BIOLOGICAL OVERVIEW

double parked (dup) is essential for DNA replication and belongs to a new family of replication proteins conserved from Schizosaccharomyces pombe to humans. Strong mutations in dup cause embryonic lethality, preceded by a failure to undergo S phase during the postblastoderm divisions. dup is required also for DNA replication in the adult ovary, establishing that dup is needed for DNA replication at multiple stages of development. Strikingly, Dup protein colocalizes with the origin recognition complex (see Orc2) to specific sites in the ovarian follicle cells. This suggests that Dup plays a direct role in DNA replication. The dup transcript is cell cycle regulated and is under the control of E2F and Cyclin E. Interestingly, dup mutant embryos fail both to downregulate S phase genes and to engage a checkpoint preventing mitosis until completion of S phase. This could be either because these events depend on progression of S phase beyond the point blocked in the dup mutants or because Dup is needed directly for these feedback mechanisms (Whittaker, 2000).

dup mutants are defective in DNA replication both in embryogenesis and in oogenesis. To analyze DNA replication in dup mutants, embryos were isolated from females heterozygous for dup^a1 that had been crossed to heterozygous males and pulse labeled with bromodeoxyuridine (BrdU). Homozygous mutant embryos were distinguished from heterozygous embryos by using a marked balancer chromosome. In the dup mutants embryonic DNA replication appears to be normal through S phase of cycle 15. This is most likely because maternal pools of Dup protein suffice for the earlier embryonic replication cycles. In contrast, BrdU incorporation is not detectable in cycle 16. The block in replication in dup^a1/dup^a1 homozygous mutant embryos occurs early in S phase, because no BrdU incorporation is seen in the nuclei (Whittaker, 2000).

In addition to its role in embryogenesis, the viable fs(2)PA77 mutation demonstrates that dup is necessary for DNA replication in adult tissues. Homozygous mutant fs(2)PA77 females lay eggs with thin egg shells, and chorion amplification levels are decreased. Follicle cell genomic replication and amplification can be visualized directly by BrdU labeling of ovaries. Between stage 9 and early stage 10A genomic replication ceases, and BrdU incorporation is no longer detectable throughout the nucleus. In stage 10B, BrdU is observed at four specific foci that are sites of amplification. Previous BrdU incorporation studies have shown that chorion amplification is decreased uniformly in the follicle cells of fs(2)PA77 homozygous mutant females (Calvi, 1998). Similarly, amplification in only a few stage 10B eggchambers is observed. Although incorporated BrdU is detectable in stage 11 eggchambers, the size of the foci is smaller, and this is indicative of reduced levels of amplification (Calvi, 1998). In addition to the amplification defects, genomic replication persists longer than normal in the fs(2)PA77 homozygous mutant females, since genomic replication is still seen in some follicle cells in late stage 10A eggchambers (Whittaker, 2000).

Occasionally fs(2)PA77/dup^a1 female flies are recovered. BrdU incorporation in ovaries from these flies has been examined and incorporation into the follicle cell genome is found to be reduced relative to wild type. This reduction is most apparent in stage 9. Moreover, no BrdU incorporation is detected at the chorion loci at any stage. These results indicate that genomic replication is decreased in dup mutants and that the initiation of amplification requires functional dup. Thus, dup is essential for replication in multiple stages of development and is required for genomic replication in both mitotic and endo cycles and also for amplification (Whittaker, 2000).

The subnuclear localization pattern of Dup at stages 10B and 11 is strikingly similar to that seen for Orc2 and Orc1, which have been shown to localize to the sites of chorion gene amplification at these stages. At stage 10B, Dup and Orc2 colocalize in the follicle cells, although the Dup staining appears more diffuse than that of Orc2. Two interesting differences are seen in the localization pattern of Orc2 and Dup. (1) At stage 10A, Orc2 has already localized to subnuclear foci. Dup, in contrast, is still diffusely localized in the nucleus. (2) Orc2 is not seen to localize to origins after stage 11. Interestingly, Dup is undiminished and remains detectable at subnuclear foci until stage 13, indicating that it may function during elongation. Alternatively, Dup could have no role in elongation but is not cleared from chromatin until after elongation has been completed (Whittaker, 2000).

