress Hid killing and would be predicted to inhibit Hid binding. These mutations change conserved cysteines in the ring domain to tyrosines. This suggests that the ring is important for Hid/Diap1 interaction. However, the region of Hid binding to Diap1 and Op-IAP has been mapped to BIR2, while the ring does not show any ability to bind to Hid. In addition, mutations in the ring, including those in conserved cysteines, have little effect on the ability of Op-IAP to protect against Hid killing. These data, together with the finding that both GOF1 mutations are cysteine-to-tyrosine changes, suggest that these mutations might have a novel ability to interfere with binding of Hid to BIR2. In addition, the observation that the GOF1 mutations slightly enhance Rpr and Grim killing suggests that these mutants are less potent inhibitors of caspases. This might result from weaker binding to caspases or from proteins that are slightly less stable. This second attribute would be predicted to enhance killing by any inducer that binds the IAP, but not to have an effect on Hid, which is unable to bind (Lisi, 2000 and references therein).

In conclusion, the data support a model where Rpr, Grim, and Hid interact with Th/Diap1 to induce apoptosis. Mutations that affect killing by Rpr and Grim or by Hid can be isolated, indicating that these inducers interact with Th/Diap1 in different ways. The GOF mutations that have been identified also provide useful tools to examine the roles of IAPs, rpr, grim, and hid during Drosophila development. The other Drosophila IAP homolog, DIAP2, has been shown to selectively inhibit Rpr- and Hid-induced but not Grim-induced death (Lisi, 2000).

In LOF th alleles, a developmental arrest occurs at the blastoderm stage and, subsequently, a synchronous apoptosis of all the nuclei. Earlier reports that homozygous th embryos show no ectopic apoptosis probably reflects the very early stage at which this apoptosis occurs. At this time, a direct requirement for th to block apoptosis cannot be distinguised from a requirement for th in another developmental process. This developmental defect could then result in secondary apoptosis. The latter possibility is reasonable, as many failures in development result in ectopic apoptosis. A BIR containing protein from Caenorhabditis elegans is required for cytokinesis in embryos. However, it is also possible that developmental arrest occurs as a result of the initiation of apoptosis, which is manifest only as DNA damage several hours later (Lisi, 2000).

Does this early requirement for th reflect a need to inhibit apoptosis induced by rpr, grim, and hid? Double mutants of th and Df(3L)H99, the deletion that removes rpr, grim, and hid, show a phenotype similar to th alone. This indicates that Th/Diap1 is not required to suppress zygotic Rpr, Grim, and Hid activity. However, hid and rpr mRNA can be seen in a subset of cells in the blastoderm embryo, as judged by in situ analysis. This may indicate that these gene products are supplied maternally. Th/Diap1 may be required to suppress maternally supplied Rpr, Grim, or Hid. Allelic differences in the stage at which apoptosis begins in the th mutants parallel the general ability of the alleles to inhibit apoptosis induced by Rpr, Hid, and Grim. The strong LOF alleles arrest at the blastoderm stage; the GOF1 alleles arrest much later, and the GOF2 allele is completely viable (Lisi, 2000).

Expression of the cell death regulatory protein Reaper (RPR) in the developing Drosophila eye results in a smaller than normal eye owing to excess cell death. Mutations in thread (th) are dominant enhancers of RPR-induced cell death. thread encodes a protein homologous to baculovirus inhibitors of apoptosis (IAPs), called Drosophila IAP1 (DIAP1). Overexpression of DIAP1 or a related protein, DIAP2, in the eye suppresses normally occurring cell death as well as death due to overexpression of rpr or head involution defective. IAP death-preventing activity localizes to the N-terminal baculovirus IAP repeats, a motif found in both viral and cellular proteins associated with death prevention (Hay, 1995).

The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, Drosophila and human genes that encode IAP-like proteins (dILP and hILP) have been isolated. Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis (Ducket, 1996).

IAPs comprise a family of inhibitors of apoptosis found in viruses and animals. In vivo binding studies demonstrate that both baculovirus and Drosophila IAPs physically interact with an apoptosis-inducing protein of Drosophila [Reaper (RPR)] through their baculovirus IAP repeat (BIR) region. Expression of IAPs block RPR-induced apoptosis and results in the accumulation of RPR in punctate perinuclear locations, which coincide with IAP localization. When expressed alone, RPR rapidly disappears from the cells undergoing RPR-induced apoptosis. Expression of P35, a caspase inhibitor, also blocks RPR-induced apoptosis and delays RPR decline, but RPR remains cytoplasmic in its location. Mutational analysis of RPR demonstrates that caspases are not directly responsible for RPR disappearance. The physical interaction of IAPs with RPR provides a molecular mechanism for IAP inhibition of RPR's apoptotic activity (Vucic, 1997a).

Members of the transforming growth factor-beta superfamily bind to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptors act as downstream components of type II receptors in the receptor complexes. Therefore, intracellular proteins that interact with the type I receptors are likely to play important roles in signaling. Drosophila inhibitor of apoptosis (Diap) 1 has been identified as an interacting protein of Thick veins (Tkv), a Dpp type I receptor. Diap1 associates with Tkv in vivo. The binding region in Diap1 is mapped to its C-terminal RING finger region. Diap2, another Drosophila member of the inhibitor of apoptosis protein family, also interacts with Tkv in vivo. These data suggest that Diap1 and Diap2 may be involved, possibly as negative regulators, in the Dpp signaling pathway, which leads to cell apoptosis. Tkv may induce apoptosis by suppressing the DIAP1 function (Oeda, 1998).

Ectopic expression of Reaper or Grim induces substantial apoptosis in mammalian cells. Reaper- or Grim-induced apoptosis is inhibited by a broad range of caspase inhibitors and by human inhibitor of apoptosis proteins cIAP1 and cIAP2. Additionally, in vivo binding studies demonstrate that both Reaper and Grim physically interacte with human IAPs through a homologous 15-amino acid N-terminal segment. Deletion of this segment from either Reaper or Grim abolishes binding to cIAPs. In vitro binding experiments indicate that Reaper and Grim bind specifically to the BIR domain-containing region of cIAPs, since deletion of this region results in loss of binding. The physical interaction has been further confirmed by immunolocalization. When co-expressed, Reaper or Grim co-localize with cIAP1. However, deletion of the N-terminal 15 amino acids of Reaper or Grim abolishes co-localization with cIAP1, suggesting that this homologous region can serve as a protein-protein interacting domain in regulating cell death. Moreover, by virtue of this interaction, it has been demonstrated that cIAPs can regulate Reaper and Grim by abrogating their ability to activate caspases and thereby inhibit apoptosis. This is the first function attributed to this 15-amino acid N-terminal domain, which is the only region having significant homology between these two Drosophila death inducers (McCarthy, 1998).

Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival in Drosophila and appear to neutralize the cell death machinery by binding to and ubiquitylating pro-apoptotic caspases. Cell death is triggered when 'Reaper-like' proteins bind to IAPs and liberate caspases from IAPs. The thioredoxin peroxidase Jafrac2 has been identified as an IAP-interacting protein in Drosophila cells that harbors a conserved N-terminal IAP-binding motif. In healthy cells, Jafrac2 resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Mature Jafrac2 interacts genetically and biochemically with DIAP1 and promotes cell death in tissue culture cells and the Drosophila developing eye. In common with Rpr, Jafrac2-mediated cell death is contingent on DIAP1 binding because mutations that abolish the Jafrac2-DIAP1 interaction suppress the eye phenotype caused by Jafrac2 expression. Jafrac2 displaces Dronc from DIAP1 by competing with Dronc for the binding of DIAP1, consistent with the idea that Jafrac2 triggers cell death by liberating Dronc from DIAP1-mediated inhibition (Tenev, 2002).

Jafrac2 was recovered as a DIAP1-interacting protein in the cell using the tandem affinity purification (TAP) system. Like Rpr, Grim, Hid, Sickle, Smac/DIABLO and HtrA2/Omi, Jafrac2 bears a conserved N-terminal IAP-binding motif (IBM) essential for IAP interaction. Jafrac2 is synthesized as a precursor protein with an N-terminal signal peptide that targets it to the ER. Upon import into the ER, the signal peptide of Jafrac2 is cleaved off, thereby exposing the IAP interacting domain that allows this mature Jafrac2 isoform to interact with DIAP1, DIAP2 and XIAP (Tenev, 2002).

