The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. A mechanism is described by which the Ebi/SMRTER corepressor complex maintains Dl expression. charlatan (chn) is repressed by Ebi/SMRTER corepressor complex by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression (Tsuda, 2006).
The corepressor complex that includes Ebi, SMRTER and Su(H) is required for expression of Dl in Drosophila photoreceptor cells. To identify genetic loci that are transcriptionally repressed by the Ebi corepressor, a screen was set up using an ectopic gene expression system (Gene Search System). Insertion of a Gene Search (GS) vector, a modified P-element carrying the Gal4 upstream activating sequence (UASG) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UASG system. GS insertions into the chn locus were identified, whose overexpression phenotype in the eye using an eye-specific Gal4 driver (GMR-Gal4) was modified by reducing ebi activity. Thus the regulation of chn by Ebi-dependent transcriptional repression was studied (Tsuda, 2006).
In third instar larval-stage eye discs, the chn transcript is highly expressed in the morphogenetic furrow (MF), where photoreceptor differentiation initiates, but is downregulated in cells in the later stage photoreceptor development. In ebi mutant eye discs, however, chn expression becomes detectable in differentiating photoreceptor cells, and its expression in the MF is increased, suggesting that Su(H) in association with Ebi and SMRTER represses chn transcription in the eye disc (Tsuda, 2006).
To reveal the role of Su(H) as an activator, chn expression was examined when the level of Su(H) expression was reduced. Removing one copy of Su(H) suppresses the loss-of-Dl expression phenotype in ebi mutants. It was found that reducing one copy of Su(H) suppresses ectopic chn expression in ebi mutants, suggesting that ectopic expression of chn in ebi mutants is Su(H)-dependent. RT–PCR analysis of chn expression in ebi- eye discs differing in the dosage of Su(H) gene also supported these results. Strong reduction of Su(H) expression alone reduced expression of chn in the MF; this expression became weaker and was slightly broader. The phenotype of ebi, Su(H) double mutants is almost the same as Su(H) single mutants , suggesting that Su(H) acts as an activator in the absence of Ebi. This might be due to dual functions of Su(H) as an activator or repressor. Hence, reducing the amount of Ebi in the corepressor complex involving Su(H) might convert Su(H) to an activator by permitting the replacement of the corepressor complex with NICD (Tsuda, 2006).
To reveal the molecular nature of transcriptional regulation of chn by Su(H), Su(H) target sites were sought in the genomic region of chn. Since Su(H) binds slightly degenerate sequences, it was not easy to identify the functional Su(H) binding region from a simple genomic search. An alternative approach was taken to map the chn genomic region, which is regulated by Su(H) in the normal chromosomal context. Ebi-mediated repression involves SMRTER, a corepressor that recruits histone deacetylases and induces the formation of inactive chromatin, which spreads from the site where Su(H) recruits the corepressor complex. Promoters near the Su(H)-binding site are thus expected to be downregulated in an Ebi-dependent manner. Four insertion lines of the GS vector were identified in the chn promoter region. All these GS lines caused ectopic expression of chn with consequent abnormal eye morphology when they were crossed with GMR-Gal4. If the effect of the Ebi/SMRTER corepressor complex reaches the UASG in those insertions, reduction of Ebi activity will derepress UASG and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebiDN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebiDN strongly enhanced the overexpression phenotype of chnGS17605 and chnGS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebiDN failed to enhance the overexpression phenotype of other GS lines (chnGS2112 and chnGS17892) that were inserted downstream (+773 and +1040, respectively) of the first exon. From these results, it is concluded that Ebi-dependent transcriptional repression is targeted to the proximity of the transcriptional initiation site of the chn promoter (Tsuda, 2006).