Because ORC is localized specifically before Dup, a test was performed to see whether ORC function is a prerequisite for Dup localization at replication foci. There is a female-sterile mutation in the orc2 gene, fs(2)293, that is defective in amplification. The effect of the orc2 mutation on ORC localization has been examined. In fs(2)293 mutants, the amount of Orc2 is greatly reduced or undetectable at specific foci during amplification. Dup is not localized to specific foci in this mutant in many follicle cell nuclei. In other follicle cells, Dup can be seen at one focus, but the levels are greatly reduced. This suggests that Orc2 is required for Dup localization. Interestingly, in some nuclei, high nuclear levels of Dup are observed. The levels are higher than those seen during stages 7-10A. Additionally, DAPI staining of the nuclei doesn't indicate that morphology of the cells is altered. This may suggest that Orc2 is required either indirectly through its requirement for replication, or directly, to prevent nuclear accumulation of Dup. It may be that Dup enters the nucleus independently of ORC, but that ORC is required to localize it to the amplifying regions (Whittaker, 2000).

To determine if Dup is necessary for continued ORC localization, Orc2 localization was examined in the fs(2)PA77 mutant. The shape and number of Orc2 foci is normal in the mutant follicle cells, but the size is smaller. One interpretation of these results is that Dup plays a role in loading or maintaining Orc2 at the amplification origins. Because the shape and number of foci seen are appropriate for the stages examined, it was thought to be more likely that the decrease in levels of Orc2 localization results from lower levels of DNA replication, causing decreased copy number of the origins. dup is not required for normal levels of the MCM (see Disc proliferation abnormal) proteins in the nuclei (Whittaker, 2000).

If S phase is blocked during the postblastoderm divisions by treatment with aphidocolin, the cell cycle arrests and mitosis does not occur. In yeast it has been demonstrated as well that arrest in S phase stops the cycle and blocks mitosis. In contrast, in yeast, mutation of the Cdc18/Cdc6 protein required to initiate DNA replication does not block mitosis even though DNA replication does not take place. Therefore, it was of interest to test whether mitosis occurs after the S phase block in dup mutant embryos to determine whether this checkpoint is functional in dup mutants. The histone H3 phosphoH3 epitope (PH3) present on condensed chromosomes was used to test for the presence of mitotic cells in dup mutant embryos. Normally, in embryos that have completed cycle 16, PH3 antibodies label mitotic cells solely in the CNS and the PNS. No labeling is seen in the G0 epidermal cells or in the endo cycling cells. In contrast, in dup^a1/dup^a1, dup^a1/dup^a3, and dup^a3/dup^a3 mutant embryos many cells in the epidermis label with anti-phospho H3 antibodies. There are also some labeled cells in the gut in addition to the CNS and PNS. This defect is first manifest at mitosis 16, after the block in S phase is observed. The mitotic block occurs after the failure of DNA replication because closer examination of the chromosomes show that they are 2N. In addition, the number of nuclei in stage 11 embryos indicates that mitosis 15 takes place in dup mutant embryos. The number of nuclei within each segment of the mutant embryos is about half that of wild type, implying failure of the last division of embryogenesis (mitosis 16) but not the last two divisions. Although the possibility that in some cells mitosis 15 does not occur cannot be excluded, it does take place in the majority of the cells (Whittaker, 2000).

The labeled cells were blocked in an abnormal mitosis 16 with unreplicated chromosomes. The chromosomes appear over-condensed and in some cells, fragmented. No telophase figures were detected, and thus the mutant cells do not complete mitosis. After stage 12, as development progresses, the number of anti-phospho H3 labeled cells decreases. Because no indication that these cells completed mitosis was seen, this may be due to cell death. However, it is also possible that these cells abort mitosis and return to G0 or G1 phase. Thus, despite the failure to undergo S phase, cells enter mitosis in dup mutant embryos (Whittaker, 2000).