In living cells Jafrac2 is compartmentalized and sequestered in the ER away from IAPs, where it exists exclusively in the processed from. This is evident because mature Jafrac2, like cytochrome c, which is compartmentalized in mitochondria, remains associated with the membrane fraction in healthy cells. Following stimulation of apoptosis by UV irradiation or ER stress-inducing agents, mature Jafrac2 is released from the membrane fraction and is present in the cytosol where it can interact with DIAP1 and DIAP2. Because the pro-apoptotic, IAP-interacting form of Jafrac2 is released only upon cell death insult, the major regulatory step for Jafrac2 appears to be its release from the ER lumen. The release of Jafrac2 from the ER of UV-irradiated cells occurs early in UV-mediated apoptosis. This is evident because Jafrac2 expression becomes diffuse in otherwise morphologically normal cells within 3-4 h following UV exposure. In similar experiments, the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi that occurs, early in apoptosis, also became apparent within 3-4 h following UV treatment. Thus, Jafrac2 resembles Smac/DIABLO and HtrA2/Omi that are similarly compartmentalized in healthy cells and that promote caspase activation after their release from mitochondria following the cell death trigger. Furthermore, analogous to Smac/DIABLO and HtrA2/Omi, Jafrac2 also requires N-terminal processing to generate its pro-apoptotic form. Hence, Jafrac2, Smac/DIABLO and HtrA2/Omi all undergo a maturation process through cleaving off their signal peptide following import into their respective organelles. This organelle-specific maturation ensures that newly synthesized Jafrac2, Smac/DIABLO and HtrA2/Omi will not promote apoptosis prior to their sequestration into organelles (Tenev, 2002).

In common with Rpr, Grim and Hid, Jafrac2 interacts genetically and biochemically with DIAP1 and is able to promote cell death. In the Drosophila eye and tissue culture cells, mature Jafrac2, like Rpr, efficiently induces cell death in a DIAP1-binding dependent manner. Recent studies have suggested that Rpr and Grim antagonize the anti-apoptotic activity of IAPs by two distinct mechanisms -- (1) by a mechanism that requires DIAP1 binding, Rpr promotes DIAP1 self ubiquitylation and proteasomal degradation and (2) Rpr and Grim were also found to repress global protein translation by a mechanism that does not rely on IAP binding. The Ub fusion technique has been used to examine whether Jafrac2 and Rpr possess apoptosis-promoting activities that are independent of IAP binding. In vivo Rpr and Jafrac2 promote cell death exclusively in an IAP-binding dependent manner because mutations that impair the binding between DIAP1 and Rpr or Jafrac2 completely abolish their ability to induce cell death in the developing eye and tissue culture cells. Thus, Rpr and Jafrac2 that fail to bind to DIAP1 also fail to induce cell death. Mutations in endogenous diap1, which greatly impair the binding of DIAP1 to Rpr or Jafrac2, suppress Rpr and Jafrac2-mediated cell killing. Together, these data argue that in common with Rpr, mature Jafrac2 promotes cell death, and this activity is contingent upon their binding to DIAP1 (Tenev, 2002).

The interaction between Jafrac2 and the DIAP1 BIR2 domain is indispensable for its pro-apoptotic function. Interestingly, Jafrac2 and Dronc share a common binding site in the BIR2 domain that is distinct from the site of interaction between the DIAP1 BIR2 domain and Rpr and Hid. The th⁴ mutation of DIAP1's BIR2 domain greatly diminishes binding to Jafrac2 and Dronc, whereas the same mutation does not affect its binding to Rpr and Hid. In addition, the th23-4 DIAP1 mutation that greatly impairs the binding of DIAP1 to Rpr and Hid does not affect the DIAP1-Jafrac2 interaction. Consistent with the biochemical data, flies carrying the th⁴ mutation, which abolishes Jafrac2 binding, display strongly suppressed Jafrac2-induced eye ablation but enhanced Rpr-induced cell death in the eye (Tenev, 2002).

Several lines of evidence show that the IBM of Jafrac2 is essential for IAP binding and induction of apoptosis. Mutations that delete or obstruct the N-terminus of mature Jafrac2 abrogate the ability of Jafrac2 to bind to DIAP1 and trigger cell death. The view that Jafrac2 harbors a bona fide IAP-binding motif is strongly supported by crystal structure analyses that have identified Ala1 of IBMs as the critical residue to anchor this motif to the BIR surface of IAPs. In addition to the requirement of Ala1, there is a strong preference for Pro3. In accordance with other IBMs, the putative IBM of mature Jafrac2 bears Ala1 and Pro3. Furthermore, the IBM of Rpr is functionally interchangeable with the IBM of Jafrac2. A chimeric Rpr mutant (AKP-Rpr) in which the IBM of Rpr was replaced with the IBM of Jafrac2, displayed the same phenotype and cell death promoting efficacy as wild-type Rpr (AVA-Rpr) in both the Drosophila developing eye and tissue culture cells. Together, these results reveal that whereas Jafrac2 and Rpr share a common IAP-binding motif, they also have some distinct DIAP1-binding requirements that presumably give these interactions their specificity (Tenev, 2002).

Physical interaction between DIAP1 and caspases is essential to regulate apoptosis in vivo because embryos with a homozygous mutation that abolishes Dronc binding die early during embryogenesis due to widespread apoptosis. Unrestrained cell death caused by loss of DIAP1 function requires the Drosophila Apaf-1 homolog DARK because a mutation in dark rescues DIAP1-dependent defects. Thus, loss of DIAP1 function allows DARK-dependent caspase activation. Although activation of downstream, effector caspases is required for normal cell death, the activation of initiator caspases, such as Dronc, is rate limiting for the activation of this cascade. The observed unrestrained cell death caused by loss of DIAP1 function is likely to be triggered by the initiator caspase Dronc because DIAP1 normally suppresses Dronc activation, which in turn is mediated by DARK. In line with the current model on caspase activation, it is argued that the DIAP1-mediated inhibition of Dronc is the key regulatory step in controlling cell death. This view is supported by the observation that flies with diap1 mutations that either abolish binding or ubiquitylation of Dronc completely fail to suppress Dronc- mediated cell death in vivo. Thus, DIAP1 suppresses Dronc activation by binding to and targeting Dronc for ubiquitylation. However, when Rpr-like molecules displace DIAP1 from Dronc, Dronc is recruited into a 700 kDa size apoptosome protein complex that results in Dronc activation. Consequently, cell death is triggered when Dronc is liberated from DIAP1. Thus, the key event in regulating the caspase cascade appears to be inhibition of Dronc by DIAP1 (Tenev, 2002).

Several lines of evidence support the notion that Jafrac2 promotes cell death by interfering with the Dronc-DIAP1 interaction, thereby displacing and liberating Dronc from DIAP1. (1) Jafrac2 and Dronc bind to the same site of the BIR2 domain of DIAP1, since the BIR2 th4 mutation of DIAP1 equally abolished Dronc and Jafrac2 binding. In contrast, Rpr and Hid binding to the th⁴ DIAP1 mutant remains unaffected. (2) Jafrac2 competes with Dronc for the binding of DIAP1, and Jafrac2 possesses a significantly higher DIAP1-binding affinity compared with that of Dronc to DIAP1, as would be expected of a protein that displaces Dronc from DIAP1. (3) Ectopic expression of Jafrac2 in the developing Drosophila eye causes a phenotype that is highly reminiscent of the phenotype observed in flies ectopically expressing Dronc. (4) Heterozygosity at the dronc locus rescued the eye-ablation phenotype induced by Jafrac2, indicates that apoptotic signal transduction initiated by Jafrac2 is mediated through Dronc. Taken together, these results indicate that Jafrac2 promotes cell death by liberating Dronc from the anti-apoptotic activity of DIAP1 (Tenev, 2002).

The observation that Jafrac2, like the apoptotic inducers Rpr, Grim and Hid, induces apoptosis through binding to DIAP1 places Jafrac2 in a potentially pivotal position to regulate apoptosis. The findings are consistent with a model whereby Jafrac2 promotes apoptosis by displacing DIAP1 from Dronc, so allowing activation of the caspase cascade and consequent cell death. The idea is favored whereby Jafrac2 function is additive to, but independent of, Rpr. The early release of Jafrac2 from the ER of UV-irradiated cells is consistent with the view that Jafrac2 is involved in the initiation of apoptosis. Thus, Jafrac2 is released from the ER at a time when other early apoptotic events occur, such as the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi in mammalian cells. Once released, Jafrac2 interacts with DIAP1 and thereby liberates Dronc, which in turn is activated by DARK. In line with the notion that Jafrac2 functions in a complementary but distinct cell death pathway to Rpr, Grim and Hid, it is found that a chromosomal deletion that includes the jafrac2 locus does not suppress the eye phenotypes caused by ectopic expression of Rpr, Grim and Hid. However, it is possible that Jafrac2 may also be part of a positive feedback mechanism, which cooperates with Rpr-like proteins to promote apoptosis in response to cellular damage. These two alternatives cannot be distinguished because no jafrac2 mutant flies are available and Jafrac2 is refractory to the effect of dsRNA interference (Tenev, 2002).

