encore: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - encore
Cytological map position - 63F1--3
Function - signaling
Symbol - enc
FlyBase ID: FBgn0004875
Genetic map position - 3-
Classification - R3H domain protein
Cellular location - cytoplasmic
In Drosophila, egg development starts at the anterior tip of the ovary, in the germarium, where the germline stem cells divide to produce a cystoblast and a self-renewing stem cell. Each cystoblast undergoes four mitotic divisions with incomplete cytokinesis. The resulting 16 cells of each egg chamber are connected by intercellular bridges called ring canals. Exit from the cell cycle at the end of these four mitotic divisions requires the downregulation of Cyclin/Cdk activity. In the ovary of Drosophila, Encore activity is necessary in the germline to exit this division program (Ohlmeyer, 2003).
In encore mutant germaria, Cyclin A persists longer than in wild type. In addition, Cyclin E expression is not downregulated after the fourth mitosis and accumulates in a polyubiquitinated form. Mutations in genes coding for components of the ubiquitin-protease pathway such as cul1, UbcD2 and effete enhance the extra division phenotype of encore. Encore physically interacts with the proteasome, Cul1 and Cyclin E. The association of three factors, Cul1, phosphorylated Cyclin E, and the proteasome 19S-RP subunit S1, with the fusome is affected in encore mutant germaria. It is proposed that in encore mutant germaria the proteolysis machinery is less efficient and, in addition, reduced association of Cul1 and S1 with the fusome may compromise Cyclin E destruction and consequently promote an extra round of mitosis (Ohlmeyer, 2003).
One of 16 cells generated during the 4 mitotic divisions of the cystoblast, the one with four ring canals, develops into the oocyte and the rest give rise to nurse cells. Each of the four mitoses is oriented and synchronized by the fusome; a germline specific organelle composed of membrane and cytoskeletal proteins. After each division, the fusome grows by fusion of ER-Golgi type vesicles and extends through the ring canals in order to connect all the cells of the cysts. The mechanism by which the number of cyst mitoses is limited to four has not been fully elucidated. However, the studies of various mutations suggest that the fusome plays a role in regulating the timing, the synchronization, and perhaps the exit from the cell cycle in the germarium. Several genes have been implicated in the regulation of germline division. Mutations in genes coding for components of the fusome such as hu-li tai shao (hts), alpha and ß spectrin, and Dynein heavy chain (Dhc64) result in egg chambers that contain less than 16 germline cells and often lack an oocyte. The integrity of the fusome is compromised and the resulting number of cells in these mutant egg chambers is variable and not always a factor of 2n as in the wild-type cyst. Mutations in genes encoding proteins that associate with the fusome such as bag of marbles (bam) or genes required for proper association of Bam to the fusome such as benign gonial cell neoplasm (bcgn) result in mutant egg chambers that are tumorous and filled with proliferating cells. Mutations in the ovarian tumor (otu) gene that produce tumorous egg chambers have fragmented fusomes (Ohlmeyer, 2003 and references therein).
Overexpression or loss-of-function mutations in a third group of genes such as Cyclin A, Cyclin B, Cyclin E and mutations in the gene encoding the E2 Ubiquitin conjugating enzyme UbcD1 lead to the production of cysts with 32 or 8 cells. These genes do not affect fusome integrity and thus timing and spatial characteristics of cell division appear to be intact. The encore gene belongs to this group of genes: its product is necessary for exit from mitosis. Loss of Encore activity results in egg chambers containing 32 rather than 16 cells (Hawkins, 1996; Van Buskirk, 2000). Mutations in the encore gene produce additional phenotypes, which show differential temperature sensitivity. encore mutant females raised at 18°C produce egg chambers with 16 cells, but they give rise to ventralized eggs (Hawkins, 1997). The extra cell division phenotype is only observed when encore mutant females are raised at high temperatures (25°-29°C). The encore gene encodes a 200 kDa protein with no homolog of a defined biochemical function (Van Buskirk, 2000). The mechanism by which Encore promotes exit from the cell cycle after four germline mitoses has been investigated (Ohlmeyer, 2003).
Cell cycle progression is controlled by a series of cell cycle dependent kinases (Cdk). Cdk activity is carefully regulated by the levels of the Cyclin subunits, by Cdk inhibitors (CKI) and by post-translational modification of the Cdk subunit through both activating and inactivating phosphorylation. Transition from G1 to S phase depends on Cdk2/Cyclin E activity, and on the timely destruction of the Cdk2/Cyclin E inhibitor p27. The Drosophila p27 homologue, Dacapo, is required for exit from the cell cycle in the embryo and eye imaginal disc. In addition, exit from the cell cycle requires destruction of the cyclins by the ubiquitin-proteasome system (UPS). The addition of ubiquitin requires three different activities; the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the ubiquitin ligase enzyme (E3). The ubiquitinated protein bound to E3 is presented to the proteasome, isopeptidase activities in the 19S-recognition particle (RP) of the proteasome cleave the ubiquitin tail, the protein is unfolded and finally destroyed by the proteasome 20S-core particle (CP) (Ohlmeyer, 2003).
There are two E3 enzyme complexes that regulate the cell cycle progression. The first, the APC/cyclosome, regulates progression from G2 to M phase transition. The second, the SCF complex regulates the G1 to S phase transition. The SCF complex is composed of Skp/Cullin/Rbx1 and F-box proteins and controls substrate ubiquitination via an interaction between the F-box component and the phosphorylated target protein. In Drosophila and mammalian systems, mutations in the Cul3 and Ago genes have been shown to cause the accumulation of Cyclin E, entry to S-phase and doubling of cell number. Thus, proper regulation of the destruction machinery is important for maintaining normal levels of Cyclin E and assuring proper cell cycle progression (Ohlmeyer, 2003).
The work presented in this study demonstrates that the encore gene product associates with the SCF-ubiquitin-proteasome system and is required for proper exit from germline mitosis. The failure to downregulate Cyclin E after four cell divisions in conjunction with an accumulation of Cyclin A protein provide the conditions to promote an extra cell division. Encore can bind to Cul1, Cyclin E-Ub(n) and the proteasome. Cul1 and the proteasome 19S-RP subunit S1 are associated with the fusome and these associations are very much attenuated in encore mutant ovaries. It is proposed that as a direct consequence, Cyclin E is not degraded properly, its activity is misregulated and the cyst undergoes one extra cell division (Ohlmeyer, 2003).