The dup mutant embryos are defective in another cell cycle process in that they fail to downregulate transcripts that were induced at the G1-S transition. This phenotype is the basis by which mutations in dup were originally isolated in a screen designed to recover mutations in G1-S transcription. PCNA transcripts remain at high levels after S phase 16 in dup mutants, rather than being downregulated. It was of interest to determine if dup mutations affect other S phase genes similarly. To test this, in situ hybridization with cyclin E and RNR2 riboprobes was done on dup mutant embryos. In all four alleles of dup, S phase transcripts fail to be down regulated in both the epidermis and the endo cycling cells. In older stage 14 embryos the level of transcript appears to decrease. Similar to staining with anti-phospho H3, this could be due to cell death occurring in the epidermis or cells reentering G0 or G1 phase. This failure to downregulate S phase transcripts is not the result of mitotic arrest in dup mutants, because pimples mutant embryos arrested at metaphase of cycle 16 downregulate transcripts properly (Whittaker, 2000). This transcriptional phenotype may indicate that dup plays a direct role in transcriptional regulation. Alternatively, the failure to down regulate S phase transcripts may simply result from a failure to complete S phase. The latter interpretation is favored for two reasons: (1) the replication phenotype precedes that of the transcription phenotype and (2) the female-sterile mutation in dup does not affect the levels of PCNA transcripts in the ovary (Whittaker, 2000).

Thus Dup is a member of a conserved family of replication proteins with homologs in organisms ranging from S. pombe to humans. Dup colocalizes with Orc2 to chorion amplification foci, suggesting that it is part of the replication machinery. The dup mutants are striking also because they lack the checkpoint that would normally prevent mitosis if replication were incomplete. This may be either because Dup is part of this regulatory pathway or because S phase does not proceed sufficiently far in the mutants for the cells to sense that replication is incomplete. Furthermore, dup mutants fail to downregulate S phase transcripts on the addition of the first G1 phase in embryogenesis, suggesting that completion of S or passage through a certain point in S phase may be required for this downregulation (Whittaker, 2000).

These results show that Dup is essential for replication at multiple stages of development: during the post blastoderm mitotic cycles (the endocycles), and the amplification that occurs during oogenesis. Dup is probably required for S phase in the early embryonic cycles as well, as the maternal pools of protein could account for why a replication defect is not detected until cycle 16. The mutant phenotypes do not distinguish whether Dup acts in initiation of DNA replication, elongation of replication forks, or both. Two results suggest that Dup acts after ORC in replication: (1) Orc2 is still localized in dup mutants; (2) Dup localization to foci does not become apparent until after Orc2 is localized. The fact that no BrdU incorporation is detectable in strong dup mutants suggests an early block in replication and a role for Dup in initiation. Because Dup foci persist longer than Orc2 foci and Dup remains localized during stages when amplification elongation rather than initiation is occurring, Dup may function in elongation as well. This does not exclude a role of Dup in initiation, since the MCM proteins are part of the preinitiation complex at origins but also appear to move with the replication fork. Further experiments will be required to determine whether Dup is present both at moving replication forks as well as the amplification origins (Whittaker, 2000).

The similarity between Dup and the S. pombe protein Cdt1 suggests a model for how Dup functions in replication. Mutations in cdt1 block DNA replication initiation (Hofmann, 1994). Recent experiments show that Cdt1 accumulates in the nucleus in G1 and is a component of the prereplicative complex (Nishitani, 2000). Cdt1 protein is in a complex with the Cdc18 protein, the S. pombe homolog of the Cdc6 initiator protein. In addition, loading of MCM proteins onto chromatin is blocked in cdt1 mutants (Nishitani, 2000). An ortholog to Cdt1 has been identified in Xenopus and shown to be required for DNA replication in vitro to load MCM proteins (Maiorano, 2000). Consistent with the results on Dup localization, Xenopus Cdt1 association with chromatin is dependent on ORC. However, in Xenopus extracts Cdt1 dissociates from chromatin after the initiation of DNA replication (Maiorano, 2000). By analogy to Cdt1, Dup may be needed in conjunction with Cdc6/Cdc18 to help load MCM proteins onto the origins, but also to maintain them at the replication fork. Because the MCM proteins are not specifically localized during amplification in the follicle cells, no effect of the dup mutations on MCM localization could be detected. Therefore, tests of this model will require biochemical approaches (Whittaker, 2000).

Interestingly, no Dup homolog has been found in S. cerevisiae. Dup may be needed for replication initiation in eukaryotes that have more complex origins of replication, and hence are not present in S. cerevisiae. Since sites of initiation in these eukaryotes are less specific than in S. cerevisiae, it is possible that Dup plays a role in helping to direct MCMs to the appropriate origins. This would be especially useful in higher eukaryotes where origin usage varies by cell type and time in development. Alternatively, S. cerevisiae may contain an analogous protein that is sufficiently diverged so as to have not been recognized in similarity searches. The MCM10 protein is needed to load MCM proteins into the S. cerevisiae prereplicative complex, so MCM10 could perform the same function as Cdt1/Dup in metazoans (Whittaker, 2000).