The data are consistent with the idea that Jafrac2, with its thioredoxin peroxidase activity and IAP-binding ability, contains two distinct functions. In healthy cells, Jafrac2 may fulfil a 'housekeeping' role through its peroxidase activity by protecting the cell from oxidative damage. Consistent with this view, members of the peroxiredoxin protein family play an important role in protecting cells against oxidative damage by scavenging intracellularly generated reactive oxygen species, such as H₂O₂. However, upon UV irradiation, mature Jafrac2 is released from the ER and competes with Dronc for the binding of DIAP1 that is independent of its peroxidase activity. Consequently, Jafrac2 liberates Dronc from DIAP1 inhibition and allows activation of the proteolytic caspase cascade, resulting in cell death (Tenev, 2002).

Members of the IAP family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper Rpr, Grim and Hid all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. In vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. These data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination (Wilson, 2002).

Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death is largely unknown. Through a genetic screen, it has been shown that a mutant of Drosophila Tumor-necrosis factor receptor-associated factor 1 (DTRAF1) is a dominant suppressor of Reaper-induced cell death. Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates DTRAF1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of DTRAF1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of DTRAF1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of DTRAF1-mediated JNK activation in Drosophila cells. Taken together, these results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin-proteasome pathway (Kuranaga, 2002).

Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Drosophila IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. Rpr, but not Hid (Head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, ubcD1 was found to suppresses rpr-induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro. These results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting its degradation (Ryoo, 2002).

In most cases, apoptotic cell death culminates in the activation of the caspase family of cysteine proteases, leading to the orderly dismantling and elimination of the cell. The IAPs (inhibitors of apoptosis) comprise a family of proteins that oppose caspases and thus act to raise the apoptotic threshold. Disruption of IAP-mediated caspase inhibition has been shown to be an important activity for pro-apoptotic proteins in Drosophila (Reaper, HID, and Grim) and in mammalian cells (Smac/DIABLO and Omi/HtrA2). In addition, in the case of the fly, these proteins are able to stimulate the ubiquitination and degradation of IAPs by a mechanism involving the ubiquitin ligase activity of the IAP itself. The Drosophila RHG proteins (Reaper, HID, and Grim) are themselves substrates for IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, regulation of Reaper by ubiquitination has been shown to be a significant factor in determining Reaper biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance (Olson, 2003a).

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. This domain, although required for IAP destabilization, is not sufficient. Moreover, a small region of Reaper, similar to the GH3 domain of Grim, has been identified that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain is deficient in these properties, these defects can be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells (Olson, 2003b).

Death executioner Bcl-2 homolog

The Bcl-2 family of proteins consists of antiapoptotic and proapoptotic members, both of which control the cell-death decision by regulating such processes as mitochondrial cytochrome c release and caspase activation through adapter protein Apaf-1 (Drosophila homolog: Apaf-1-related-killer), and/or by neutralizing the effects of opposing Bcl-2 family members. The first Drosophila Bcl-2 protein to be described has been termed it Debcl (pronounced debacle) for Death executioner Bcl-2 homolog. After screening a number of UAS-debcl lines, two lines (UAS-debcl#26 on chromosome III and UAS-debcl#18 on chromosome II) were found that, when crossed to GMR-GAL4, give rise to adults with severely ablated eyes. To use this phenotype to examine genetic interactions, a stock was generated containing GMR-GAL4 (2nd chromosome) and UAS-debcl#26. To examine whether the rough eye phenotype is due to the activity of caspases, GMR-p35 was crossed to these flies and the eye phenotype of the progeny was examined. GMR-p35 significantly improves the severe rough eye phenotype of GMR-GAL4; UAS-debcl#26 eyes. These results confirm that Debcl functions in a caspase-dependent fashion upstream of caspase activation (Colussi, 2000).

To determine the involvement of rpr, hid, or grim in the GMR-GAL4; UAS-debcl#26 eye phenotype, these flies were crossed to a deficiency that removes all three genes (Df(3L)H99). If rpr, hid, or grim are rate limiting for Debcl function, then suppression of the GMR-GAL4; UAS-debcl#26 eye phenotype would be expected. However, no significant suppression of this phenotype was observed, suggesting that the GMR-GAL4; UAS-debcl#26 eye phenotype is not dependent on the gene dosage of rpr, hid, or grim (Colussi, 2000).

Next, a test was performed to see whether the inhibitor of apoptosis (IAP) homolog, diap1, genetically interacts with debcl, by examining the GMR-GAL4; UAS-debcl#26 eye phenotype when the dosage of diap1 is halved. Halving the dosage of diap1, using two different deficiencies, results in a strong enhancement of the GMR-GAL4; UAS-debcl#26 eye phenotype. Furthermore, there is a significant reduction in the number of flies expected to contain either of the diap1 deficiencies and GMR-GAL4; UAS-debcl#26. This is possibly due to leaky expression of the GMR-GAL4; UAS-debcl#26 construct in other tissues during development and the enhancement of this effect by reducing the dose of diap1. Thus, diap1 genetically interacts with debcl. No genetic interaction between debcl and diap2 was observed when a diap2 deficiency was crossed with GMR-GAL4; UAS-debcl#26 (Colussi, 2000).

Shaker K+ channel

Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. Voltage-dependent Shaker (Sh) K+ channels open in response to depolarization and subsequently undergo N-type inactivation by a "ball and chain" mechanism. The 20 N-terminal residues of the ShB channel form the inactivation "ball," which is tethered to membrane-spanning channel domains by the following ~200-residue "chain." Inactivation occurs when the N-terminal inactivation ball physically occludes the inner pore of the channel from the cytoplasmic side. Stability of the inactivated state is enhanced by the hydrophobicity of approximately the first 10 residues of the inactivation ball, whereas positively charged amino acids within the following 10 residues promote entry into the inactivated state via electrostatic interactions. Deletions in the distal N terminus of the channel disrupt inactivation, which can be reversed by application of a 20-residue synthetic peptide corresponding to the initial N-terminal sequence of the channel. Ancillary beta subunits in some K+ channel complexes serve to produce N-type inactivation by a similar mechanism. The conserved N-terminal sequences of Reaper, Grim, and Hid resemble those N-terminal Sh K+ channel domains that are involved in inactivation. This sequence similarity led to the hypothesis that Reaper, Grim, and Hid facilitate initiation of apoptosis by inducing inactivation of K+ channels. Sustained inactivation of K+ channels will result in chronic membrane depolarization that may lead to the initiation of the caspase-dependent apoptotic program, perhaps by increasing the level of cytosolic free Ca2+. Synthetic Reaper and Grim N terminus peptides are shown to induce fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that is qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlates with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein causes near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels (Avdonin, 1998).

Head involution defective

Deletion of chromosomal region 75C1,2 blocks virtually all programmed cell death (PCD) in the Drosophila embryo. A second gene in this region, head involution defective (hid) plays a similar role in PCD. hid mutant embryos have decreased levels of cell death and contain extra cells in the head. The hid gene expression is sufficient to induce PCD in cell death defective mutants. The hid gene encodes a novel 410-amino-acid protein, and its mRNA is expressed in regions of the embryo where cell death occurs. Ectopic expression of hid in the Drosophila retina results in eye ablation. This phenotype can be suppressed completely by expression of the anti-apoptotic p35 protein from baculovirus, indicating that p35 may act genetically downstream from hid (Grether, 1995).

Interleukin-1 beta converting enzyme (ICE)-like proteases

Genetic studies indicated that the Drosophila protein Reaper controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulates apoptosis. RPR-mediated apoptosis is blocked by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1 beta converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis is associated with increased ceramide production that is also blocked by Z-VAD-fmk, suggesting that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1 (Pronk, 1996).

While Caenorhabditis elegans has only a single identified caspase, CED-3, whose activity is absolutely required for all developmental programmed cell deaths, most mammalian cell types express multiple caspases with varying specificities. The fruit fly possesses two known caspases: DCP-1 and drICE. The role of drICE was examined in in vitro apoptosis of the D. melanogaster cell line S2. Cytoplasmic lysates made from S2 cells undergoing apoptosis induced by either reaper expression or cycloheximide treatment contain a caspase activity with DEVD specificity that can cleave p35, lamin DmO, drICE and DCP-1 in vitro, one that can trigger chromatin condensation in isolated nuclei. Immunodepletion of drICE from lysates is sufficient to remove most measurable in vitro apoptotic activity; re-addition of exogenous drICE to such immunodepleted lysates restores apoptotic activity. It is concluded that, at least in S2 cells, drICE can be the sole caspase effector of apoptosis (Fraser 1997).

ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central role in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. The CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protects cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduces the cell death induced by ced-3 in Drosophila SL2 cells. Apoptosis of SL2 cells induced by reaper, a Drosophila cell-death gene, is partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems for dissecting the main machinery of cell death (Hisahara, 1998).

The cytoplasmic region of Fas, a mammalian death factor receptor, shares a limited homology with Reaper, an apoptosis-inducing protein in Drosophila. Expression in Drosophila cells of either the Fas cytoplasmic region (FasC) or reaper causes cell death. The death process induced by FasC or reaper is inhibited by crmA or p35, suggesting that in both cases the death process is mediated by caspase-like proteases. Both Ac-YVAD aldehyde and Ac-DEVD aldehyde, specific inhibitors of caspase 1- and caspase 3-like proteases, respectively, inhibited the FasC-induced death of Drosophila cells. However, the cell death induced by Reaper is inhibited by Ac-DEVD aldehyde, but not by Ac-YVAD aldehyde. A caspase 1-like protease activity that preferentially recognizes the YVAD sequence gradually increases in the cytosolic fraction of the FasC-activated cells, whereas the caspase 3-like protease activity recognizing the DEVD sequence is observed in the Reaper-activated cells. Partial purification and biochemical characterization of the proteases indicates that there are at least three distinct caspase-like proteases in Drosophila cells that are differentially activated by FasC and Reaper. The conservation of the Fas-death signaling pathway in Drosophila cells, which is distinct from that for Reaper, may indicate that cell death in Drosophila is controlled not only by the Reaper suicide gene, but also by a Fas-like killer gene (Kondo, 1997).

Scythe: a Xenopus Reaper-binding protein

Reaper is a central regulator of apoptosis in D. melanogaster. With no obvious catalytic activity or homology to other known apoptotic regulators, Reaper's mechanism of action has remained obscure. Recombinant Drosophila Reaper protein induces rapid mitochondrial cytochrome c release, caspase activation and apoptotic nuclear fragmentation in extracts of Xenopus eggs. This paper reports a 150 kDa Reaper-interacting protein from Xenopus egg extracts, named Scythe. Scythe is highly conserved among vertebrates and contains a ubiquitin-like domain near its N-terminus. Other than this conserved domain, Scythe shows no other resemblence to proteins in the database. Immunodepletion of Scythe from extracts completely prevents Reaper-induced apoptosis without affecting apoptosis triggered by activated caspases. Moreover, a truncated variant of Scythe lacking the N-terminal domain induces apoptosis even in the absence of Reaper. Since Reaper requires cooperating cytosolic factors to trigger mitochondrial cytochrome c release, it was hypothesized that Scythe might be a cytochrome c-releasing factor. Indeed, addition of truncated Scythe to crude egg extracts accelerated release of cytochrome c from the mitochondria, relative to controls. Truncated Scythe is also able to trigger cytochrome c release when added to a mixture of isolated cytosol and mitochondria. Unlike truncated Scythe, full-length Scythe does not induce mitochondrial cytochrome c release in either crude extract or isolated cytosol; in several experiments, some suppression of cytochrome c release by the full-length Scythe protein has been observed. In contrast to the results obtained in the presence of cytosol, truncated Scythe does not promote cytochrome c release from isolated mitochondria in buffer (in the absence of other cytosolic proteins), even in the presence of recombinant Reaper. These data suggest that other accessory cytosolic factors are required to promote cytochrome c release. Efforts by many researchers have failed to identify a vertebrate reaper homolog using standard molecular cloning techniques. Given the findings reported here, it is hypothesized that Drosophila Reaper triggers apoptosis in Xenopus egg extracts by mimicking an endogenous vertebrate Scythe-activating factor. By analogy to Reaper, such a Scythe-activating factor might be transcriptionally induced in response to external stimuli or in response to developmental cues. Therefore, using Scythe as a bait to search for Reaper-like factors in extracts from appropriately staged or irradiated embryos may provide a means to isolate Reaper-like factors that may not be well conserved at the primary sequence level. It will be equally interesting to determine whether there are Scythe-related proteins acting downstream of Reaper in Drosophila. It is theoretically possible that Reaper accesses an apoptotic pathway in Xenopus egg extracts that is distinct from the one used in flies. It is concluded that Scythe is a novel apoptotic regulator that is an essential component in the pathway of Reaper-induced apoptosis (Thress, 1998).

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper that is detectably homologous to Grim and Hid is dispensable for Scythe binding (Thress, 1999).

Apaf-1-related-killer

Genetic studies of cell death in Drosophila have led to the identification of three apoptotic activators: rpr, head involution defective (hid), and grim. The deletion of all three genes blocks apoptosis in the Drosophila embryo, and overexpression of any one of them is sufficient to kill cells that would normally live. The products of these genes appear to activate one or more caspases, because cell killing by rpr, hid, and grim is blocked by the caspase inhibitor p35. If Apaf-1-related-killer (Ark) actually acts as a caspase activator, like Apaf-1/CED-4 in adult flies, the downstream pathways of one or more of these three gene products should depend on Ark function to activate caspases. dpf^K1/dpf^K1 flies were crossed to the GMR-rpr and GMR-hid strains to examine whether or not there are any genetic interactions. Compared with GMR-rpr adult flies in a wild-type background, GMR-rpr flies homozygous for dpf^K1 show significantly improved eye morphology, but no obvious influence on hid-activated cell killing was observed in this case. drICE and Dredd appear to be activated downstream of Rpr in Drosophila S2 cells, and drICE is an essential caspase in rpr-induced cell death, consistent with observations that Dapaf-1L and Dapaf-1S activate drICE in S2 cells. These results suggest that Ark is involved in the rpr-induced cell death pathway, and the contribution of Ark against hid-induced cell death may not be as high as that of rpr (Kanuka, 1999).

The genetic evidence that Ark interacts with rpr and the observation that Dapaf-1L contains WDRs strongly imply that cyt c might act as an initiator for Dapaf-1-mediated caspase activation. Overexpression of rpr and treatment with staurosporine or cycloheximide causes rapid caspase activation and increase of cyt c in digitonin-extracted lysates of Drosophila S2 cells. In S2 cells, immunoprecipitation experiments reveal that released cyt c by rpr directly interacts with Dapaf-1L, but Dapaf-1S, which lacks WDRs, binds to cyt c weakly. These observations suggest that one candidate for the internal signaling molecule between Rpr and Ark could be cyt c, and the target of cyt c would be Dapaf-1L, a structural homolog of mammalian Apaf-1 (Kanuka, 1999).

In mammals and Drosophila, apoptotic caspases are under positive control via the CED-4/Apaf-1/Dark adaptors and negative control via IAPs (inhibitor of apoptosis proteins). However, the in vivo genetic relationship between these opposing regulators is not known. In this study, it has been demonstrated that a dark mutation reverses catastrophic defects seen in Diap1 mutants and rescues cells specified for Diap1-regulated cell death in development and in response to genotoxic stress. dark function is required for hyperactivation of caspases that occurs in the absence of Diap1. Since the action of dark is epistatic to that of Diap1, these findings demonstrate that caspase-dependent cell death requires concurrent positive input through Apaf-1-like proteins together with disruption of IAP-caspase complexes (Rodriguez, 2002).

Mutations in dark cause a variety of developmental defects, including an enlarged larval CNS. In the absence of dark function, cells specified to die actually survive and also differentiate. The embryonic CNS midline glia is a well-studied cell lineage in the fly embryo and, though only this particular lineage was studied, it is likely that supernumerary cell types in other lineages also persist in dark mutants. Several important conclusions derive from these observations. (1) The persistence of extra cells excludes trivial explanations for reduced TUNEL labeling in dark mutants (e.g. cell death in the absence of TUNEL labeling or redirected cell fates that prevent the specification of PCD) and reinforces models that favor a fundamental role for dark in embryonic PCD. (2) As shown in C.elegans and in studies of Reaper interval Drosophila mutants, rescue from PCD can uncover a cryptic differentiation program. (3) Most important, the same midline glial cells that fail to die in dark mutants also require the induced action of reaper, grim and hid. There is a requirement for dark function in normal PCD when reaper, grim and/or hid are expressed at physiological levels. It is noteworthy that Diap1 and at least two of the cell death initiators (i.e. reaper and hid) are expressed in pools of progenitor cells, which give rise to the embryonic midline glia and also play an important role in specifying the death of these cells. Thus, although midline glia presumably receive death signals from reaper and hid leading to the disruption of Diap1-caspase interactions, these cells still fail to die and are even capable of differentiating normally in the absence of dark function. This inference supports conclusions from epistasis studies indicating that a disruption of caspase-Diap1 interactions alone is insufficient for apoptosis, and suggests that dark functions as a co-effector of cell death signaling along with reaper, grim and/or hid. It is worth noting that the mammalian ortholog of dark, Apaf-1, functions downstream from the point at which mitochondrial factors including cytochrome c are released from mitochondria. If the same holds true for the fly protein, it follows that embryonic midline glia (and perhaps other cell types) can survive and differentiate beyond this mitochondrial 'point of no return' (Rodriguez, 2002).