Chn is a 1108-amino-acid protein with multiple C2H2-type zinc-finger motifs. Although no highly homologous gene within the mammalian genome could not be detected using BLAST, a small sequence of similarity between the N-terminal zinc-finger motif of Chn and the fifth zinc-finger of human NRSF/REST was found. Chn has several structural and functional similarities to human NRSF/REST, as follows. First, Chn and NRSF/REST each contain an N-terminal region with multiple zinc-finger motifs (five motifs in 264 residues in Chn and eight motifs in 251 residues in NRSF/REST), followed by a cluster of S/T-P motifs (serine or threonine followed by a proline) and a single zinc-finger motif at the C terminus. Second, the C-terminal region of NRSF/REST binds a corepressor, CoREST, which serves as an adaptor molecule to recruit a complex that imposes silencing activities. The Drosophila homolog of CoREST (dCoREST) (Andres, 1999; Dallman, 2004) can associate with the C-terminal half of Chn in cultured S2 cells. Finally, NRSF/REST binds to NRSE/RE1, a 21-bp sequence located in the promoter region of many types of neuron-restricted genes, via the N-terminal zinc-finger motifs. It was found that a recombinant protein containing the N-terminal zinc-fingers of Chn bound specifically to the NRSE/RE1 sequence in vitro. Thus, the structural similarity to NRSF/REST, binding to dCoREST and the DNA-binding specificity of Chn suggest that it is a candidate for a functional Drosophila homolog of NRSF/REST (Tsuda, 2006).
If Chn acts as a regulator of neural-related functions, as suggested for NRSF/REST, then Chn would be expected to bind to a regulatory region common to many types of neural-related genes in Drosophila. Numerous sequences similar to NRSE/RE1 were identified in the Drosophila genome, and their binding to Chn was assessed by EMSA. Using these sequences, a consensus binding sequence for Chn (Chn-binding element (CBE), 5'-BBHASMVMMVCNGACVKNNCC-3') was derived. 26 CBEs were identified within 10 kb of annotated genes from the Drosophila genome. Binding to Chn was confirmed for 18 CBEs using EMSA competition assay. Genes containing the CBE include dopamine receptor 2 (DopR2) and the potassium channel, ether-a-go-go, for which the mammalian homologs are target genes of NRSF/REST. These results suggest that the CBE is a good indicator of Chn binding sites and that Chn regulates many types of neural-related genes, as is implicated for NRSF/REST. However, it was found that divergent forms of CBE adjacent to hairy and extramacrochaetae were bound specifically by Chn. Likewise, some of the CBE sites failed to bind to Chn. Thus, a further refinement will be necessary to predict a definitive set of Chn binding sites in the Drosophila genome (Tsuda, 2006).
Although it has been established that mammalian NRSF/REST is a key regulator of neuron-specific genes, attempts to isolate invertebrate homolog of NRSF/REST have so far failed to identify a true homologous factor in invertebrates. The properties of Chn, including the similarity in DNA-binding specificity, association with CoREST and transcriptional repressor activity, suggest that Chn is a strong candidate for a functional Drosophila homolog of NRSF/REST. chn was originally identified by its requirement in the development of the PNS. This study identified a number of candidate target genes of Chn, a large fraction of which is implicated in neural function and/or gene expression. It is expected that further analysis of these candidates will provide valuable information about chn function in vivo, which may be extended to the understanding of NRSF/REST (Tsuda, 2006).
The Chn mutation blocks eye development by preventing the initiation of MF, a process requiring Notch signaling. This phenotype is likely owing to a loss of Notch function, because elevated Dl expression is known to block Notch signaling. The function of Chn during the early stage of eye development might be to regulate Notch signaling at an appropriate level by downregulating Dl. It is possible that Chn-mediated modulation places a variety of Notch functions in eye under the influence of EGFR signaling and provides flexibility in its regulation (Tsuda, 2006).
Although chn is expressed in the MF, genetic analyses show that small clones of chn mutant cells permit progression of the MF and photoreceptor differentiation. It is speculated that the repressive effect of Chn is overcome by other signals in the MF, such as hedgehog signaling, which strongly induces Dl (Tsuda, 2006).