The dup mutations uncouple the dependency of mitosis upon completion of S phase. This may be because dup is part of the machinery that implements this checkpoint. However, precedents in yeast support the hypothesis that the failure to engage this checkpoint in dup mutants is a consequence of replication not initiating and not producing stalled intermediates that can be recognized. In both S. cerevisiae and S. pombe, mutations that block initiation of DNA replication do not block progression into mitosis, whereas arrest during elongation stages of DNA replication does. Therefore, if Dup is indeed critical for initiation of DNA replication, the absence of DNA synthesis may account for the checkpoint failure without any direct involvement of Dup in the machinery that signals the checkpoint (Whittaker, 2000 and references therein).

Mutations in dup also reveal a likely requirement for the initiation of S phase for the subsequent downregulation of S phase transcripts. Interestingly, mutations in string that block in G2 phase and prevent S phases 15 and 16 do not prevent downregulation of transcripts, nor does arrest in metaphase by mutations in three rows or pimples. Therefore, the failure to downregulate the level of S phase transcripts in dup mutants is not the consequence of a simple block in the cell cycle or complete prevention of S phase. It is logical that a cell would not downregulate the transcripts encoding products required for DNA replication until initiation had occurred, possibly reflecting a strategy to drive S phase to completion by maintaining high levels of the replication machinery. Interestingly, in S. pombe, cdc18 transcript is not downregulated if S phase is blocked with hydroxyurea, indicating that downregulation may require the completion, not merely the initiation, of DNA replication (Whittaker, 2000 and references therein).

Knockdown of SCF^Skp2 function causes Double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes

In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, data was considered from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like (Cul-1), SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF^Skp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type (Kroeger, 2013).

The generation of an eaterGal4; UAS-GFP strain allowed identification the functional importance of SCF complex members for the plasmatocyte blood cell lineage by a RNAi knockdown approach. Using this technique, several genes belonging to the core SCF complex were identified that, when knocked-down, caused a very distinctive giant cell phenotype. Importantly, as eater was bing used as a driver to identify complex components, it was confirmed that these enlarged cells were plasmatocytes by anti-P1 plasmatocyte-specific antibody staining. This suggested, as proof-of-principle, that knockdown of gene function in mature plasmatocytes could elicit aberrant phenotypes dependent on the functional requirement of an essential gene/gene complex (Kroeger, 2013).

Previous research has shown that there are several Drosophila genes that may be involved in SCF complexes in order to determine specificity for a substrate. The F-box is thought to convey specificity of this complex by recruiting the substrate, however activation of the Cullin by neddylation factors also plays a role in ubiquitation of the substrate. A comprehensive list of all known and predicted complex members was used to identify the remaining members of the specific SCF complex that function in Drosophila hematopoiesis, as knockdown of only one of each of the core components caused enlarged plasmatocytes. lin19, SkpA and Roc1a likewise play a role in the hematopoietic niche, the PSC of the larval lymph gland. Knockdown of these genes caused a decrease in the number of PSC cells, as well as an increase in the size of these cells. These data, along with the findings in this current study, suggest that the SCF complex has a significant role in multiple aspects of Drosophila larval hematopoiesis (Kroeger, 2013).