This study shows that the fusome is a regulator of cell division during early oogenesis. Some of the functions ascribed to the fusome are to synchronize cyst mitosis and to provide the scaffold for the transport system necessary for oocyte determination. Limiting the number of cell divisions in the germarium could be achieved by regulating the association of proteins such as the cyclins and/or other cell cycle regulators with the fusome. The expression pattern of Cyclin A, Cul1, P-Cyclin E and 19S-S1 proteins in the germarium supports the idea that the fusome plays an important role in the regulation of mitosis. Indeed, Cyclin A association with the fusome is transient and occurs only during cyst division. In encore mutant germaria, Cyclin A remains associated with the fusome after cell division has stopped. Cul1 localization to the fusome suggests that the rest of the SCF complex also associates with the fusome and that substrate ubiquitination may happen at the fusome. The SCF component Cul1 is mainly associated with the fusome in the wild-type germaria. In encore mutant germaria, Cul1 localization to the fusome is very poor, leading to a proposal that this may be one reason why Cyclin E is not degraded properly. This also suggests that the degradation of Cyclin E and perhaps of other proteins degraded by the SCF-UPS may occur at the fusome. The association of P-Cyclin E supports this idea. The localization of P-Cyclin E in the wild type seems to be dynamic, consistent with the idea that the phosphorylated substrate is localized to the fusome, and then rapidly degraded via the SCF-UPS. In encore mutant germaria, the poor localization of Cul1 may result in an inefficient assembly of SCF complexes at the fusome. P-Cyclin E is localized to the fusome, but its degradation is compromised and as a result a consistent expression of P-Cyclin E is observed at the fusome. The partial association of the proteasome 19S-RP subunit S1 to the fusome supports the idea that proteolysis may occur at the fusome. The proteasome 19S-RP would recognize the polyubiquitinated substrate and recruit the rest of the proteasome to the fusome (Ohlmeyer, 2003).
The results suggest that Encore can associate with the SCF ubiquitin-proteasome system machinery and assists with the degradation of Cyclin E and perhaps other SCF substrates. Since the mutant Encore protein can still interact with SCF-UPS components, the mutant protein may form complexes but these might be inactive and/or the mutant protein poisons the degradation machinery. Consistent with such a hypothesis, the encore extra cell division phenotype is milder in hemizygous versus homozygous females at 25°C (Hawkins, 1996). Encore is required for the proper localization of Cul1, P-Cyclin E, S1 and presumably the rest of the proteolysis complex to the fusome. This localization may be more crucial at 29°C, whereas at lower temperatures a less efficient degradation system may have enough time for normal cell cycle regulation. encore mutations do not affect the 20S-Core Particle activity as measured by the rate of degradation of a fluorogenic peptide. It is not known whether Encore retains Cul1 at the fusome or whether Encore directly or indirectly modifies Cul1 in order to promote its localization at the fusome. Cul1 is known to be modified by the addition of Nedd8; however, Cul1 seems to be equally neddylated in encore and wild-type ovary extracts (Ohlmeyer, 2003).
In summary, the results suggest that the Encore protein assists with proper cell cycle progression in the Drosophila germarium by ensuring that Cul1 and the proteolysis machinery is localized at the mitosis coordination center, the fusome (Ohlmeyer, 2003).
Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Half pint (Hfp; FlyBase designation Poly-U-binding splicing factor) regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp. hfp is also required for efficient splicing of gurken mRNA and in the splicing of eukaryotic initiation factor 4E (eIF4E), which binds to the seven-methylguanosine cap at the 5' end of messenger RNAs and is a limiting factor in translation initiation (Van Buskirk, 2002).
The encore (enc) gene is also required for proper grk mRNA localization (Hawkins, 1997). However, in enc mutant egg chambers, Grk protein levels are reduced, and these females lay ventralized, not dorsalized, eggs. Thus it appears that enc is not only required for proper grk mRNA localization but for its translation as well. enc encodes a large, cytoplasmic protein of unknown function that becomes concentrated at the dorsal side of the oocyte in stage 9 egg chambers, where it colocalizes with grk mRNA (Van Buskirk, 2000). While the DV defects of enc are cold sensitive, enc females raised at higher temperatures show a highly penetrant extra mitosis in the germline, producing oocytes that are associated with 31 nurse cells instead of the normal 15 (Hawkins, 1996). However, the effectors that mediate this extra mitosis are not known (Van Buskirk, 2002 and references).
Half pint was isolated in a yeast two-hybrid screen with Enc; mutations in enc affect grk RNA localization and germline mitosis. Half pint is so named based on the mutant phenotype, which includes the production of short eggs and the formation of egg chambers that contain eight instead of sixteen germline cells. Surprisingly, half pint encodes the Drosophila homolog of human PUF60, an RNA binding protein characterized as a general splicing factor. Drosophila Hfp does indeed affect splicing: it specifically regulates the alternative splicing of a subset of genes within the ovary (Van Buskirk, 2002).
The encore (enc) gene is required for the regulation of germline mitosis, karyosome formation, and establishment of dorsoventral (DV) polarity of the Drosophila egg and embryo (Hawkins, 1996; Hawkins, 1997; Van Buskirk, 2000). enc encodes a large cytoplasmic protein with a single conserved region that contains an R3H motif (Van Buskirk, 2000), which is implicated in RNA binding. A portion of the Enc protein containing this conserved domain was used as bait in a two-hybrid screen for interacting proteins. A single factor that shows a specific interaction with Enc was recovered, and this interaction has been confirmed through coimmunoprecipitation. The gene encoding this interacting protein has been named half pint (Van Buskirk, 2002).
Both hfp and enc affect the regulation of germline mitosis. While hfp mutant egg chambers often contain eight germline cells, enc mutations result in an extra round of division, resulting in egg chambers with 32 germline cells. Overexpression of Otu104 can rescue the division defect of hfp and reduction of Otu104 activity can suppress the enc extra division. However, though Otu104 levels are strongly decreased in hfp, no converse overproduction of this isoform is detected in enc mutants, and, thus, enc does not appear to antagonize Hfp's splicing activity. It is possible that enc regulates germline mitosis through a pathway independent of hfp. However, the observed interaction between Hfp and Enc proteins raises the possibility that these genes function in a common pathway. If Enc does not antagonize Hfp's splicing activity, though, under what circumstances do these proteins interact? It may be useful to consider the Hfp-Enc interaction in terms of the roles these genes play in DV patterning. hfp and enc mutants both show defects in grk RNA localization, and enc is required for the accumulation of Grk protein (Hawkins, 1997). hfp plays a role in grk splicing. As has been observed for other splicing factors, it is possible that Hfp remains associated with its targets during nuclear export and perhaps delivers the spliced grk transcript to Enc in the cytoplasm. Enc may in turn be required for both anchoring and translation of the grk message. It is thus proposed that Hfp may have two roles: one in splicing regulation and one in the subcellular targeting of RNAs (Van Buskirk, 2002).