Developing photoreceptor cells are exposed to the EGFR ligand, Spitz, and the Notch ligand, Dl, and each cell must assess the level of the two signals and respond appropriately to perform each task of photoreceptor cell specification and induction of non-neural cone cells. This question was investigated by studying the expression of Dl in photoreceptor cells. chn was identified as a direct target of Ebi/SMRTER-dependent transcriptional repression and as a repressor of Dl expression. The abrogated expression of Dl in ebi mutants was recovered by reducing one copy of chn, suggesting that the negative regulation of chn by ebi is indeed prerequisite for photoreceptor cell development (Tsuda, 2006).
Genetic data suggest that Su(H) may activate or repress chn expression. This idea is supported by data showing that Ebi/SMRTER and NICD are recruited to the promoter region of chn. The Ebi/SMRTER complex formed in this region did not contain any detectable level of the intracellular domain of Notch (NICD), suggesting that the binding of Ebi/SMRTER and NICD to this region may be mutually exclusive, and therefore it is expected that a regulatory system controls the balance between the active and repressive states of Su(H). Taken together, these results suggest that chn is a key factor in the crosstalk between two major signal transduction pathways: the EGFR-dependent pathway and the Notch/Delta-dependent pathway (Tsuda, 2006).
In the mammalian system, competition between SMRT and NICD for interaction with RBPJkappa determines the state of RBPJkappa-dependent transcriptional activity. Extracellular signaling may modulate this competition; diverse signaling pathways modulate the functions of N-CoR/SMRT. The current findings would prompt investigations of potential interaction of two repression systems of NRSF/REST and N-CoR/SMRT, and their regulation by Notch and EGF signaling in mammalian neuronal differentiation (Tsuda, 2006).
The strong genetic interaction between the LOF conditions for chn and ac/sc, together with the presumed activation of chn by ac/sc, led to an examination of whether chn might in turn stimulate ac/sc expression. Initially examined was whether the overexpression of chn affects Sc accumulation in third instar wing discs. In these discs, ac and sc are coexpressed in a stereotyped pattern of well-resolved proneural clusters from which SOPs emerge. With the MS248-Gal4 driver, UAS-chn promotes strong and generalized expression of sc in most of the domain of expression of the driver, namely, the medial and part of the lateral prospective notum. Many SOPs arose from this enlarged region of Sc accumulation, as detected by the Sens marker, consistent with the additional macrochaetae that develop on the notum of these flies. With the MS1096-Gal4 driver, which is expressed most strongly in the dorsal part of the wing anlage, there is also ectopic expression of sc and emergence of extra SOPs in the wing territory. Interestingly, expression of UAS-chn disrupts the characteristic double row expression of sc and sens at the wing margin, suggesting interference with its formation. This is also consistent with the presence of small, crumpled adult wings that carry many bristles and other types of sensilla. Overexpression of UAS-chn with the ubiquitous wing disc driver C765-Gal4 activates sc but fails to stimulate atonal, a proneural gene which is not a member of the ASC and is normally expressed in a few cells at the presumptive tegula and ventral radius. Conversely, overexpression of atonal does not stimulate chn in the wing disc (Escudero, 2005).
The expression of ac/sc in proneural clusters is controlled by a series of separable enhancer elements in the ASC. Each enhancer is responsible for expression in one or in a few proneural clusters. Thus, whether the ectopic activation of sc could be mediated by the overexpression of UAS-chn acting upon these enhancers was examined. UAS-chn strongly stimulates the activity of a construct in which the lacZ gene is under the control of the ASC L3/TSM enhancer [construct 2.3-lacZ; MS1096-Gal4 driver], which directs expression at the wing vein L3 and the twin sensilla of the wing margin proneural clusters. Similar observations were made with the dorsocentral (DC) enhancer [construct AS1.4DC=DC-lacZ; C765-Gal4 ubiquitous driver], which promotes expression in the central part of the notum. It also activates expression directed by the ANP enhancer. Since the DC-lacZ construct bears the heterologous hsp70 promoter, these data indicate that the sc endogenous promoter is dispensable for the stimulation by UAS-chn. The Ac and Sc proneural proteins are also not essential for the increased activity of the enhancers; DC-lacZ expression is strongly increased by Chn in an In(1)sc10.1 background. By contrast, the sc SOP-dedicated enhancer (SRV-lacZ construct), which is responsible for the strong accumulation of Sc in SOPs, is only clearly activated by UAS-chn (C734-Gal4 driver) in the presence of ac/sc, and this stimulation occurred in individual cells. This observation suggests that the upregulation of this enhancer results from the formation of ectopic SOPs by the UAS-chn-induced overexpression of sc, rather than from a direct effect of Chn on the enhancer. Still, the possibility remains that Chn and Sc cooperate in the activation of this enhancer (Escudero, 2005).