Using fluorescence microscopy, it was noted that the enlarged cells caused by the SCF knockdown had a significant excess of DNA in the nuclear region. To investigate the hypothesis that DNA re-replication was occurring in plasmatocytes with the SCF complex knockdown, anti-gamma- Tubulin staining of centrioles was performed. Previously, it was shown that knockdown of Gem elicits DNA re-replication, therefore this study used it as a positive control. It was evident that the lin19 knockdown had multiple centrioles in one giant plasmatocyte, similar to plasmatocytes from the gem RNAi samples. It was also clear that the DNA had replicated many times, without any cellular division as indicated by BrdU-positive, but phospho-Histone H3-negative enlarged cells. These data support the idea that plasmatocytes from SCF knockdown animals undergo DNA re-replication, thus the SCF complex is necessary for Dup degradation. Additionally, previous research had identified a number of proteins that when misexpressed or knocked-down cause an enlarged cell phenotype with excess DNA replication. Several papers have shown that misregulation of Cyclin E can cause aberrant DNA synthesis. Research has also suggested that knockdown of Gem can cause this excessive DNA phenotype. In the current experiments, antibody staining identified that the subcellular localization of both these proteins changed between control samples and the lin19 knockdown. Importantly, Dup is necessary for DNA replication, but it must be degraded to prevent re-replication. As the main role of Gem is to inhibit Dup, and Gem was no longer found in the nucleus in the knockdown, this is suggestive that Gem had complexed with Dup, removing it from the nucleus. Conversely, Cyclin E was found in the nucleus. This is notable because Cyclin E is known to phosphorylate Dup marking it for ubiquitination, leading to its nuclear localization. It is also known that SCF^Skp2 degrades Cyclin E. This is another explanation for the accumulation of Cyclin E in the nucleus of SCF knockdown hemolymph samples. These data suggest that Dup may be the target substrate for the SCF complex being studied, with a secondary target possibly being Cyclin E. Previous research in human cells has shown that SCF^Skp2 regulates the degradation of Cdt1 (the homolog of Drosophila Dup)(Li, 2003). It has also been shown that the activated SCF^Skp2 complex plays a role in murine hematopoiesis, by ubiquitinating proteins necessary for proper cell cycle, such as Cyclin E. There are still many questions to be answered about SCF regulation in blood cells, as some of these results are contradictory (Kroeger, 2013).

In addition to these data, protein localization in the knockdown of Cyclin E showed that Gem had been removed from the nucleus, again consistent with the notion that it was titrated away from the nucleus by binding Dup. This is plausible because the SCF complex can recognize its substrates due to phosphorylation state. Since Cyclin E was knocked-down, Dup was not properly phosphorylated, and it was not recognized as the substrate by the SCF complex, therefore never being ubiquitinated nor degraded. Furthermore, in the Cyclin E knockdown, Dup localized to the nucleus similar to its localization in the SCF knockdown. This would make it necessary for Gem to inhibit Dup, causing Gem to take on a non-nuclear localization, while Dup would have a nuclear localization, if Dup was in excess. Taken together, these lines of investigation support the hypothesis that Cyclin E is necessary to phosphorylate Dup, allowing the SCF complex to recognize and ubiquitinate it. Dup that remains in the nucleus after degradation must be bound by Gem for the cell cycle to progress properly. DNA re-replication will occur if Dup remains in the nucleus. It is highly suggestive that knockdown of Cyclin E or the SCF complex perturbs this mechanism, causing Dup accumulation in the nucleus, and the cells to re-initiate DNA replication. Furthermore, others have shown there must be a balance of Gem and Dup in the nucleus for proper progression through the cell cycle. This research shows that there is a lack of Gem and an accumulation of Dup in the nucleus, which leads to excessive DNA replication and additional centriole replication in five percent of the plasmatocyte population (Kroeger, 2013).

Although re-replication is one mechanism to explain the SCF loss-of-function phenotype, a similar non-canonical process, known as endoreplication, could also account for the over-replicative system in these cells. Endoreplication is a cycle in which cells undergo S phases that are separated only by gap phases but not an intervening mitosis. However, endoreplication is not known to occur in wild-type Drosophila plasmatocytes. Further, Drosophila plasmatocytes are most similar to mammalian macrophages, which also do not endoreplicate. Since several of the proteins studied in this paper have been implicated in re-replication with phenotypes including enlarged cells, increased DNA content, and multiple centriole replication, the hypothesis is favored that re-replication is triggered in plasmatocyte development in the absence of SCF complex activity (Kroeger, 2013).