Additional evidence that PCD requires coordinated dark-dependent caspase activation in conjuction with the release of IAP-mediated caspase inhibition comes from tests of dark mutants under conditions of stress that elicit apoptotic responses by the embryonic cell death initiators. Like the Reaper interval mutants, dark mutants also exhibit profound failures in cell death in response to ionizing radiation. Interestingly, the dark mutation itself does not interfere with the radiation induction of reaper mRNA. Therefore the possibility that resistance to damage-induced apoptosis evident in irradiated dark mutants is caused by a disruption of upstream elements in the signaling pathway can be excluded. Instead, the results raise the possibility that dark, like reaper, may function as an important effector of Drosophila p53-mediated apoptosis. In addition, these findings reinforce an apoptotic requirement for dark (even when reaper is transcriptionally induced) and provide evidence for models where cell death signals do not converge on Diap1 alone. In future studies, it will be interesting to determine the range of damage signals that can engage dark activity, since this locus might also function in a broader range of damage signals beyond those provoked by radiation (Rodriguez, 2002).

In flies, Diap1 is thought to function as a rate-limiting brake on apoptotic cell death. If, however, Diap1 were the most proximal rate-limiting regulator of apoptosis, then the presence or absence of dark function should have no influence on Diap1-dependent effects. If, however, dark and Diap1 exert interdependent functions, then the opposite outcome is predicted. The results from these studies provide consistent and compelling evidence favoring the latter scenario since, wherever tested, the influence of Diap1 upon apoptotic signaling is heavily dependent upon intact dark function. In the dark^CD4 mutant, for instance, the Diap1⁵ mutation fails to enhance grim- and hid-induced cell killing in the eye. These experiments examined Diap1 in the heterozygous condition, but the same outcome is also true when Diap1 is tested in the homozygous state. Early in embryonic development, Diap1 homozygotes exhibit catastrophic phenotypes including morphological arrest soon after gastrulation, extensive TUNEL-positive nuclei and hyperactivation of caspases. Each of these defects is profoundly dependent upon dark function since each is dramatically reversed by the dark mutation. In dark^CD4; Diap1⁵ double mutant embryos, no evidence is found for widespread apoptosis and, in fact, the large majority of these double mutants proceed through stages of embryogenesis well beyond the point at which single Diap1 mutants arrest. Thus, loss of dark not only suppresses apoptotic deaths that would otherwise occur, but definitively reverses a profound morphogenetic arrest that ensues in the absence of Diap1. Consequently, homozygosity at dark not only prevents the onset of apoptotic markers (i.e. DNA fragmentation and/or caspase activation), but actually preserves cells that are otherwise fated to die when normal checks upon caspases are removed. Rescue from inappropriate apoptosis in this instance is consistent with what was observed in the midline glia but, rather than preserving cells fated for programmed death, rescue occurs even when signaling from IAP antagonists in the Reaper region is bypassed. A similar result, with similar implications, was obtained in the ovary, where ~90% of heteroallelic Diap1^6/8 mutants show abnormal degeneration of early staged egg chambers and associated sterility. Again, loss of dark function not only suppresses the pathological effect, but actually reverses these degenerative defects to the extent that half of the dark^CD4; Diap1^6/8 double mutant females produce normal egg chambers and are fertile. Thus, in both the ovary and the embryo, loss of dark rescues functional cells from apoptosis caused by misregulated Diap1 (Rodriguez, 2002).

Reversal of Diap1-dependent defects by dark is also evident when caspase activity is directly assayed in early embryos. In contrast to the 'hyperactivated' caspase levels detected in Diap1 single mutant lysates, Diap1⁵; dark^CD4 lysates show levels of caspase activity that are suppressed >90% relative to Diap1 mutants. The unusually high caspase activity detected in Diap1 mutant embryos is thought to reflect the action of these proteolytic enzymes unimpeded by native inhibitors. However, studies here demonstrate that removing a negative regulator alone is not sufficient to achieve caspase hyperactivation, since dark is clearly required for this unrestrained activity. It is worth mentioning that the basal DEVDase activity in double mutants is still somewhat elevated compared with control embryos. This might indicate dark-independent caspase activity due to caspase auto-activation in the absence of Diap1 or, since it is possible that the dark^CD4 allele used here is not a complete null, it could reflect hypomorphic dark function. Alternatively, since a role for Diap1 in cytokinesis has not been ruled out, it is possible that this residual caspase activity ensues because of secondary developmental defects leading to cell death. Nevertheless, these enzymatic data extend the analyses to the biochemical level in a manner fully consistent with phenotypic studies (Rodriguez, 2002).

Interestingly, since Apaf-1/Dark adaptor proteins act upon procaspases while IAPs preferentially inhibit processed caspases, these results further suggest that pre-existing levels of processed caspases in most cells are probably not high enough to achieve an apoptotic threshold. Instead, a positive cell death stimulus from dark is required for the unusually high levels of caspase activation seen in Diap1^-/- embryos. It is also evident that Dark-dependent 'basal' levels of DEVDase are detected in these assays several hours before the onset of embryonic PCD, and therefore authentic effector caspase (e.g. DrICE, Dcp-1) activity occurs even in the absence of overt apoptotic signals. These data indicate that constitutive levels of active effector caspases, not derived from autoproteolysis but instead promoted by Apaf-1-like adaptor proteins, may exist in many and perhaps all viable cells (Rodriguez, 2002).

Taken together, a strict Diap-1-caspase 'liberation' model does not explain sufficiently the evidence described. The action of dark is epistatic to that of Diap1, demonstrating an order of gene action whereby Dark functions either downstream or parallel to Diap1. Therefore, simple derepression of caspases via an IAP inhibitory bridge does not account adequately for epistasis between Diap1 and dark. Put another way, these results support the notion that apoptotic cell death in vivo results from the simultaneous activation of caspases by dark and the derepression of caspases by reaper, grim and/or hid. Accordingly, the findings are inconsistent with models that presume that Diap1 is the sole effector of reaper, grim and hid and that cells are 'pre-loaded' with sufficient levels of IAP-inhibited processed caspases to achieve cell killing. Instead, a 'gas and brake' model is favored whereby positive input from Apaf-1/Dark adaptors, together with removal of IAP inhibition, drives caspase activation to levels that exceed a threshold necessary for apoptosis (Rodriguez, 2002).

RHG motif of Reaper confers distinct cell death-inducing abilities

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH2 -terminal Reaper-Hid-Grim (RHG) motif. The importance of the RHG motifs in Reaper and Grim have been examined for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicates that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retains an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions are not efficiently blocked by the cell death inhibitors Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, a second region of sequence similarity was identified in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities (Wing, 2001b).

Analyses of R/Grim and G/Reaper chimeras have indicated that the closely related RHG motifs of Reaper and Grim are not functionally interchangeable. Instead, the four amino acid substitutions between their RHG motifs help determine the unique cell killing abilities of Reaper and Grim. For example, unlike Grim, R/Grim resembles Reaper and is unable to induce cell death in the CNS midline. In contrast, one P[UAS-g/reaper] strain induces significant midline cell death, implying that the presence of the Grim RHG motif can confer Grim-like cell killing abilities on Reaper. It is important to note, however, that the identity of the RHG motif does not completely transform the cell killing properties of the chimeras, indicating that other regions of Reaper and Grim proteins are also crucial for their distinct actions. In this regard, like Grim, both R/Grim and G/Reaper are able to act synergistically with Reaper to induce CNS midline cell death (Wing, 2001b).