UAS-chn upregulates the activity of these enhancers, but it does not lead to a generalized expression of lacZ in all the domains of UAS-chn expression. These data indicate that despite the elevated activation, the enhancers are still dependent on the prepattern factors that define their spatial domains of activity. This fact was verified by the observation that the overstimulation of DC-lacZ is strongly dependent on its prepattern activator, the transcription factor Pnr. Moreover, 2.3-lacZ, which is active only in the wing pouch, is not stimulated by the overexpression of UAS-chn in the prospective notum (MS248-Gal4); this indicates that the sc promoter present in this construct was not responsive to UAS-chn (Escudero, 2005).
Next examined were mosaic wing discs to determine whether removal of chn function affects expression of sc or enhancer-lacZ constructs in proneural clusters. Homozygous chnECJ1 cells generally display reduced expression of sc or ß-galactosidase under the control of proneural enhancers, when compared with neighboring heterozygous chnECJ1/+ cells. Note however, that the expression is not completely abolished. Similar decreased expression of sc is observed by misexpressing UAS-chniS in cell clones. These effects appear to be cell-autonomous. While SOPs can still emerge from homozygous chnECJ1 cells with reduced levels of Sc, SOPs were often missing, in agreement with the partial suppression of macrochaetae observed within the chnECJ1 clones. When both homozygous and heterozygous cells were near a position where an SOP emerged, a heterozygous cell appeared to be preferentially selected. These findings clearly indicate that chn+ is required for proneural proteins to accumulate in proneural clusters at levels sufficient to ensure SOP selection. Moreover, the observation that expression of enhancer-lacZ constructs is reduced in chn- cells and increased in chn overexpressing cells indicates that the effect of chn+ is not due to an enhanced perdurance of the Sc protein, but to the increased transcription of the sc gene (Escudero, 2005).
To analyze whether Chn is a direct regulator of the ASC enhancers, the ability of the Chn protein to bind to the DC enhancer was assayed in vitro. A fragment of the Chn protein containing the five zinc fingers was produced in and purified from E. coli. The enhancer DNA was divided into six partially overlapping fragments of approximately 300 bp each, and each of them was assayed in gel retardation experiments. Only the fragment that comprised the proximal-most region of the enhancer (fragment DC6) shows binding of the Chn polypeptide. Interestingly, the DC6 fragment is included within the PB0.5DC sequence, the smallest subfragment of AS1.4DC, which still retains enhancer activity. For unclear reasons, the PB0.5DC enhancer drives expression only in the PDC SOP. Still, misexpression of UAS-chn expands this expression to many cells of the posterior notum. This suggests that the binding of Chn protein to the DC6 region of the DC enhancer may prompt its response to Chn in vivo (Escudero, 2005).
The patterns of expression of chn in embryos and imaginal discs were examined using in-situ hybridization. In early blastoderm stages, the expression of chn is ubiquitous, but before stage 5, chn mRNA disappears from the poles of the embryo and faint stripes become visible. At stage 5, chn mRNA also accumulates in the dorsal region, cephalic furrow and in the presumptive mesoderm. At stage 11, chn mRNA is found mostly in the mesoderm, and in ectodermal patches between the tracheal pits, where neurons of the PNS appear. Older embryos (stage 15) show strong expression, which is mostly restricted to the central nervous system (CNS) and PNS. In the latter case, the pattern suggests that expression occurs in many of the neurons of the ventral, lateral and dorsal clusters of neurons (Escudero, 2005).