It is intriguing that only five percent of the cells display the re-replication phenotype. One explanation is that the smaller cells have arrested. There are many intrinsic mechanisms to ensure proper cell cycle progression preventing re-replication and ultimately cancer. It is possible these enlarged cells have escaped these mechanisms, causing the cell to replicate their DNA many times without going through mitosis, while the smaller cells arrest, to prevent this phenotype. It is also possible that only five percent of these cells are going through cell division during the time the RNAi is functionally knocking down the gene. Previous research has suggested that during mid-to-late third instar larval stages, only one to two percent of cells are going through mitosis at a given time. eaterGal4 is activated during second instar, however there is likely a latent period between activation of Gal4 and protein knockdown by the RNAi. This is consistent with only five percent of cells having an active cell cycle, and becoming enlarged through re-replication. A final possibility is that there are partially redundant mechanisms for the regulation of Dup. As previously described, the SCF complex has been shown to be involved in the ubiquitination and subsequent degradation of Dup, and Gem will inhibit the remainder of the Dup that may be in the nucleus. There may be additional mechanisms which ubiquitinate or inhibit Dup, therefore avoiding re-replication. The smaller cells may have activated one of these mechanisms to aid the cell in proper cell cycle, ultimately avoiding cancer. The regulation of Dup is of vast importance, and there are several possibilities of alternate mechanisms to prevent the re-replication phenotype elicited by cells which have excess Dup in the nucleus (Kroeger, 2013).

To further implicate the necessity of Dup regulation in the proper cell cycle of plasmatocytes, a rescue experiment was performed by overexpressing the Dup inhibitor, Gem. By overexpressing this inhibitory protein, it was hypothesized that the nuclear localization of Gem would increase, the protein would bind Dup, and therefore decrease the re-replication that is observed in SCF complex knockdown. Performing immunohistochemistry experiments identified that there was an increase in nuclear Gem and a decrease in Dup. Additional experimental evidence supports this hypothesis as there is a decrease in size of plasmatocytes with genotype pxnGal4>UAS-Gem⁴³>UAS-lin19 RNAi^HM05197 compared with pxnGal4>UAS-lin19 RNAi^HM05197. There is a drastic decrease in the number of giant cells, which are larger than 25.1 μm, in pxn>UAS-Gem⁴³>UAS-lin19 RNAi^HM05197 (8/100) plasmatocytes compared with SCF knockdown hemocytes (45/100). It was also noted that there was a significant decrease in the average size of plasmatocytes in hemolymph samples from Gem overexpression in the SCF knockdown background (p<0.001). These lines of evidence are all suggestive that knockdown of the SCF complex increased nuclear Dup leading to re-replication. By over-expressing its inhibitor, Gem, it is possible to partially rescue this enlarged cell phenotype generated by excess nuclear Dup. These data suggest the regulation of Dup is important in the proper cell cycle progression of plasmatocytes. Furthermore, these data support the hypothesis that the SCF^Skp2 complex is responsible for the ubiquitination of Dup, allowing plasmatocytes to proliferate properly. Although this study provides substantial genetic evidence that the SCF^Skp2 complex is necessary to ubiquitinate Dup allowing for proper hematopoietic cell cycle progression, future studies using biochemical techniques to show physical interactions are needed to support the model proposed here (Kroeger, 2013).

Furthermore, there are two ubiquitin ligase complexes known to be involved in the ultimate degradation of Dup: The SCF^Skp2 complex, described in this manuscript, and the Cul4-DDB1-CDT2-PCNA (Cul4^CDT2) complex. To vastly decrease the possibility that the Cul4^CDT2 complex was responsible for the enlarged cell phenotype, both DDB1 and PCNA were knocked-down via RNAi and Cul4 mutants were also analyzed. Although DDB1 functional knockdown elicited a small number of enlarged cells, these cells had a different morphology than the SCF^Skp2 knockdown. Additionally, none of the other analyses elicited any enlarged cells as observed when the SCF^Skp2 complex was knocked-down. This further implicates the necessity of the SCF^Skp2 complex in the proper plasmatocyte cell cycle (Kroeger, 2013).

To summarize, this manuscript identifies the SCF ubiquitin ligase complex as a novel regulator of plasmatocytes. Genetic evidence is presented that suggests that Dup is the main target for the SCF^Skp2 complex. It is proposed that the SCF^Skp2 complex plays an integral role in Drosophila hematopoiesis by ubiquitinating Dup, which is necessary for proper cell cycle progression. Knockdown of the SCF complex causes an accumulation of Dup in the nucleus, inducing the cell to undergo multiple rounds of replication without an intervening mitosis or cytokinesis. This causes some plasmatocytes to become vastly enlarged, with multiple centrioles and excessive DNA content. Taken together, these findings provide evidence that the SCF complex is necessary for proper cell cycle progression during plasmatocyte development in Drosophila. As the SCF complex is conserved from Drosophila to humans, these findings implicate the importance of the roles of ubiquitin ligase complexes in the cell cycle and their potential malfunctions in blood cell cancers (Kroeger, 2013).