While the Grim-Reaper proteins do not contain defined structural domains, they each share sequence similarity in the 14 amino acids at their NH2-termini. This RHG (Reaper, Hid, Grim) motif is most similar between Reaper and Grim (71.4% identity), and least similar between Hid and Grim (21.4% identity). The RHG motif plays a key role in interactions between Grim-Reaper proteins and members of the Inhibitor-of-Apoptosis-Protein (IAP) family, including Drosophila Diap1 and Diap2. Like other IAPs, Diap1 and Diap2 both contain related baculovirus IAP repeat (BIR) motifs, as well as a Really Interesting New Gene (RING) finger. Diap1 is an essential cell death regulator and diap1 mutants exhibit early embryonic lethality due to massive ectopic cell death. The functions of Diap2 in regulating cell death are less clear; however, it does share a number of functional properties with Diap1. Diap1 can directly bind caspases and repress their proteolytic activities. Significantly, caspase inhibition by Diap1 is antagonized by Hid, suggesting a double-repression model where the Grim-Reaper proteins promote cell death by binding to Diaps, suppressing their ability to inhibit caspases. Recent studies have indicated that the vertebrate Diablo/SMAC protein also promotes cell death activation by binding to IAPs and suppressing their death inhibitory activities. Thus, IAP suppression may be an evolutionarily conserved cell death regulatory mechanism. In this regard, while Grim-Reaper orthologs have not been identified, the expression of each protein can induce vertebrate cells to die, implying that they may suppress vertebrate IAPs (Wing, 2001b).

As with Reaper or Grim, P[GMR-gal4]-targeted expression of R/Grim or G/Reaper is very effective at inducing cell death. However, the actions of the chimeras are distinct from those of Reaper or Grim. In particular, the cell death phenotypes resulting from R/Grim or G/Reaper expression are completely blocked by Diap1 and partially blocked by Diap2. In contrast, both Diap1 and Diap2 completely block the effects of Reaper expression, but do not affect cell death induced by Grim. Thus, as a result of the presence of the Reaper RHG motif, R/Grim exhibits an increased sensitivity to repression by the Diaps compared with Grim. Similarly, the presence of the Grim RHG motif in G/Reaper results in decreased sensitivity to repression by Diaps compared with Reaper. These results indicated that the sequence differences in the RHG motifs of Reaper and Grim may strongly influence functional interactions with the Diaps (Wing, 2001b).

Diap1, like Diap2, exhibits distinct abilities to repress cell death induced by Reaper, Hid, or Grim. In the CNS midline, Diap1 more effectively blocks Grim-induced cell death than cell death induced by Reaper and Hid. In contrast, when examined in the adult eye, Diap1 is most effective at blocking Reaper-induced cell death, moderately effective against Hid, and ineffective against Grim. Similar results were obtained using the th^sl gain-of-function diap1 mutant allele, which represses Reaper-induced eye cell death more effectively than death induced by Hid or Grim. Importantly, these data indicate that Diap1 has distinct, tissue-specific effects on cell death induced by Grim-Reaper proteins, and that these effects differ from those of Diap2. The basis for these functional distinctions are not yet clear. One possibility is that the associations between each Diap and Grim-Reaper protein may differ in strength, or be influenced by specific ancillary factors. Differences have been noted between Diap1 and Diap2 in their ability to bind and repress the actions of certain caspases, and Reaper, Hid, and Grim can act through different downstream caspases. Taken together, these findings suggest potentially complex functional interactions between Grim-Reaper proteins, Diaps, and caspases. It is likely that distinct activities of individual Grim-Reaper and Diap proteins provide enhanced capabilities for regulating cell death processes in different developmental and physiological contexts (Wing, 2001b).

Do Reaper, Hid, and Grim share RHG-independent functions? Both truncated ReaperC and GrimC proteins induce cell death in developing tissues, indicating that regions outside the RHG motif also have death-inducing activities. Surprisingly, it was found that cell death induced by GrimC or ReaperC is only partially repressed by p35, suggesting a distinct mode of action compared with native Reaper or Grim. Similar to Reaper, Hid and Grim, GrimC does apparently act through the p35-insensitive caspase, Dronc, as GrimC-induced death is partially suppressed by a dominant/negative Dronc^C318S protein. However, the persistence of some eye cell death in the presence of Dronc^C318S indicates that GrimC and ReaperC also act through alternate pathways. Perhaps GrimC acts through pro-apoptotic Drosophila Bcl-2 orthologs that may induce cell death which is not blocked by p35. Another interesting possibilty is that GrimC might act via a Drosophila ortholog of Scythe, a Xenopus cell death regulator that binds Reaper, Hid, and Grim independently of the RHG motif (Wing, 2001b and references therein).

A second region of sequence similarity, the 30 amino acid Trp-block, has been identified that is present once in Reaper and Grim, and four times in Hid. The Trp-blocks may be important for the cell death activation capabilities of GrimC and ReaperC, as well as for potentially shared RHG motif-independent activities of native Grim-Reaper proteins. This additional sequence similarity also suggests a modular organization of the Grim-Reaper proteins, where distinct functions may be afforded by the RHG motif and Trp-block. Taken together, the sequence similarities of the Grim-Reaper proteins, as well as the organization and chromosomal location of the corresponding genes, imply that the grim-reaper genes arose from duplication of a common ancestor and have diverged to assume overlapping yet distinct cell death activation functions. It will be of interest to determine the representation of grim-reaper orthologs in other species, information that could provide important insights into the evolution of cell death control mechanisms. This is of particular relevance given that inhibition of IAP activity is likely to constitute a conserved mechanism to regulate cell death activation (Wing, 2001b).

Ceramide and Slipper acts upstream of Reaper

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity have not been identified. This study identifies ceramide as the activator of Drosophila MLK (Slipper) and ceramide and TNF-alpha are identified as agonists of mammalian MLK3. Slipper and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of Slipper and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of Slipper and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha (Sathyanarayana, 2002).

There are very few known kinases that are direct targets of ceramide. PKC-zeta, Raf-1, and CAPK (ceramide activated protein kinase) have been shown to be activated by ceramide. These results show that Slipper and MLK3 are targeted by ceramide. Reaper, a Drosophila protein known to cause ceramide generation, induces apoptosis during normal Drosophila development. In addition, overexpression of reaper induces apoptosis in S2 cells. It is therefore speculated that reaper may lead to apoptosis, at least in part, via ceramide-mediated activation of Slipper (Sathyanarayana, 2002).

In conclusion, ceramide is a potent agonist of Drosophila MLK and mammalian MLK3. The specificity of Slipper and MLK3 in mediating only ceramide-induced JNK activation without affecting ceramide-induced activation of ERK and p38 suggests an intriguing mechanism by which a specific MAPKKK can regulate different MAPK pathways in response to various physiological and pathological stimuli. These results also suggest that MLK3 plays a role in TNF-alpha-induced JNK activation. Studies showing that overexpression of MLK3 causes apoptosis and that neuronal cell death can be prevented by inhibition of the MLK family of kinases suggest a role for MLKs in apoptosis of neuronal cells. Since both ceramide and TNF are important triggers of cell death, these studies also indirectly suggest a role for MLK3 in modulating apoptosis. It is speculated that further elucidation of the role of MLKs, and specifically of MLK3, in apoptosis may ultimately facilitate the development of a targeted pharmacological intervention in neurodegenerative disorders such as idiopathic Parkinson's disease and Alzheimer's disease, both of which are associated with dysregulation of apoptosis (Sathyanarayana, 2002).

Reaper protein induces ceramide

Rpr expression induces generation of the lipid second messenger ceramide, and through use of the peptide caspase inhibitor N-benzyloxycarbonyl-VAD-fluoromethylketone(zVAD.fmk ) ceramide generation has been ordered downstream of caspases in SL2 cells. This study evaluates these events in SL2 cells transfected with cDNA for Rpr, with or without the baculovirus caspase inhibitor p35, under the control of the metallothionein promoter. Following copper addition, Rpr protein is detected at 1.5 h and is maximal at 2.5 h. Ceramide generation and caspase activation occurs nearly simultaneously; each is detectable at 2-2.5 h and is maximal at 6 h. Ceramide levels increase from a base line of 5 pmol/nmol lipid phosphorus to a maximum of 10 pmol/nmol lipid phosphorus. Apoptosis, first detected at 4 h, is maximal at 16 h. Co-expression of p35 did not affect Rpr-induced ceramide generation, whereas caspase activation and apoptosis are abolished. In contrast, zVAD.fmk inhibits ceramide generation and apoptosis. These data show that Rpr-induced ceramide generation is upstream or independent of p35-inhibitable caspases and demonstrate differences in the actions of peptide and p35 caspase inhibitors (Bose, 1998).