At higher temperatures, the rate of the ubiquitin-proteasome dependent proteolysis decreases and polyubiquitinated proteins may require the activity of ancillary proteins for proper substrate presentation and destruction by the proteasome. The results suggest that encore has a role in facilitating the destruction of Cyclin E and suggest a possible physical interaction between Encore, Cyclin E and components of the SCF pathway. Immunoprecipitation assays followed by Western blots were performed using antibodies directed against the Drosophila Cyclin E, Encore, Cul1 and against the mammalian proteasome 19S-RP subunit S1 and the proteasome 20S-CP subunit LMP7. Encore can immunoprecipitate Cyclin E and antibodies against Ubiquitin recognize this protein. Unexpectedly, antibodies against Encore can immunoprecipitate Cyclin E in encore mutant extracts. These immunoprecipitation experiments were performed using the point mutant allele encQ4 and the P-element insertion allele encBB. Both encore mutant alleles produce protein. Encore and Cul1 antibodies can immunoprecipitate the same Cyclin E-Ubn and there is more Cyclin E-Ubn immunoprecipitated in the encore lanes compared with the wild-type lanes. Significantly, Cyclin E and Cul1 can associate with Encore. These observations suggest that Cyclin E-Ubn, Cul1 and Encore can form a complex. By contrast, the anti-Encore antibody did not precipitate Fizzy, Cyclin A or B. Antibodies against the S1 and LMP7 proteins immunoprecipitate several Cyclin E-Ubn forms of the same (and higher) molecular weight than the Cyclin E-Ubn associated with Cul1 and Encore. LMP7 seems to bring down more Cyclin E-Ubn in wild-type extracts compared with the encore mutant extracts. Antibodies against S1 and LMP7 can immunoprecipitate several Encore forms, suggesting that Encore may be part of a complex formed by the SCF-proteasome system and that Encore may be a substrate for the ubiquitin-proteasome system. The fact that Encore can be seen in the mutant and wild-type lanes suggests that the defective protein can still form a complex with SCF-proteasome components. At the moment no protein null encore mutation is available and therefore it is not known whether Encore is required for complex formation in the germarium (Ohlmeyer, 2003).
During Drosophila oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal–ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal–anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation (Clouse, 2008).
This study has taken a direct approach to identify proteins that interact with Sqd protein in ovaries. Using an Sqd antibody, immunoprecipitations out of ovarian extracts were performed, proteins were isolated that specifically interacted with Sqd, and those proteins were identified by mass spectrometry. Four of the Sqd-interacting proteins were positively identified in the mass spectrometry analysis: Cup, PABP55B, Imp, and Hrb27C/Hrp48. The remaining bands were not identified with certainty. Imp and Hrb27C/Hrp48 are two factors that have previously been shown to be involved in RNA localization, and both Hrb27C/Hrp48 and Imp bind to grk mRNA. The identification of these two factors confirmed that the immunoprecipitation method could successfully identify functional Sqd interactors (Clouse, 2008).
One of the Sqd interactors identified in the mass spectrometry analysis was the novel 150-kDa protein Cup. cup mutants display egg chambers with nurse cell nuclear morphology defects and eggs with open chorions. Cup interacts with several factors known to be required for osk localization and translation, such as Exu, Yps, eIF4E, Me31B, and Bruno and independent studies have shown that osk mRNA is prematurely translated in cup mutants. Cup co-localizes with the cap-binding protein, eIF4E, and eIF4E is not properly localized to the oocyte posterior pole in cup mutants. Cup competes away eIF4G, another translation initiation factor, for binding to eIF4E, thereby repressing translation. Together, these data are consistent with the following model for Cup-mediated translational repression; Cup represses the translation of RNAs containing BREs through interactions with Bruno. In this complex, Cup binds directly to eIF4E and interferes with eIF4G binding to eIF4E. Because eIF4G binding to eIF4E is a prerequisite for translation initiation, Cup represses translation by blocking this interaction. Direct biochemical data supporting this model have recently been obtained (Chekulaeva, 2006). It is proposed that Cup represses grk translation by a similar mechanism prior to its localization to the dorsal–anterior of the oocyte (Clouse, 2008).
Cup activity is used by several transcript-specific factors to mediate translational repression of that RNA in a developmentally appropriate context. For instance, Cup is required to mediate the translational repression of the nanos (nos) transcript. Cup has been shown to interact with Nos protein and co-localizes with Nos in the germarium. cup and nos also interact genetically, as heterozygosity for cup suppresses nos-induced phenotypes in early oogenesis. Later in development, Cup binds to Smaug, a factor that specifically binds to nos RNA and is required for its translational repression in embryos. In this example, Cup is required for Smaug to interact with eIF4E and mediate nos repression. Consistent with this biochemical model, Smaug-mediated translational repression is less efficient in cup mutants (Clouse, 2008).
This study as shown that Cup is also required for grk translational repression. This contrasts with previous reports that grk expression is normal in cup mutants, but these earlier reports used relatively weak cup alleles and monitored Grk levels by immunofluorescence. In contrast, in this study alleles were used that allowed assessment of the eggshell phenotype in cup mutants, providing the most sensitive assay for defects in Grk levels. These analyses showed that the different cup alleles vary greatly in phenotypic strength and range of phenotypes (Clouse, 2008).
Using two different alleles of cup from two distinct genetic backgrounds, it was shown that cup mutants lay dorsalized eggs, display defects in Grk protein accumulation, and display less efficient grk mRNA localization. Furthermore, Cup interacts biochemically with Sqd and Hrb27C/Hrp48 in ovarian extracts. Finally, heterozygosity for cup is able to enhance the moderate dorsalization observed in weak allelic combinations of sqd. Together, these data strongly support a model in which Cup functions with Sqd and Hrb27C/Hrp48 to mediate the translational repression of the grk message (Clouse, 2008).
Once grk mRNA is properly localized to the future dorsal/anterior of the oocyte, translational control must be switched from repressive to promoting. In many cellular situations, this activation is accomplished by binding of PABP to polyA tails of transcripts. In fact, PABP55B contains four RNA-recognition motifs (RRMs) that directly bind to polyA tails. PABP55B also has a C-terminal polyA domain that is used for oligomerization of PABP55B on polyA tails. Once PABP55B is bound to RNA, it binds to eIF4G, and this interaction helps to increase the affinity of eIF4G for eIF4E. With this increased affinity, eIF4G is able to effectively compete with Cup for binding to eIF4E, and translation is able to begin (Clouse, 2008).
There are at least three polyA-binding proteins in the Drosophila genome (CG5119 at 55B, CG4612 at 60D, and CG2163 at 44B), which are predicted to function as general translation factors, so it is conceivable that PABP55B could regulate a subset of RNAs. CG2163 has also been designated as PABP2 and has been shown to have essential roles in germ line development and in early embryogenesis (Benoit, 2005). This study has shown that PABP55B mediates the translational activation of fully localized grk mRNA. Specifically, heterozygous pAbp55B mutants lay ventralized eggs in certain genetic combinations, and heterozygosity for pAbp55B also enhances the weakly ventralized phenotype of grk heterozygotes, consistent with a role in translational activation of grk (Clouse, 2008).
PABP55B binds to Enc in ovarian extracts, and that this interaction may be direct and not bridged by an RNA molecule. Furthermore, heterozygosity for pAbp55B is able to enhance the weakly ventralized phenotype of enc mutants raised at 25 °C. Taken together, the biochemical and genetic interactions suggest that PABP55B and Enc function together to mediate the translational activation of grk mRNA once it is localized to the dorsal–anterior of the oocyte (Clouse, 2008).
Previously, Enc has been shown to be required for activation of grk translation in mid-oogenesis. An effect on osk mRNA localization has also been previously observed in enc mutants, but it is unclear at what level this process is affected, or whether this effect is direct. In addition, Enc has been shown to interact with subunits of the proteasome early in oogenesis. Because of its large size and its ability to interact with several different proteins, Enc may play multiple roles during oogenesis. Considering the function of Enc in grk translational activation and its localization to the dorsal–anterior region of the oocyte, It is hypothesized that Enc could function as a scaffolding protein that helps to mediate the transition from translational repression to activation of grk mRNA (Clouse, 2008).