Molecular determinants of phase separation for Drosophila DNA replication licensing factors

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, it was shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. In this study, using D. melanogaster (Dm) Cdt1 as a model initiation factor, it was shown that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori (Parker, 2021).

Previous work has shown that several factors used to initiate DNA replication in metazoans - in particular the Orc1 subunit of ORC, as well as Cdc6 and Cdt1 - possess an N-terminal IDR that promotes protein phase separation upon binding DNA. When these initiator proteins are combined and DNA is added, they form comingled condensates that exclude noncognate phase-separating proteins and are active for the ATP-dependent recruitment of the Mcm2-7 helicase. The biophysical mechanisms underlying LLPS by replication initiators have remained unknown (Parker, 2021).

This study used Drosophila Cdt1 as a model system to dissect the molecular basis for DNA-dependent phase separation by initiation factors. These studies show that this condensation mechanism has several distinctive features compared to other proteins characterized to date that also undergo LLPS. Two types of intermolecular interactions synergize with each other to drive phase separation by Cdt1. One set of interactions occur between Cdt1 and DNA. These associations appear to be nonspecific and electrostatic in nature, with DNA serving as a polyanion that bridges the cationic IDRs of multiple Cdt1 molecules. A second set of interactions that occur between the IDRs of different Cdt1 protomers were also identified; these appear to be salt-insensitive and primarily hydrophobic in nature. Importantly, it was found that inter-IDR interactions can drive Cdt1, ORC, and Cdc6 LLPS in the absence of DNA. When these interactions in Cdt1 were abolished, DNA is insufficient to induce phase separation. Thus, inter-IDR interactions provide the primary force behind initiator LLPS, with electrostatic DNA-bridging interactions contributing an additional adhesive force (Parker, 2021).

In the absence of a crowding reagent, DNA is required to induce initiator phase separation at physiological salt and protein concentrations. This dependency suggests that DNA serves as an essential nucleating factor that determines where Cdt1 can form condensates, ensuring that initiator LLPS is restricted to a chromatin context. Interestingly, other anionic biopolymers, such as RNA and heparin, are also capable of inducing phase separation by Cdt1. This type of scaffold 'promiscuity' has been observed in noninitiator systems that undergo LLPS, such as for the nucleolar protein fibrillarin. Given that the cytosol contains multiple anionic biopolymers, future work will need to answer how replication initiators are specifically targeted to chromatin, a question that is similarly relevant for RNA-dependent cellular bodies. The selection of an appropriate nucleic acid scaffold across different phase-separating systems is likely to rely on both general mechanisms, such as the sequestration of alternative, inappropriate scaffolds by proteins with different or nonexistent LLPS properties, and by system-specific mechanisms, such as a requirement for particular sequences or secondary structure (Parker, 2021).

A growing body of work has demonstrated the importance of aromatic residues in driving protein phase separation, along with a related role for π-π and π-cation interactions. In parallel, many studies have shown that condensates formed through aromatic residue interactions are readily dissolved by 1,6-HD. Cdt1 LLPS is both resistant to treatment with 1,6-HD and does not rely on aromatic residues within its IDR for phase separation. ORC and Cdc6 phase separation is also resistant to 1,6-HD. Although this study has not explicitly investigated the role of aromatic residues within these proteins, it is noted that the fraction aromatic residues of Orc1 (0.019) and Cdc6 (0.012) is lower than that of Cdt1 (0.027), suggesting that aromaticity is dispensable for LLPS by replication initiation factors in general. Similarly, charged residues have an established role in facilitating homomeric inter-IDR interactions that lead to phase separation, yet the inter-IDR interactions that drive initiator phase separation are salt insensitive (Parker, 2021).