Loss of mir-14 enhances Reaper-dependent cell death

MicroRNAs (miRNAs) are small regulatory RNAs that are between 21 and 25 nucleotides in length and repress gene function through interactions with target mRNAs. The genomes of metazoans encode on the order of several hundred miRNAs, but the processes they regulate have been defined for only a few cases. New inhibitors of apoptotic cell death were sought by testing existing collections of P element insertion lines for their ability to enhance a small-eye phenotype associated with eye-specific expression of the Drosophila cell death activator Reaper. The Drosophila miRNA mir-14 has been identified as a cell death suppressor. Loss of mir-14 enhances Reaper-dependent cell death, whereas ectopic expression suppresses cell death induced by multiple stimuli. Animals lacking mir-14 are viable. However, they are stress sensitive and have a reduced lifespan. mir-14 mutants have elevated levels of the apoptotic effector caspase Ice, suggesting one potential site of action. Mir-14 also regulates fat metabolism. Deletion of mir-14 results in animals with increased levels of triacylglycerol and diacylglycerol, whereas increases in mir-14 copy number have the converse effect (Xu, 2003).

The two C. elegans miRNAs with known functions, lin-4 and let-7, are thought to regulate development by binding to the 3'untranslated region of target transcripts and thereby repressing the translation of their products. In these examples, the analysis of genetic interactions provides important clues as to the identity of targets. In the absence of this sort of information, it is difficult to predict miRNA targets in animals. This is because base pairing between the mature miRNA and its target is imperfect and the rules that govern which base pair interactions are important are unknown. Potential Mir-14 binding sites were sought in a number of apoptotic regulators, including Dronc, Rpr, Hid, and Grim. Potential target sites were identified in the transcripts of several genes, including Ice, Dcp-1, Scythe, SkpA, and Grim (however, the Grim target is present in the 3'UTR, which was absent in the GMR-Grim transgene). Of these, Ice, an apoptotic effector caspase, is of particular interest. Ice is required for at least some cell deaths and is activated by Dronc, which promotes cell death induced by Rpr, Hid, and Grim. Ice levels in adults were measured by using an anti-Ice antibody. Ice is elevated in mir-14Δ¹ flies as compared to the wild-type, and this increase is suppressed in the presence of two copies of the mir-14-containing 3.4 kb genomic DNA fragment. Whereas these observations alone do not prove that Ice is a direct target of Mir-14, they do suggest that Ice is regulated, either directly or indirectly, by Mir-14 levels (Xu, 2003).

Reaper is involved in ROS-induced apoptosis caused by impairment of the selD/sps1 homolog in Drosophila

The cellular antioxidant defense systems neutralize the cytotoxic by-products referred to as reactive oxygen species (ROS). Among them, selenoproteins have important antioxidant and detoxification functions. The interference in selenoprotein biosynthesis results in accumulation of ROS and consequently in a toxic intracellular environment. The resulting ROS imbalance can trigger apoptosis to eliminate the deleterious cells. In Drosophila, a null mutation in the selD gene (homologous to the human selenophosphate synthetase type 1) causes an impairment of selenoprotein biosynthesis, a ROS burst and lethality. This mutation (known as selD^ptuf) can serve as a tool to understand the link between ROS accumulation and cell death. To this aim, the mechanism by which selD^ptuf mutant cells become apoptotic was analyzed in Drosophila imaginal discs. The apoptotic effect of selD^ptuf does not require the activity of the Ras/MAPK-dependent proapoptotic gene hid, but results in stabilization of the tumor suppressor protein p53 and transcription of the Drosophila pro-apoptotic gene reaper (rpr). Genetic evidence supports the idea that the initiator caspase DRONC is activated and that the effector caspase DRICE is processed to commit selD^ptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selD^ptuf mutant cells. These observations indicate that selD^ptuf ROS-induced apoptosis in Drosophila is mainly driven by the caspase-dependent p53/Rpr pathway (Morey, 2003).

Drosophila IAP antagonists form multimeric complexes to promote cell death

Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. This study shows that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. The domain involved in self-association has been defined and it was demonstrated to be critical for cell-killing activity in vivo. In addition, Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. These results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis (Sandu, 2010).

This study shows that IAP antagonists undergo self-association and hetero-association that is essential for their full killing activity. Specifically, the physical association between Rpr, Hid, and Grim involves the central helical domain of Rpr. Disrupting this protein-protein interface leads to a significant loss of Rpr’s ability to induce cell death in vivo. The importance of Rpr self-association was revealed by generating enforced Rpr dimers in which the central helical domain of this protein is replaced by defined dimerization motifs. These experiments revealed that enforced parallel, but not anti-parallel dimerization of Rpr (RprLZ) can induce cell death very efficiently in transgenic Drosophila. The resulting cell death occurred by apoptosis and was rescued by the overexpression of the caspase inhibitor p35, or through Rpr-insensitive diap1 alleles. Furthermore, mutants that inhibit the self-association of Rpr have reduced pro-apoptotic activity, providing independent support for the importance of Rpr multimerization. Because an anti-parallel Rpr dimer (RprProP) was not efficiently inducing cell death in transgenic animals, it appears that the IBM motifs of multimeric Rpr have to be in a specific conformation, or at least in close proximity for efficient DIAP1 inactivation. This may occur, for example, by engaging both BIR domains of one DIAP1 molecule in a similar fashion to how SMAC can engage XIAP (Sandu, 2010).

The association of Rpr with the other IAP antagonists Grim and Hid is also reported. Hid is the only IAP antagonist that has a defined mitochondrial targeting sequence at its C terminus and is targeted to the mitochondria by itself; therefore, focus was placed particularly on the interaction between Rpr and Hid. Consistent with previous reports, it was found that Hid consistently localizes to the mitochondria in both human and Drosophila cells. Although it has been previously reported that Rpr localizes to the mitochondria through the GH3-lipid interaction, the current results support an alternative view that Rpr’s ability to translocate to the mitochondria is an indirect consequence of associating with Hid. Specifically, in support of this model, it was shown that Rpr is uniformly distributed in cells when transfected alone in heterologous cells, translocating to the mitochondria only when cotransfected with Hid. It was further shown that the GH3 mutant F34AL35A, unlike wild-type Rpr, does not coimmunoprecipitate with Hid. This is in agreement with previous observations that a GH3 mutant failed to localize to the mitochondria in Drosophila S2 cells (Sandu, 2010).

Rpr induces ubiquitination of DIAP1 in vitro and in HEK293 cells. Unlike Rpr, Hid is not able to perform this function. Thus, the significance of Rpr-Hid interaction might be to bring Rpr at the mitochondrial surface to degrade DIAP1. Although both Rpr and Hid belong to the IAP antagonists family, share a conserved IBM motif, bind DIAP1, and induce cell death, their role in induction of cell death seems to be distinct. In many paradigms Hid appears to be a more potent inducer of cell death than Rpr. It is possible that the primary role of Hid is to assemble a complex at the mitochondrial membrane that recruits Rpr as one the players. The role of Rpr in this complex is to induce DIAP1 ubiquitination. Inability of Hid itself to induce DIAP1 degradation might be related to its larger size (410 amino acids) as compared with Rpr (64 amino acids) or even Grim (138 amino acids). Potentially, the bulkier Hid might interfere with conformational changes in DIAP1 or with the ubiquitin-related transfer process (Sandu, 2010).

In addition, evidence is provided that Rpr is more potent at inducing apoptosis when present at the mitochondrial membrane. When Rpr was fused to the mitochondrial targeting sequence from Hid and expressed in Drosophila eyes, strong cell killing and pupal lethality were observed. Flies dissected from the pupal cases show severely ablated eyes that are reduced to black spots. Even the inactive GH3 mutant F34AL35A, when artificially targeted to the mitochondria using the Hid MTS, induces significant eye ablation. Therefore, Rpr is more potent when present at the mitochondrial membrane. Two possible explanations are considered for this enhanced pro-apoptotic activity: First, Rpr may be more active at the mitochondrial surface because of increased protein stability. Consistent with this idea, cytoplasmic Rpr is not very stable and it was found that Rpr accumulates to higher protein levels when the presence of Hid permits mitochondrial localization. The resulting high local concentration of Rpr may be critical for DIAP1 ubiquitination. As predicted by this model, it was found that Rpr-induced cell death is less efficient when Hid is depleted by RNA knockdown. The model is also in agreement with several previous observations. For example, it has been reported that Rpr and Hid localize to mitochondria and can induce changes of the mitochondrial ultrastructure. This study also showed that inhibition of Rpr localization to mitochondria significantly inhibits cell killing, and that Rpr and Hid act in concert with caspases to promote mitochondrial disruption and Cyt C release. In addition, overexpression of both rpr and hid is required to induce cell death in midline cells of the nervous system, and neither of them kills well individually. This is consistent with the observation that more than one IAP antagonist is expressed and they act synergistically in the dying midline glia cells. Finally, Drosophila salivary gland cell death is preceded by the expression of both rpr and hid, and RNAi knockdown of hid alone is sufficient to block the death of these cells. The second, and not mutually exclusive explanation is that Rpr may be more active at the mitochondria because of local concentration of apoptosis regulators that operate at this surface. It has been previously shown that Dronc and active Drice are present at the mitochondrial membrane, and more recently that mammalian XIAP can translocate to the mitochondrial surface in response to apoptotic stimuli. In addition, mitochondrial proteins involved in energy metabolism have been recently described to modulate caspase activity and cell death in Drosophila cells. Recently, it was shown by coimmunoprecipitation experiments in fly cell culture that Grim interacts with the Bcl-2 family proteins Debcl and Buffy. Thus, Rpr may be part of a higher-order complex at the mitochondria to locally regulate IAP turnover and caspase activity (Sandu, 2010).