Cup functions with Sqd in a protein complex that mediates the translational repression of grk mRNA before it is properly localized. It is clear from the analysis of mutants such as spn-F and encore, in which mislocalized grk mRNA is translationally silent, that these two steps can be uncoupled. It is proposed that once the RNA has reached the future dorsal–anterior region of the oocyte, PABP, Sqd, and Enc facilitate the translational activation of grk mRNA, PABP is shown associating with the complex once it is fully localized; however, it is possible that PABP associates with the grk transport complex in an inactive form that is remodeled following its anchorage at the dorsal–anterior of the oocyte (Clouse, 2008).
Previous studies have shown that Bruno (Bru) binds directly to Cup protein and is required for the translational repression of osk. Bru binds to specific sequence elements in the osk 3′ UTR called Bruno Response Elements (BREs), and mutations in these BREs have been shown to reduce Bru binding and result in ectopic Osk accumulation in the oocyte. Similarly, Bru has also been shown to bind to grk mRNA and to Sqd protein. Overexpression of bru cDNA leads to ventralization of the eggshell, consistent with reduced Grk protein expression in the oocyte. Furthermore, disrupting bru expression in certain genetic contexts has been shown to result in excess Grk protein in the oocyte, consistent with Bru being required to mediate grk translational repression. In light of the results presented in this study, it is proposed that this phenotype is the result of Bru-mediated repression of grk translation by Cup (Clouse, 2008).
The mechanism of grk translation and the trans-acting factors required for translational control largely parallel the mechanism employed by osk RNA, so an important question to be answered is how these two different RNAs are differentially transported and translationally regulated in distinct parts of the oocyte at the appropriate stage in oogenesis. Since the same group of trans-acting factors is involved in the expression of both RNAs, the specificity could be provided by cis-acting sequences within the RNA molecules themselves that affect the activity of common trans-acting factors. Alternatively, RNA-specificity could be generated by as-yet unidentified trans-acting factors. Given that Enc functions in grk translational activation, but is not required for osk translational activation, it is possible that Enc is providing some degree of specificity to the commonly used machinery that mediates translational control of multiple, unrelated transcripts. Currently, Enc is the only factor known to function uniquely in the translational activation of grk mRNA, and these results provide the first evidence of a link between this factor and the general translational control machinery that is used by multiple RNAs in oogenesis (Clouse, 2008).
The mitotic phenotype of enc mutants suggests that enc acts to restrict cystocyte division. Thus it seemed reasonable that Enc expression within the germarium might coincide with exit from the mitotic cycle, at the formation of the 16-cell cyst. However, Enc is detected in all germline cells of the germarium, including the stem cells and dividing cystocytes. Thus Enc expression alone cannot inhibit cystocyte division. Enc begins to accumulate preferentially within the future oocyte shortly after formation of the 16-cell cyst. In midoogenesis, Enc can be seen transiently at the posterior edge of the oocyte, but by stage 9 assumes an anterior localization, and appears to be more concentrated at the dorsal side of the oocyte, above the oocyte nucleus. This pattern of localization is similar to that seen for grk RNA and protein, though Enc is not as tightly restricted to the dorsal side. In a double labeling experiment, Enc protein is seen to colocalize with grk RNA at the dorsal-anterior corner of the oocyte. While the localization of Enc protein around the oocyte nucleus is not dependent upon the presence of grk mRNA, the localization of grk mRNA is perturbed in enc mutants, such that the RNA is less tightly restricted to the dorsal side of the oocyte. Since Enc does not contain known RNA-binding motifs, it is unlikely that it binds directly to the grk mRNA, though this possibility cannot be ruled out. It is possible that Enc is part of a complex of proteins required for the localization and translation of grk, or it could act indirectly, affecting the activity of a factor involved in these processes (Van Buskirk, 2000).
Within the male germline, four rounds of mitosis occur to generate 16 primary spermatocytes in a manner similar to that seen in female cystocyte division. It was thus of interest to see whether enc might also be expressed in the dividing cystocytes of males. Enc is expressed at the tip of the testis, where germline mitosis occurs. The possible function of enc in spermatogenesis has not been investigated, but male fertility does not appear to be affected in enc mutants (Van Buskirk, 2000).
encore is involved both in regulating the number of germline mitoses and in the process of oocyte differentation. Mutations in encore result in exactly one extra round of mitosis in the germline. Genetic and molecular studies indicate that this mitotic defect may be mediated through the gene bag-of-marbles. The isolation and characterization of mutiple alleles of encore reveals that they were all temperature sensitive for this phentoype. Mutations in encore also affect the process of oocyte differentiation. Egg chambers are produced in which the oocyte nucleus has undergone endoreplication often resulting in the formation of 16 nurse cells. It is argued that these two phenotypes produced by mutations in encore represent two independent requirements for encore during oogenesis (Hawkins, 1996).
Although enc egg chambers contain twice the normal number of germline cells, only a single oocyte develops. The oocyte now possesses five ring canals instead of four. Since the oocyte develops from one of two cells with the maximum number of rings canals instead of a cell with four ring canals, it is not the absolute number of ring canals that is a determining factor in oocyte identity. Previously, it has been observed that oocytes could develop from cystocytes with fewer than four ring canals. However, this is the first demonstration that more than four ring canals are compatible with oocyte development. The exact doubling in the number of germline cells, and the presence of an additional ring canal on the oocyte has led to the conclusion that the enc phenotype is due to one extra round of mitosis in the germline. Therefore, enc appears to be required to regulate the number of cystocyte divisions (Hawkins, 1996).
The enc phenotype is unique. No other mutations have been isolated that result in precisely one extra round of mitosis in the germline. Mutations in genes that may have a role in regulating germline mitosis such as hu-li tai shao (hts) and bam produce phenotypes very distinct from enc. Mutations in hts result in egg chambers with too few nurse cells, which rarely develop an oocyte, while mutations in bam produce an ovarian tumor phenotype. In contrast, an extra nurse cell phenotype is Delta (Dl) and pipsqueak (psq), however, the mechanism involved is different from enc. This group of mutants affect follicle cell populations necessary for the correct separation of adjacent 16 cell cysts, resulting in egg chambers containing multiple 16 cell cysts. Unlike enc, the total number of nurse cells in these compound egg chambers is variable and additional oocytes often develop (Hawkins, 1996).
Bipolar egg chambers, which are most prevalent in the original P-element insertion allele encBB, also have a doubling in the number of germline cells. In these egg chambers, the oocyte also has 5 ring canals. Thus, the bipolar phenotype does not arise from a defect in oocyte specification. Instead, there is an inability of the oocyte to assume its correct position at the posterior pole of the egg chamber. In the bipolar egg chambers, the oocyte might be impeded from assuming its correct position in the egg chamber due to the presence of extra nurse cells. However, in certain enc allele combinations that produce almost 100% extra nurse cell egg chambers, the frequency of bipolar egg chambers is very low; generally less than 1%. Even for encBB, the frequency of this class varies depending on genetic background. Thus, enc is not likely to be directly involved in positioning of the oocyte (Hawkins, 1996).