In third instar wing discs , expression of chn is observed in rows of cells on either side of the prospective anterior wing margin and in groups of cells that coincide with proneural clusters of ac/sc expression. This pattern suggests that chn may be positively regulated by ac/sc. Indeed, expression of chn in proneural clusters is abolished in discs null for ac/sc (In(1)sc10.1). Moreover, overexpression of a UAS-sc transgene in the posterior compartment of the disc (en-Gal4 driver) induces ectopic expression of chn. Note that chn is also expressed independently of ac/sc in certain areas of the disc, such as the postnotum and posterior dorsal proximal wing. chn is also expressed in proneural clusters of the leg discs and in the eye/antenna disc. In the latter case, the pattern seems very similar to that of the proneural gene atonal (ato), and includes the region of the morphogenetic furrow, and the presumptive cephalic capsule and second antennal segment (Escudero, 2005).
When driven by en-Gal4, the EPIL6 insertion induces the overexpression of both chn and bda transcripts in wing imaginal discs. Hence, flies were created carrying either a UAS-chn or a UAS-bda transgene. Overexpression of UAS-bda using several drivers (MS1096-Gal4, C765-Gal4, ap-Gal4 and MS248-Gal4) does not cause noticeable phenotypic effects. By contrast, overexpression of UAS-chn with these and other drivers gives rise to extra bristles. Ubiquitous expression with the C765-Gal4 driver causes the appearance of many macrochaetae near wild-type bristles, and it increases the density of microchaetae [141±10 microchaetae per female hemi-notum versus 103±7 in Oregon R controls (averages of 10 hemi-nota). However, the bristles were always separated by epidermal cells, suggesting that Notch-mediated lateral inhibition is still active. With earlier-expressing drivers such as MS248-Gal4, overexpression gives rise to many bristles. In addition, this early expression reduces the size of the hemi-nota and interfers with their dorsal fusion. With the appropriate drivers many extra bristles appear on other regions of the fly body, including wings (C765-Gal4, MS1096-Gal4 and nub-Gal4), head (MS248-Gal4) and the metathorax (MS1096-Gal4) (Escudero, 2005).
Formation of additional bristles by overexpression of chn depends on the presence of the proneural genes ac/sc. Halving the dose of these genes sharply reduces the number of extra macrochaetae induced by the overexpression of chn. Removal of both ac and sc completely suppresses bristle formation. The genetic interaction between sc and chn is also manifested by the synergism of their overexpression in bristle formation, which gives rise to many macro- and mesochaetae in ectopic positions, such as the anterior notum (Escudero, 2005).
To examine the effects of the removal of chn, new LOF alleles were obtained by generating imprecise excisions of the Pl(2)42/18 insertion. One of these, chnECJ1, is probably a null, since the excision removed at least part of the promoter region of chn, and homozygous embryos lack the chn mRNA (as detected by in-situ hybridization) and die as embryos. In keeping with the expression of chn in the cells of the PNS, chnECJ1 embryos display conspicuous anomalies in PNS cells. These include the absence of many neurons, especially in the dorsal and ventral clusters, and an abnormal morphology of chordotonal lateral neurons, which appear bunched and lack the typical apical dendrites. Individually identifiable neurons such as the v'chn1 and the dbp are generally absent. Some of these defects are similar to the phenotype described for the original Pl(2)42/18 insertion but are more severe. Ubiquitous expression of UAS-chn in the epidermis (69B-Gal4 driver) largely rescues many of the missing neurons and the morphological defects of the lateral chordotonal neurons. However, overexpression of UAS-chn in a wild-type background (using da-Gal4, 69B-Gal4 and 1407-Gal4 drivers) does not appreciably affect the larval PNS (Escudero, 2005).
In the adult, the effects of chnECJ1 were examined through clonal analysis. Clones of cells that lack chn often fail to generate macrochaetae. These were observed to be missing in all notum positions, but scutellar bristles were the most sensitive. However, the penetrance was far from complete: clones including the dorsocentral or the posterior postalar positions lost approximately 25% of the bristles, while in scutellar clones about 45% of the bristles were removed. It was also observed, with low frequency, that the bristle shaft was missing, but not the tormogen cell, suggesting a role for chn in formation of the sensory organ. No defects were seen in the pattern of notum microchaetae (Escudero, 2005).