Taken together, this study uncovered the role of the Rpr helical domain in self-association and interaction with Hid and Grim. The mechanism of Rpr recruitment to the mitochondria by interaction with Hid was revealed. Most importantly, this study has provided a new concept with respect to IAP antagonist activity in fly, which acts cooperatively by physical interaction rather than by additive cell death output (Sandu, 2010).

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. This study reports the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc (Chai, 2003).

Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death

In most multicellular organisms, the decision to undergo programmed cell death in response to cellular damage or developmental cues is typically transmitted through mitochondria. It has been suggested that an exception is the apoptotic pathway of Drosophila melanogaster, in which the role of mitochondria remains unclear. Although IAP antagonists in Drosophila such as Reaper, Hid and Grim may induce cell death without mitochondrial membrane permeabilization, it is surprising that all three localize to mitochondria. Moreover, induction of Reaper and Hid appears to result in mitochondrial fragmentation during Drosophila cell death. Most importantly, disruption of mitochondrial fission can inhibit Reaper and Hid-induced cell death, suggesting that alterations in mitochondrial dynamics can modulate cell death in fly cells. This study reports that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. These in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or gamma-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. These findings are consistent with a growing body of work describing a role for mitochondrial fission and fusion machinery in the decision of cells to die (Thomenius, 2011).

Tango7 regulates cortical activity of caspases during reaper-triggered changes in tissue elasticity

Caspases perform critical functions in both living and dying cells; however, how caspases perform physiological functions without killing the cell remains unclear. This study identified a novel physiological function of caspases at the cortex of Drosophila salivary glands. In living glands, activation of the initiator caspase Dronc triggers cortical F-actin dismantling, enabling the glands to stretch as they accumulate secreted products in the lumen. Tango7 (Eukaryotic translation initiation factor 3 subunit m), not the canonical Apaf-1-adaptor Dark, regulates Dronc activity at the cortex; in contrast, dark is required for cytoplasmic activity of dronc during salivary gland death. Therefore, tango7 and dark define distinct subcellular domains of caspase activity. Furthermore, Tango7-dependent cortical Dronc activity is initiated by a sublethal pulse of the inhibitor of apoptosis protein (IAP) antagonist Reaper. The results support a model in which biological outcomes of caspase activation are regulated by differential amplification of IAP antagonists, unique caspase adaptor proteins, and mutually exclusive subcellular domains of caspase activity. Caspases are known for their role in cell death, but they can also participate in other physiological functions without killing the cells. In this study the authors show that unique caspase adaptor proteins can regulate caspase activity within mutually-exclusive and independently regulated subcellular domains (Kang, 2017).

Principles that govern the activation and function of caspases have fallen short in providing an understanding for how these enzymes can be activated to perform both delicate intracellular remodeling in living cells and total destruction in dying cells. This paper provides new insights into the mechanisms that regulate caspase activation by comparing two completely different biological outcomes in the same tissue that both require caspase function. The Drosophila homolog of caspase-9, dronc, is required for dismantling of the cortical F-actin cytoskeleton during salivary gland development -- a role that is distinct from its known function in the salivary gland death response during metamorphosis. By systematically dissecting the regulation of dronc function at the cortex, this study showed that cortical functions of dronc are regulated independently from its cytoplasmic functions. The cytoplasmic functions of activated dronc require the canonical adaptor protein Dark, while the cortical roles of dronc require tango7. In this manner, tango7 and Dark restrict the function of dronc to distinct subcellular domains. Moreover, this study also showed that these two functions can be initiated independently through differential amplification of IAP antagonist expression, providing a model for how lethal and vital roles of caspases can be differentially activated in the same cell. Finally, a new non-apoptotic function was identified for caspases in the control of tissue elasticity to accommodate buildup of secreted products in the lumen of secretory tissues, facilitating their timely release (Kang, 2017).

The results demonstrate that caspases can be activated in distinct, mutually exclusive subcellular domains within a single cell, and that these subcellular domains are generated by use of unique caspase adaptor proteins. Local activation of caspases, as detected by staining with antibodies to activated caspases, has been reported before; however, this study demonstrates that local activation is achieved by targeting caspases to subcellular domains, and this targeting is necessary for subcellular functions of these caspases. Importantly, this study shows that caspases can be activated specifically in one domain without being activated in another, providing a mechanism that allows control of caspase activity with a previously unknown level of subcellular precision. However, the mechanisms that restrict caspase cascades to distinct subcellular compartments remain unclear. It is possible that caspase expression levels are intentionally kept low during non-lethal responses, and localized enrichment mediates subcellular domain-specific activation. For example, if most of the Dronc protein present in the cell localizes to the cortex, then this specific localization may restrict caspase functions to the cortical compartment. This model fits with the results at the end of larval development; however, in dying glands, caspases are independently activated in cortical and cytoplasmic compartments, suggesting that additional mechanisms are in play to restrict caspase activity to the appropriate subcellular compartment. For example, it is possible that caspase cascades occur within a physical complex consisting of initiator caspases, their adaptor proteins, effector caspases, and their substrates. In this model, only one of these proteins, likely the initiator caspase, would need to be subcellularly localized in order to generate a compartment-specific caspase cascade. However, resolution of this possible mechanism will require further studies. This subcellular domain-specific model for caspase activation contrasts with the commonly held belief that activated caspase cascades passively perpetuate themselves and spread throughout the cell, and also opens the possibility that caspases, through specific subcellular localization mediated by adaptor proteins, may play a role in many yet-to-be-identified biological processes (Kang, 2017).

This study demonstrates that differential amplification of IAP antagonists at specific developmental stages determines lethal vs. non-lethal outcomes of caspase activation. In the system used in this study, differential amplification is accomplished through the use of transcription factors that function downstream of a steroid hormone signal. However, caspases must have an ability to 'sense' the magnitude of the IAP antagonist pulse, ensuring that they initiate the appropriate lethal or non-lethal responses. One possible 'sensing' mechanism may involve the aforementioned selectivity of initiator caspase adaptor proteins, like was observed with tango7 and dark. In this model, some adaptor protein complexes would require a lower IAP antagonist threshold for initiator caspase activation than others. However, elucidation of the detailed molecular mechanisms mediating 'sensing' of IAP antagonist expression levels will require further study. Finally, the results indicate that small pulses of IAP antagonist expression are tissue specific, raising the possibility that many more of these pulses are generated in other tissues and developmental stages that have not yet been detected or characterized. The data suggests that non-lethal, physiological functions of caspases may be more widespread than previously thought (Kang, 2017).

These results show that caspases play a novel role during the secretion of glue proteins. Glue proteins are essential to allow a newly formed prepupa to adhere to a solid surface; however, when cortical F-actin dismantling fails, glue precociously 'leaks' onto the surface of the animal. Although precocious expulsion of glue does not appear to have a deleterious effect in the lab, in the wild, it may adversely affect fitness by inhibiting larval movement or reducing the ability of the animal to stick securely to a surface during metamorphosis. Additionally, the results raise the question of whether other exocrine tissues in different species, such as the mammary gland, may utilize caspases in a similar manner to accommodate large amounts of secreted luminal products prior to their release (Kang, 2017).

In conclusion, systematic analysis of vital and lethal responses to caspase activation in the same cells has revealed mechanisms that allow caspases to be activated without killing the cell. The results demonstrate that caspases can be activated in mutually exclusive subcellular domains, where activation of caspases in one domain does not trigger activation of caspases in another domain. These subcellular domains were shown to. e generated by different caspase adaptor proteins. It is likely that yet-to-be-identified adaptor proteins define other subcellular domains and, in so doing, help regulate the many physiological functions of caspases. Moreover, the results demonstrate that some of these subcellular domains have lower thresholds for activation of caspases, thereby allowing sublethal pulses of IAP antagonists to selectively initiate physiological functions of caspases. Together, these results outline a simple conceptual framework for controlling caspase activation during normal development and physiology (Kang, 2017).