Mutations in enc also produce egg chambers with a reduction in the number of nurse cells. Like the bipolar class, this phenotype was most frequent in the original P-element induced allele, encBB, but was also observed at a frequency of approximately 1% in the outcrossed encBB line and the EMS induced alleles. The reduction in the number of germline cells is not due to a decrease in number of cystocyte divisions. Instead, this phenotype appears to be due to a mispackaging by the follicle cells of a small group of nurse cells that were originally derived from a cyst that had undergone an extra round of mitosis (Hawkins, 1996).
All the enc alleles are temperature sensitive for the mitotic defect. This high incidence of temperature sensitive alleles is highly unusual. It has been estimated that on average only 5% to 10% of EMS induced mutations are temperature sensitive. It would be remarkable if all the enc alleles resulted in thermolabile proteins. More likely, enc could be participating in an intrinsically temperature sensitive process. This hypothesis is supported by the occurrence of temperature sensitive mutations in at least three other genes required for cyst formation: ovarian tumor (otu), fs(1)1621 (san fille) and fs(2)B (fes). In the case of otu, 15 alleles have been shown to be temperature sensitive (Hawkins, 1996).
From the genetic analysis it remains unclear if any of the enc alleles are null. Both loss-of-function and recessive gain-of-function alleles have beeen generated that produce an identical phenotype, one extra round of mitosis in the germline. The main difference between these two groups of alleles is the penetrance of the phenotype. The loss-of-function alleles are relatively weak, while the gain-of-function alleles produce nearly 100% mutant egg chambers as homozygotes. The gain-of-function alleles have been classified as recessive antimorphs since the phenotype of these alleles improves when placed in trans to a deficiency. The relatively weak phenotype produced by the loss-of-function alleles suggests that enc function might be partially redundant and that the antimorphic alleles produce a 'poison' product that antagonizes a wild-type pathway resulting in a stronger phenotype (Hawkins, 1996).
The mitotic defect of enc appears to be mediated through its effect on bam expression. In wild-type ovaries, bam transcript is detected in the cystobast and 2 cell cysts. In enc ovaries, it has been shown that the domain of bam expression is significantly expanded. This expansion of bam expression could merely be the consequence of early changes in cell fate due to the reiteration of the stem cell to cystoblast division or the cystoblast to two cell cyst division. This might result in additional cells having acquired a cystoblast or 2 cell cyst fate and as a consequence, the expression of a bam transcript occurs. However, since a null allele of bam acts as a dominant suppressor of the enc mitotic defect, the misexpression of bam transcript is not simply a consequence of the extra division but is required to produce the extra division (Hawkins, 1996).
In addition to enc's role in germline mitosis, there is a second requirement for enc during oogenesis in the process of oocyte differentiation. In wild-type egg chambers, the oocyte nucleus is arrested in meiosis I with a DNA content of 4C, while the nurse cell nuclei begin endoreplication as the cyst exits the germarium. In enc mutants, the oocyte frequently acquires a partial nurse cell identity in that the oocyte nucleus undergoes endoreplication. Usually the extent of endoreplication lags behind that of the adjacent nurse cell nuclei, though egg chambers were observed in which the oocyte nucleus was indistinguishable from the nurse cell nuclei. This phenotype is similar to that produced by a small subset of ovarian tumor (otu) alleles that allow formation of a nurse cell/oocyte syncytium. Oocytes derived from weak otu alleles have been observed in which the oocyte nucleus had undergone endoreplication. Like enc, the extent of endoreplication of the oocyte laggs behind that of its sibling nurse cells. Two other genes have been identified that are required for oocyte determination. Females mutant for recessive alleles in Bicaudal-D (Bic-D) or egalitarian (egl) produce egg chambers comprising 16 nurse cells and no oocyte. Egg chambers develop until mid oogenesis and then degenerate prior to vitellogenesis. In contrast to Bic-D and egl, none of the enc alleles produce a completely penetrant phenotype for this defect although the strongest allele, encD6, produces very few post vitellogenic egg chambers and results in no eggs being laid. Many other enc alleles produce an intermediate phenotype in which post vitellogenic egg chambers are observed with a polyploid oocyte nucleus. Since this phenotype is less penetrant than in Bic-D or egl, enc might be involved in the maintenance of oocyte identity as opposed to the initial establishment of oocyte identity (Hawkins, 1996).
The role of enc in oocyte determination appears to represent an independent requirement for enc and is not simply a secondary consequence of the mitotic defect. Two lines of evidence support this assumption. (1) The oocyte nucleus defect occurs almost exclusively in egg chambers that have not undergone an extra round of mitosis. (2) This phenotype is cold sensitive, unlike the mitotic defect, which is temperature sensitive. enc therefore has at least two separable functions in oogenesis. It is required for the regulation of germline mitosis and it is subsequently involved in the process of oocyte differentiation (Hawkins, 1996).
Establishment of anterior-posterior and dorsal-ventral polarity within the Drosophila egg chamber requires signaling between the germline and the somatic cells of the ovary. The gene gurken (grk) encodes a TGFalpha-like protein that is localized within the developing oocyte and is thought to locally activate torpedo/Egfr (top/Egfr), the Drosophila homolog of the EGF receptor that is expressed throughout the follicular epithelium surrounding the oocyte. grk- Egfr signaling is required early in oogenesis for specification of posterior follicle cell fate and later in oogenesis for dorsal follicle cell fate determination, thus establishing the axes of the egg shell and embryo. Previous studies have shown that these patterning processes are highly sensitive to changes in the levels and localization of grk mRNA. Post-transcriptional regulation of Grk protein levels is required for correct pattern formation. encore (enc), a gene that functions in the regulation of germline mitosis and maintenance of oocyte identity, is also required for the accumulation of Grk protein during oogenesis. Evidence is presented that enc regulates Grk post-transcriptionally to ensure adequate levels of signaling for establishment of the anterior-posterior and dorsal-ventral axes (Hawkins, 1997).
A specific role for enc in the grk/Egfr signaling pathway was revealed by examination of Grk protein in enc mutant ovaries. This analysis shows that enc is involved in the posttranscriptional regulation of grk. By Northern analysis, grk RNA levels are similar to wild type while, by Western blot analysis, there is a signficant reduction in the amount of Grk protein at 18°C. However, even at 18°C there is not a complete absence of Grk protein. Since the strength of the enc ventralization is weaker than that produced by a strong grk mutant, the presence of a low level of Grk protein was expected. By antibody staining of enc mutant ovaries raised at 18°C, little or no Grk protein was detected at any stage of oogenesis. Thus, enc is required during all stages of oogenesis for maintaining wild-type Grk protein levels. At the permissive temperature of 25°C, in which >95% of the enc mutant egg shells are phenotypically wild type, Grk protein is correctly localized and, by Western blot analysis, the levels of protein are similar to wild type (Hawkins, 1997).
The reduction in Grk protein is unlikely to be an indirect effect of weak mislocalization of the grk RNA observed in enc mutant ovaries. Previous analysis of genes required for grk RNA localization has suggested that correct localization is not necessary for translation. In K10 and sqd mutants in which grk RNA is mislocalized along the anterior margin of the oocyte, wild-type levels of protein are observed. In addition, the mislocalization of the grk RNA in enc mutant ovaries is considerably less pronounced, with only a fraction of the egg chambers exhibiting detectable mislocalization, whereas the reduction in Grk protein levels is observed in all egg chambers (Hawkins, 1997).