The effects of the removal of chn in macrochaetae formation were examined with the help of an RNA-interference construct -- UAS-chni. A strong (UAS-chniS) and a weak (UAS-chniW) expressing line were used. UAS-chniS clearly antagonized chn function, because it largely rescued the extra bristle and heminota fusion phenotypes of MS248-Gal4; UAS-chn. No rescue was observed by replacing UAS-chniS by UAS-lacZ or UAS-GFP, which indicated that the effect was not caused by reduced expression of UAS-chn in the presence of an additional UAS transgene. Moreover, overexpression of UAS-chniS with MS1096-Gal4 sharply decreased accumulation of the endogenous chn mRNA in the wing margin These experiments indicate that the UAS-chniS acts as a LOF allele of chn. With the drivers ap-Gal4 or sca-Gal4 (the latter promotes expression in proneural clusters), UAS-chniS moderately removes notum macrochaetae while microchaetae are not affected. With the MS248-Gal4 driver, the macrochaetae in the medial part of the notum, dorsocentrals and scutellars, were often missing, but, similar to the homozygous chnECJ1 clones, in no case was the phenotype fully penetrant (Escudero, 2005).
The effectiveness of both UAS-chniS and UAS-chniW in removing macrochaetae was increased when the accumulation of ac/sc in proneural clusters was compromised. Thus, flies overexpressing UAS-chniW with C765-Gal4 or pnr-Gal4 had, respectively, wild-type or almost wild-type phenotypes, but a sizable number of macrochaetae were lost in the heterozygous In(1)sc10.1/+ genetic background. The N allele AxM1 has been shown to reduce ac/sc expression in proneural clusters. Hence, in AxM1/+ individuals, several notum macrochaetae are missing. UAS-chniS almost completely eliminates the remaining bristles in AxM1/+ with the exception of the ANP and APA that are always present. The pnr prepattern gene is necessary for the formation of the dorsocentral and scutellar macrochaetae. In the hypomorphic pnrV1/pnr-Gal4 background, expression of ac/sc is diminished and these bristles are partially removed. In this genetic background, UAS-chniW almost completely eliminates all these bristles. Interestingly, UAS-chniW was able to partially suppress the extra macrochaetae that are generated by the expression of UAS-sc driven by C765-Gal4 -- the number of macrochaetae per notum decreased in the dorsocentral area from 12±1.2 to 7.5±1.2 and, in the scutellum, from 18±1.3 to 10.2±1.3. Moreover, macrochaetae that appeared away from the normal macrochaeate-bearing regions, such as the anterior notum, were almost completely eliminated (from 5.4±1.3 to 1±1.2). This suggested that these extra macrochaetae have a stronger requirement for chn than the extant bristles. Taken together, these and the data obtained from the chnECJ1 clones indicate that chn is not essential for macrochaetae formation, but that it facilitates the process (Escudero, 2005).
A genome-wide RNA interference screen was performed to systematically identify regulators of apoptosis induced by DNA damage in Drosophila cells. Forty-seven double- stranded RNAs were identified that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. These data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death (Yi, 2007).
The genes that are specifically involved in caspase-dependent cell death were classified. Substantial induction of caspase activity was observed 8 h after treatment with a topoisomerase II inhibitor, doxorubicin (dox), to induce dose-dependent cell death. Any RNAi suppressing this activity implicates the target gene in early regulation of caspase activation. In addition to dcp-1 RNAi, knockdown of dronc and jra (the Drosophila homolog of c-Jun) significantly suppressed caspase-3/7-like activity in the presence of dox, whereas the negative control, RNAi against calpain A, a calcium-dependent cysteine protease, did not affect this pathway (Yi, 2007).
