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).

GENE STRUCTURE

cDNA clone length - 6858 bases

Bases in 5' UTR - 683

Exons - 12

Bases in 3' UTR - 1328

PROTEIN STRUCTURE

Amino Acids - 1548

Structural Domains

The encore cDNAs isolated from the poly(dT)-primed library fell into two main classes, which differed in the length of their 3' UTRs. Using a probe common to both classes of cDNAs in Northern analysis, transcripts of 7 and 8 kb were detected in wild-type ovarian RNA. Using a probe unique to the longer class of cDNAs, only the 8 kb transcript is detected, strongly suggesting that the two transcripts differ only in the length of their 3' UTRs. These transcripts are not detected in the enc^BB mutant using a probe that lies downstream of the BB129 P-element insertion. A truncated transcript is observed in this mutant when a probe upstream of the insertion is used. The enc cDNA sequence is in total 6858nt, with a 3' end corresponding to the 8 kb transcript, and hence falls short of the expected size. Since primer extension analysis suggests that the 5' end may lie approximately 400 nt further upstream, bringing the total transcript size to only 7.2 kb, a discrepancy still exists between this cDNA and the observed transcript size. This may arise from an aberrant migration of the RNA in Northern analysis, from undetected splicing events, or from an inability to isolate the true 5' end of the gene (Van Buskirk, 2000)

Though primer extension analysis indicates that the 5' end of the enc transcript has not been isolated, analysis of the cDNA sequence strongly suggests that the translational start site has been isolated. The 5'-most exon of enc contains multiple stop codons in all frames, and the second and third exons each begin with potential initiator methionine codons, in-frame and separated by 426 bp. The second methionine, unlike the first, is preceded by a consensus translational start sequence, suggesting that the second methionine may be used for translational initiation. Furthermore, a transgene that excludes the first methionine can rescue the enc phenotypes. Thus while it is possible that both sites are used for translational initiation, the first appears dispensable for function. Hence all references to amino acid number assume the latter as the translational start site, corresponding to nucleotide 684 of the enc cDNA sequence, predicting an open reading frame of 1548 amino acids. There are no motifs in the protein sequence indicative of a particular biochemical function. Interestingly, however, database searches reveal homology to a group of closely related human cDNAs of unknown function that are over 40% identical with each other and most likely represent paralogous genes. The most similar of these to enc is KIAA0029 (GenBank accession Q15032). While the overall simlarity is low, a highly conserved domain within the N-terminal region is apparent: over 157aa, (aa 273-429 of Enc) these proteins are 47% identical and 67% similar. This domain is also present in a C. elegans predicted open reading frame (AAF39995). Thus encore encodes a large protein of unknown function with at least one domain that has been highly conserved (Van Buskirk, 2000).

date revised: 5 September 2004

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