There are two other requirements for enc during oogenesis. It is involved both in regulating the number of germline mitoses and in the process of oocyte differentiation (Hawkins, 1996). Although it is conceivable that either a doubling in nurse cell number or abnormal oocyte differentiation could affect the production of Grk, this analysis demonstrates that the ventralized phenotype observed in enc mutants is not a secondary consequence of either defect. The mitotic defect is temperature sensitive, in contrast to the D/V patterning defect, which is cold sensitive. At 18°C, the non-permissive temperature for the D/V defect, most egg chambers contain the normal number of nurse cells. In addition, at 25°C, egg chambers were observed which contained extra nurse cells but there was no visible ventralization of the follicle cells as assayed with the BB142 enhancer trap line. The ventralized phenotype is also unlikely to be an indirect result of a defect in oocyte differentiation. Although this phenotype is also cold sensitive, it was only observed in a subset of the enc alleles while ventralized eggs are produced by all the alleles. In addition, if egg chambers containing a polyploid oocyte nucleus developed into mature eggs, presumably they would never produce embryos upon fertilization. However, mutations in enc, in addition to producing eggs with ventralized egg shells, also produce ventralized embryos. These embryos must have been derived from eggs containing a normal haploid oocyte nucleus. Finally, the allele that produces the strongest ventralized phenotype, encR17, does not exhibit other oogenesis defects. Thus, a requirement for enc in the grk/Egfr signaling pathway represents a third independent requirement for enc during oogenesis (Hawkins, 1997).
Mutations in the encore cause one extra round of mitosis in the germline, resulting in the formation of egg chambers with extra nurse cells. In addition, enc mutations affect the accumulation of Gurken protein within the oocyte, leading to the production of ventralized eggs. enc mutants also exhibit abnormalities in karyosome morphology, similar to other ventralizing mutants such as okra and spindle B. Unlike these mutants, however, the defects in Gurken accumulation and karyosome formation do not result from activation of a meiotic checkpoint. Furthermore, the requirement for enc in these processes is demonstrated to be temporally distinct from its role in germline mitosis. Cloning of the enc locus and generation of anti-Enc antibodies reveal that enc encodes a large novel protein that accumulates within the oocyte cytoplasm and colocalizes with grk mRNA. It is argued that the enc mutant phenotypes reflect a role for Enc in the regulation of several RNA targets (Van Buskirk, 2000).
In wild-type egg chambers, the oocyte chromosomes condense early in oogenesis to form a compact structure, the karyosome, which persists until late oogenesis. In enc mutant egg chambers, however, in addition to the previously described polyploid oocyte nucleus phenotype, more subtle defects are found in the formation of the karyosome. In all alleles examined, a fraction of the egg chambers contain oocytes in which the karyosome has a less compact appearance. Often the nuclear material is split into two or three lobes or can be seen to line the inside of the nuclear membrane. While it is possible that these karyosome defects are related to the polyploid oocyte nucleus phenotype, homozygous encR17 females, which show severe defects in karyosome formation, do not display the previously described oocyte differentiation defect, suggesting that these phenotypes represent perturbations of separate processes (Van Buskirk, 2000).
grk-Egfr signaling is required not only for the establishment of dorsal follicle cell fates, but also earlier in oogenesis for the determination of the posterior follicle cells. A signal from these cells is in turn responsible for the polarization of the oocyte along the anterior-posterior (AP) axis. Thus in the absence of grk signaling early in oogenesis, the AP polarity of the oocyte is not correctly established, and oskar RNA, which is normally found at the posterior of the oocyte, becomes localized to an internal site within the oocyte. In addition, egg chambers were frequently observed in which Osk could be found at both sites. Thus the distribution of Osk protein is similar to the pattern of osk RNA localization in these mutant egg chambers, and hence enc is not required for Osk translation (Van Buskirk, 2000).
It was surprising to find that even the mislocalized osk RNA was translated in enc, given that in grk2B6 mutants, in which osk RNA is found mislocalized in 99% of egg chambers, Osk protein is never detected. The lack of Osk protein expression in grk mutants, and in other mutants with abolished posterior localization of Osk, has been shown, and thus it has been suggested that proper osk RNA localization is required for its translation. Recently, however, mutant situations have been found in which this is not the case. In egg chambers mutant for par-1, or those with Laminin A posterior follicle cell clones, osk RNA and protein can be found tightly localized to the center of the oocyte. These results with enc confirm that localization of osk RNA to the posterior of the oocyte is not absolutely required for its translation. Furthermore, sufficient Osk is present at this noncortical site so that Vasa, whose localization is dependent upon Osk, can be recruited (Van Buskirk, 2000).
Mutations in the encore gene cause the accumulation of Cyclin A protein in the Drosophila germarium: In wild-type ovaries, Cyclin A protein is expressed in a cell cycle-dependent manner. The Cyclin A protein is detected in the stem cells, and in dividing cystoblasts. Its expression declines rapidly in post-mitotic cysts. In encore mutant females raised at 29°C Cyclin A protein expression lingers longer than in wild-type germaria and is detected in cysts located in a posterior area not associated with Phospho-histone3 expression. This suggests that Cyclin A protein remains present after mitoses have stopped. The persistence of Cyclin A expression in the germarium may indicate that proper Cyclin A protein turnover is defective. In the Drosophila embryonic cellular blastoderm Cyclin A distribution is very dynamic and accumulates in the cytoplasm during interphase, in the nucleus during prophase and is degraded during metaphase. Immunohistochemical assays also show a dynamic Cyclin A subcellular localization in the germarium, which seems to depend on the cell cycle stage. Cyclin A levels in the cytoplasm increase to fill the cyst completely. In these cysts, Cyclin A is also observed in transient association with the fusome during late prophase/metaphase of the cell cycle. In dividing cells, Cyclin A is degraded at metaphase and there is no detectable Cyclin A in anaphase and telophase. Cytoplasmic accumulation and nuclear localization of Cyclin A in encore mutant germaria is comparable with wild type. The transient association of Cyclin A with the fusome, however, is prolonged; Cyclin A is observed in the fusome in a posterior position of the germarium. The accumulation of Cyclin A protein in encore mutant germaria can also be observed in Western blots. Increased levels of Cyclin A protein are observed in germaria-enriched extracts from encore mutant ovaries compared with wild type. These data suggest that encore mutations at the restrictive temperature of 29°C promote the accumulation and/or prevent the timely turnover of Cyclin A protein in the germarium (Ohlmeyer, 2003).