This analysis was expanded to all of the genes identified in the initial RNAi screen and 20 dsRNAs were discovered that suppressed caspase activation induced by DNA damage. Interestingly, 12 of these genes were found to be epistatic to diap1 (Yi, 2007).
diap1 epistatic analysis was performed to further categorize the genes. DIAP1, the fly orthologue of the mammalian inhibitors of apoptosis proteins, is a direct inhibitor of caspases, and deficiency in DIAP1 leads to rapid caspase activation and apoptosis in vivo. Thus, apoptosis induced by the loss of DIAP1 presents an alternative apoptotic assay independent of DNA damage. Silencing of genes that regulate activation of the core apoptotic machinery may provide protection against apoptosis induced by both DNA damage and the loss of DIAP1. RNAi against dcp-1 partially suppressed cell death induced by the depletion of DIAP1 in Kc cells. Also, dronc RNAi potently protected cells against apoptosis induced by deficiency in DIAP1. Altogether, 32 of the genes confirmed from the primary screen provided significant protection against cell death induced by the silencing of DIAP1 (Yi, 2007).
Interestingly, 12 dsRNAs suppressed caspase-3/7-like activity after dox treatment and protected against cell death induced by diap1 RNAi, suggesting that these genes are required for apoptosis induced by multiple stimuli. To confirm that these genes are necessary for the full activation of caspases, it was determined whether these dsRNAs could suppress spontaneous caspase activity induced by diap1 RNAi. Maximal induction of caspase activity by diap1 RNAi was observed after 24 h, and this effect was completely suppressed by dsRNA against dcp-1. Importantly, ablating 10/12 dsRNAs resulted in the significant suppression of caspase activity compared with diap1 RNAi only (Yi, 2007).
In addition to dronc RNAi, dsRNAs targeting chn and dARD1 provided the strongest suppression of spontaneous caspase activity. Consistent with the observation that RNAi against chn protects against DNA damage-induced cell death, the mammalian orthologue neuron-restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) was recently identified as a candidate tumor suppressor in epithelial cells (Westbrook, 2005). Previous work indicates that Chn and NRSF/REST function as a transcriptional repressor of neuronal-specific genes (Chong, 1995; Schoenherr, 1995; Tsuda, 2006), suggesting that cellular differentiation may render cells refractory to caspase activation and apoptosis. Also, several metabolic genes, CG31674, CG14740, and CG12170, were identified that may be involved in the general regulation of caspase activation. It has been demonstrated that NADPH produced by the pentose phosphate pathway regulates the activation of caspase-2 in nutrient-deprived Xenopus laevis oocytes. Together with these results, these observations provide further evidence for an intimate link between the regulation of metabolism and induction of apoptosis (Yi, 2007).
To further explore the significance of these findings, whether silencing the mammalian orthologues of the fly genes identified from the RNAi screen confers protection against dox-induced cell death was investigated in mammalian cells. A set of mammalian orthologues was selected that are believed to be nonredundant. The list includes the orthologues of dMiro, which functions as a Rho-like GTPase; dARD1, which functions as an N-acetyltransferase; CG12170, which functions as a fatty acid synthase; and Chn, which functions as a transcriptional repressor (RHOT1, hARD1, OXSM, and REST, respectively; FlyBase). In addition, Plk3, a mammalian orthologue of Polo, was tested since dsRNA targeting polo potently protected against dox treatment (Yi, 2007).
The ability of siRNAs targeting a gene of interest to protect against DNA damage was tested in HeLa cells. As a positive control, cells were transfected with siRNAs targeting Bax or Bak, two central regulators of mammalian cell death. Indeed, silencing of Bax or Bak resulted in significant protection against dox- induced cell death. It was observed that plk3 RNAi provided partial protection against dox treatment, which is consistent with previous studies implicating Plk3 in stress-induced apoptosis. Interestingly, the knockdown of hARD1 dramatically enhanced cell survival in the presence of dox to levels similar to that of Bak. This protective effect was also evident at the morphological level. In cells transfected with a nontargeting control siRNA, dox treatment resulted in typical apoptotic morphology, including cell rounding and membrane blebbing. In direct contrast, cells transfected with siRNAs against hARD1 maintained a normal and healthy morphology and continued to proliferate in the presence of dox (Yi, 2007).