Reduction of Cyclin A protein by overexpression of the Cyclin A inhibitor Roughex suppresses the encore extra division phenotype: Overexpression of a Cyclin A transgene under the control of heat shock promoter results in an extra mitotic division in only 3% of the egg chambers. However, expression of a stable form of Cyclin A increases to 17% the number of egg chambers containing 32 rather than 16 cells. The expression of the HS-Cyclin A transgene causes an extra mitotic division in 4% of egg chambers. Given these results and the observed accumulation of Cyclin A protein in encore mutant germaria, it was of interest to find out whether Cyclin A is responsible for the extra mitotic division phenotype. Reduction of Cyclin A gene dose by half in an encore mutant background does not suppress the extra division phenotype. One reason for this result could be that one copy of the Cyclin A gene might produce enough protein to allow an extra cell division. Another possibility is that reduction of Cyclin A gene dose is compensated by turning on feedback mechanisms that affect the production or stability of Cyclin A. In order to circumvent this problem, a different approach to decreasing the amounts of Cyclin A protein was taken by over-expressing the Cyclin A inhibitor Roughex (Rux). The rux gene product binds to Cyclin A and this complex is then transported to the nuclei where it is destroyed via the ubiquitin protease system (UPS). Overexpression of Rux using a HS-rux transgene gives rise to a reduced number of mitotic divisions. Similarly, in Drosophila embryonic ectoderm and imaginal discs, expression of the HS-rux transgene at 37°C reduces the number of mitoses. The expression of the HS-rux transgene alone or in an encore heterozygous mutant background at 25°C had no effect on mitosis. Flies expressing the HS-rux transgene in an encore mutant background were raised at 25°C. In this experiment about 55% of the encore mutant control ovaries contained 32 cell egg chambers. The mild expression of Rux resulted in the suppression of the extra mitotic division phenotype such that only 25% of the egg chambers showed the encore phenotype. The extent of the HS-rux suppression did not vary significantly when in addition to expressing HS-rux, cyclin A gene dose was reduced by half. Thus, it seems that the extra Cyclin A protein present in the encore mutant germarium contributes to the promotion of an extra mitotic division (Ohlmeyer, 2003).
Cyclin E protein expression is altered in encore mutant germaria: In hypomorphic mutations of the Cyclin E gene, 30% of the egg chambers have only eight cells. Conversely, expression of a heat inducible cyclin E transgene induces entry to S phase and results in an extra round of mitosis in the Drosophila embryo and eye imaginal disc. When the HS-cyclin E transgene is expressed, a modest 6% of egg chambers produce an extra cell division. To assess Cyclin E protein expression in encore mutant ovaries, immunolocalization experiments were carried out on flies raised at 29°C. In wild-type germaria, Cyclin E protein is expressed in the nuclei of the germline stem cells, cystoblasts and dividing cysts. The protein levels are dramatically reduced after mitosis ends. There is no Cyclin E expression in postmitotic cysts of region 2A and it is absent in region 2B of the germarium. Cyclin E protein expression resumes in region 3 and persists throughout the rest of oogenesis. However, this second phase of Cyclin E protein expression is no longer synchronized since not all the cells in the egg chamber express Cyclin E simultaneously. This pattern of expression is in accordance with BrdU incorporation experiments that indicate the requirement for Cyclin E during S phase of the mitotic cycle and of the endocycle. In encore mutant ovaries, Cyclin E protein is expressed throughout the germarium, indicating that the mechanism of downregulation of Cyclin E at stage 2A and 2B is defective. Cyclin E is degraded during S phase in the cell cycle. Thus, in wild-type germaria, its expression oscillates and not all the cysts in region 1 express Cyclin E simultaneously. In encore mutant germaria, all cysts express some Cyclin E, indicating that at each cell division Cyclin E degradation is affected. The unsynchronized expression of Cyclin E in region 3 and later is comparable with wild-type ovaries. The Cyclin E protein expression in wild-type germaria suggests that cessation of mitosis in the ovary requires Cyclin E downregulation (Ohlmeyer, 2003).
Whether the persistent expression of Cyclin E in encore mutant germaria can promote the extra division phenotype was tested. Double mutant females homozygous for encore and heterozygous for Cyclin E were raised at 29°C. Indeed the encore extra division phenotype is suppressed from 70% to 10% in the double mutant females ovaries. The suppression of the encore phenotype is more pronounced when Cyclin E gene dose is reduced than when Cyclin A activity is reduced. It has been shown that Cyclin E overexpression can promote the accumulation of Cyclins A and B in the Drosophila embryonic ectoderm without affecting RNA levels. As S-phase progresses and Cyclin E expression increases, the CycE/Cdk2 complex promotes the destruction of the Rux protein and allows Cyclin A to accumulate in the cell. Whether the accumulation of Cyclin A in encore mutant germaria is a consequence of the abnormal expression of Cyclin E was tested. Reduction of cyclin E gene dose by half in an encore mutant background clearly reduces the accumulation of Cyclin A protein. Given these results, it is proposed that the persistent expression of Cyclin E in the encore mutant germaria causes the accumulation of Cyclin A. Reducing Cyclin E dose brings Cyclin A expression down to more normal levels resulting in suppression of the extra mitotic division. Because reducing Cyclin A activity levels has only a partial effect in suppressing the extra division, it is believed that the extra round of mitosis produced by Encore is promoted by the joint effects of accumulating Cyclin E and Cyclin A proteins (Ohlmeyer, 2003).
Cyclin E protein turnover is defective in the encore mutant germarium: An important feature of the cell cycle is the tight regulation of the Cyclin/Cdk complexes by the rapid and timely destruction of the cyclin partner. It was of interest to test whether the persistence of Cyclin E protein in encore mutant germaria results from improper degradation. Female flies were raised at 29°C and Western blots of extracts enriched for germaria and previtellogenic egg chambers were performed. These experiments revealed some differences between the expression of Cyclin E in the wild-type and encore mutant ovaries. In Drosophila, the Cyclin E transcript encodes two proteins: the zygotic and the maternal forms of Cyclin E, which are products of differential splicing. The predicted molecular weight for the maternal Cyclin E protein is 78 kDa and for the zygotic Cyclin E is 60 kDa. Western blots using polyclonal antibodies against Cyclin E show that extracts from encore mutant ovaries accumulate high molecular weight forms of Cyclin E. Extracts made in the absence of protease inhibitors show little protein and no difference in Cyclin E expression between the encore mutant and wild-type extracts. The addition of protease inhibitors results in a stronger signal in both wild-type and encore and reveals a clear difference in Cyclin E levels between encore mutant and wild-type extracts. Cyclin E accumulates as high molecular weight protein in the encore mutant compared with wild-type. This observation suggests a slower rate of Cyclin E degradation in the encore mutant extract. The addition of isopeptidase inhibitors further protects these high molecular weight forms of Cyclin E. NEM and Llnl inhibit the action of the proteasome 19S-RP isopeptidases that de-ubiquitinate substrates. In order to confirm that the high molecular weight bands are Cyclin EUbn, germaria-enriched extracts were prepared in the presence of protease inhibitors, NEM and Llnl. Immunoprecipitation assays were performed using antibodies against Cyclin E followed by immunoblot (IB) with antibodies against Ubiquitin. Cyclin E in the wild-type extract and the accumulated Cyclin E in the encore mutant extract are recognized by antibodies against ubiquitin. The reciprocal immunoprecipitation using antibodies against Ubiquitin followed by IB with antibodies against Cyclin E confirms that the rate of degradation of Cyclin E-Ubn in the encore mutant extract is compromised (Ohlmeyer, 2003).
Mutations in genes encoding SCF pathway components enhance encore's mitotic phenotype: These results suggest that proper destruction of Cyclin E requires Encore activity. In order to test this idea further, double mutant flies were generated using encore mutations and mutations in genes that encode for components of the SCF ubiquitination pathway. Mutations in the cul1 and archipelago (Ago) genes result in the accumulation of Cyclin E in mammals and in the Drosophila eye. As expected, reduction of the cul1 gene dose in an encore mutant background enhances the extra division phenotype from 27% to 65% at the mildly restrictive temperature of 25°C. Moreover cul1 mutations enhance the encore phenotype at room temperature from 3% to 44%. Thus, the reduction of Encore activity sensitizes the system such that even at room temperature, the proteolysis machinery can no longer ensure the destruction of Cyclin E when cul1 gene dose is reduced. Similar results were obtained with mutations in the Ubiquitin ligase component UbcD2 and effete (UbcD1). Reducing cul1 gene dose in an encore heterozygous background produces only 16-cell egg chambers. It is proposed that the reduction of Cul1 results in decreased degradation efficiency and accumulation of Cyclin E. In this situation, Encore is required to facilitate the destruction of the surplus Cyclin E. As expected for a component of the SCF-UPS, the Drosophila Cul1 protein is a nuclear protein and is expressed throughout oogenesis. Cul1 protein expression in the germaria of wild-type females raised at room temperature or 29°C, shows strong localization to the fusome. Unlike Cyclin A, Cul1 association with the fusome is not transient. Cul1 protein is observed in the germline stem cells in association with the spectrosome. During cystoblast division and up to region three of the germarium Cul1 can be seen in association with fusome. In encore mutant germaria of flies raised at 29°C, the association of Cul1 with the fusome is disrupted. There is more nuclear Cul1 staining in encore mutant germaria compared with the wild type. Western blots were performed using germarium-enriched extract and no difference was observed between the levels of Cul1 in wild-type and encore mutant extracts. This suggests that Encore activity is required for proper Cul1 localization to the fusome and that Cul1 association with the fusome may be important for proper Cyclin E processing. It also indicates that possibly degradation of this important cell cycle regulator occurs at the fusome (Ohlmeyer, 2003).
The proteasome 19S subunit S1 expression is defective in encore mutant germaria: The localization of Cul1 to the fusome suggests that perhaps Cyclin E and other SCF-UPS substrates may be degraded at the fusome. To test whether the proteasome is also localized to the fusome, immunostaining assays were performed using antibodies against the proteasome 19S-RP subunit S1. Indeed the 19S-S1 colocalizes with the fusome in wild-type germaria. 19S-S1 association with the fusome is incomplete, since not all the fusomes are associated with S1. Unlike Cul1, S1 seems to be associated only with some areas of the fusome. Moreover there is also S1 accumulation in a granular appearance in the rest of the germarium. In encore mutant ovaries of flies raised at the restrictive temperature of 29°C, S1 expression is very much reduced. However some S1 protein can still be seen localized to the fusome. Thus, it seems that Cul1, 19S-S1 and presumably the rest of the 19S-RP associates with the fusome. The strong reduction of Cul1 localization to the fusome in encore mutant germaria could result in inefficient recruitment of the 19S-RP and the rest of the proteolytic machinery (Ohlmeyer, 2003).
Phosphorylated Cyclin E association with the fusome is defective in encore mutant germaria: Cyclin E/Cdk2 activity is regulated by auto-phosphorylation of its regulatory subunit, Cyclin E. Phosphorylation of Cyclin E results in the disassembly of the Cdk2/Cyclin E complexes, follow by ubiquitination and destruction via the SCF-UPS. The results predict that phosphorylated Cyclin E (P-Cyclin E) would be degraded at the fusome. Using an anti-P-Cyclin E antibody it was found that P-Cyclin E is expressed at the tip of the wild-type germarium in region 1 and it associates with the fusome. Some cysts contain very high levels of P-Cyclin E that fill the cyst completely, other cysts express intermediate levels or no P-Cyclin E. Unlike expression of Cul1, P-Cyclin E association with the fusome is not observed in all cysts suggesting some periodicity. In encore mutant germaria of flies raised at 29°C, P-Cyclin E is present in most of the fusomes observed in a given germarium. These observations suggest that in encore mutant germaria, P-Cyclin E localization to the fusome occurs and because the degradation process is inefficient, more P-Cyclin E accumulates at the fusome (Ohlmeyer, 2003).
The Ubiquitin-proteasome pathway requires Encore activity for proper protein turnover: The data shows that Cyclin E can be ubiquitinated in encore mutant germaria and that the defect resides in the destruction of Cyclin E-Ubn. Polyubiquitinated proteins are recognized by the proteasome 19S-RP, deubiquitinated by resident isopeptidases and unfolded before being destroyed by the proteasome 20S-CP. In order to test whether encore mutations affect the activity of the proteasome 20S-CP subunit, the rate of proteolysis was measured in wild type and encore mutant germaria-enriched extracts. The peptidase activity was monitored by the hydrolysis of the fluorescent-labeled peptide Suc-LLVYMCA. The results show that rate of proteolysis in encore mutant extract of females raised at 29°C or room temperature is comparable to that of wild type extracts of flies raised at 29°C. These results suggest that Encore does not affect the peptidase activity of the proteasome and that the defect may reside in substrate recognition, the formation of an inactive complex and/or in subcellular localization of the proteolysis machinery. Whether the requirement of Encore for proper proteolysis was also tested using an exogenous mammalian protein. Commercially available histidine-tagged P27 was ubiquitinated in vitro. Deubiquitination and destruction of p27 was compromised when encore mutant extracts were used. In the wild-type situation, after 20 minutes of incubation most of the ubiquitinated p27 had disappeared. By contrast, after 1 hour, ubiquitinated p27 can still be detected in the reaction using encore mutant extracts (Ohlmeyer, 2003).
Search PubMed for articles about Drosophila encore
Clouse, K. N., Ferguson, S. B. and Schüpbach, T. (2008). Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila. Dev. Biol. 313(2): 713-24. PubMed citation: 18082158
Hawkins, N. C., Thorpe, J. and Schüpbach, T. (1996). encore, a gene required for the regulation of germ line mitosis and oocyte differentiation during Drosophila oogenesis. Development 122: 281-290. 8565840
Hawkins, N. C., Van Buskirk, C., Grossniklaus, U. and Schüpbach, T. (1997). Post-transcriptional regulation of gurken by encore is required for axis determination in Drosophila. Development 124: 4801-4810. 9428416
Ohlmeyer, J. T. and Schüpbach, T. (2003). Encore facilitates SCF-Ubiquitin-proteasome-dependent proteolysis during Drosophila oogenesis. Development 130: 6339-6349. 14623823
Van Buskirk, C., Hawkins, N. C and Schüpbach, T. (2000). Encore is a member of a novel family of proteins and affects multiple processes in Drosophila oogenesis. Development 127: 4753-4762. 11044391
Van Buskirk, C. V. and Schüpbach, T. (2002). half pint regulates alternative splice site selection in Drosophila. Developmental Cell 2: 343-353. 11879639
date revised: 30 May 2008
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