To examine whether the protection provided by siRNAs targeting hARD1 and plk3 is associated with the suppression of caspase activation, caspase activity was measured in these cells treated with dox. RNAi against plk3 provided partial suppression of caspase activity, again supporting the observed protection phenotype. Interestingly, the depletion of REST resulted in some suppression of caspase activity in the presence of dox even though the protection against cell death was not statistically significant. Consistent with the viability assay, complete suppression of caspase-3/7 activity was observed in cells transfected with hARD1 siRNA. These results indicate that hARD1 is required for caspase-dependent cell death induced by DNA damage. Furthermore, all four siRNAs targeting hARD1 were individually capable of providing robust protection against cell death, strongly suggesting that these siRNAs target hARD1 specifically (Yi, 2007).
Because the silencing of hARD1 dramatically suppressed activation of the downstream caspases, whether activation of the upstream caspases in response to dox treatment is also perturbed was also examined. Remarkably, hARD1 RNAi inhibited the cleavage of caspase-2 and -9 in cells treated with dox, whereas caspase cleavage was readily detected in control cells. Thus, it is proposed that hARD1 regulates the signal transduction pathway apical to the apoptotic machinery in the DNA damage response itself or the activation of upstream caspases (Yi, 2007).
Consistent with the results of the caspase-3/7 assay, silencing of hARD1 completely inhibited the appearance of activated caspase-3 induced by dox. This assay was used for a hARD1 complementation experiment to demonstrate the proapoptotic role of hARD1 in response to DNA damage. A new siRNA pool was used, targeting the 5' untranslated region of hARD1 (5'si); this treatment inhibited caspase-3 cleavage induced by dox treatment. Furthermore, caspase-3 cleavage was observed in reconstituted hARD1 knockdown cells. Because six out of six siRNAs against hARD1 provided strong protection against DNA damage–induced apoptosis and complementation of hARD1-sensitized cells to caspase activation, it is concluded that the functional role of ARD1 for dox-induced apoptosis is evolutionally conserved from Drosophila to mammals (Yi, 2007).
In summary, this study used an unbiased RNAi screening platform in Drosophila cells to identify genes involved in promoting DNA damage-induced apoptosis. Forty-seven dsRNAs were isolated that suppress cell death induced by dox. These genes encode for known apoptotic regulators such as Dronc, the Drosophila orthologue of the known proapoptotic transcriptional factor c-Jun, and an ecdysone-regulated protein, Eip63F-1, thereby validating the primary screen. Furthermore, this study implicates a large class of metabolic genes that were previously not suspected to have a role in modulating caspase activation and apoptosis, such as genes involved in fatty acid biosynthesis (CG11798), amino acid/carbohydrate metabolism (CG31674), citrate metabolism (CG14740), complex carbohydrate metabolism (CG10725), and ribosome biosynthesis (CG6712). These results support the proposal that the cellular metabolic status regulates the threshold for activation of apoptosis and thus plays a critical role in the decision of a cell to live or die (Yi, 2007).
Of particular interest is the identification of ARD1. Evidence is presented that RNAi against ARD1 provides protection against cell death and leads to the suppression of caspase activation induced by DNA damage in fly cells and HeLa cells. Furthermore, deficiency in dARD1 renders fly cells resistant to the spontaneous caspase activity and cell death associated with loss of Diap1. Importantly, substantial evidence is provided that hARD1 is required for caspase activation in the presence of DNA damage in mammalian cells. Cleavage of initiator and executioner caspases are suppressed in hARD1 RNAi cells treated with dox, suggesting that hARD1 functions further upstream of caspase activation, and the complementation of hARD1 knockdown cells restores caspase-3 cleavage. These data indicate that ARD1 is necessary for DNA damage-induced apoptosis in flies and mammals (Yi, 2007).
ARD1 functions in a complex with N-acetyltransferase to catalyze the acetylation of the Nα-terminal residue of newly synthesized polypeptides and has been implicated in the regulation of heterochromatin, DNA repair, and the maintenance of genomic stability in yeast. These studies suggest that ARD1 may be involved in regulating an early step in response to DNA damage. It is anticipated that future studies will focus on determining whether ARD1 functions in similar processes in mammals. The diversity of genes identified in our screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).
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date revised: 20 February 2007
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