Instead of relying on aromatic-mediated π-π/π-cation interactions or charge-charge interactions, the data show that interactions between Cdt1 IDRs are instead facilitated by branched hydrophobic residues. Although hydrophobic sequences have been shown to be important for the phase separation of other factors, such as the extracellular matrix protein elastin and the nephrin intracellular domain (NICD), important mechanistic distinctions exist with respect to metazoan replication initiators. For example, both NICD and Cdt1 bear an identical FCRs (FCR = 31%) and rely on a counterion for phase separation. However, inter-IDR interactions within NICD appear to rely primarily on sequence aromatics (11% aromatic content), with hydrophobic residues playing only a supporting role in the self-association of this protein. By contrast, elastin is more like metazoan replication initiators in that it is relatively devoid of aromatic residues (3.9%) and contains a similar content of branched hydrophobic amino acids that underly phase separation (elastin and Cdt1 fraction-L,I,V = 0.21 and 0.19, respectively); however, elastin also differs from initiators in that it readily forms condensates in the absence of a counterion. Elastin's hydrophobic sequences are additionally contained within highly repetitive 'VPGVG' and 'GLG' sequences, and scrambling or shuffling these motifs negatively impacts elastin phase separation. For their part, initiator IDRs lack such repeats and (for Cdt1 at least) LLPS is insensitive to sequence order. It is speculated that the inter-IDR interactions which occur in initiators are weaker than those of Elastin due to the nonrepetitive, distributed nature of their hydrophobic residues, a property that necessitates additional counterion-mediated interactions to promote Cdt1 self-assembly (Parker, 2021).

Understanding the distinctive molecular interactions of different phase-separating systems is essential for developing in silico approaches for predicting the cellular partitioning of a given IDR from primary sequence alone. Such efforts will ultimately require an understanding of both the specific and general features of condensates' scaffolding components (e.g., repetitive motifs vs. amino acid composition). The present work highlights the importance of the general sequence features of initiator IDRs (as opposed to strict amino acid order) in promoting LLPS. Strikingly, this study found that the Cdt1 IDR can be fully scrambled without losing the ability to form condensates and that condensates formed from the scrambled variant can recruit wild-type Cdt1 into droplets. Regions of NICD can similarly be scrambled without losing the ability to form cellular condensates, as can the LCDs of certain transcription factors without affecting promoter targeting. These studies demonstrate the importance of sequence composition in not only driving phase separation but also in facilitating interactions with partner proteins, and is consistent with recent work showing that proteins with similar functional annotation have disordered sequences with similar, evolutionary conserved physical-chemical features (pI, composition, FCR, kappa, etc.). The observations involving the cocondensation properties of Cdt1, ORC, and Cdc6 - three proteins with IDRs of similar amino acid composition but no direct sequence homology - provide experimental support for this 'like-recruits-like' concept. Future work will need to address whether heteromeric inter-IDR interactions (e.g., Orc1IDR-CdcIDR) are indeed governed strictly by sequence composition or whether linear sequence motifs facilitate specific interactions, as was recently suggested for human Orc1 and Cdc6. Further, while metazoan Cdt1 orthologs have no identifiable sequence identity across their disordered domains, they do have a similar amino acid composition. This led to a prediction that the capacity to phase-separate was likely conserved across metazoan initiators, and this was confirmed for human Cdt1. These predictions were further validated for human Orc1 and Cdc6 with the demonstration that these proteins form condensates in the presence of DNA. These results suggest that certain classes of disordered domains can have conserved functionality in the absence of linear sequence identity (Parker, 2021).

Although amino acid composition is the primary determinant underpinning LLPS by replication initiators, short sequence motifs can nonetheless play a critical role in modulating condensation behavior and controlling biological function. Sequence information within the Orc1 and Cdt1 IDR's is high, and while saltatory leaps seem to have occurred between phyla, the conservation of sequence homology within the Drosophila genus, over millions of years, is remarkable and indicates an evolutionary pressure on sequence order for unknown function. A conserved sequence feature within the IDRs of Orc1, Cdt1, and Cdc6 are CDK phosphorylation sites which are known to regulate replication initiation in vivo. It has been previously shown that the phosphorylation of these sites - which are distributed throughout the initiator IDRs - by CDKs abrogates the ability of the Drosophila initiators to form condensates, and a recent finding shows that phosphorylation impedes inter-IDR interactions between human ORC1 and CDC6 as well. Notably, consensus motifs for CDK-dependent phosphorylation (full site = [S/T]PX[R/K] and minimal site = [S/T]P) are conserved in a majority of metazoan Cdt1s and in all sequenced Drosophilidae Cdt1 orthologs, leading to a prediction that phospho-tunable phase separation is a broadly conserved mechanism for regulating metazoan replication licensing. Given that a scrambled IDR can still support LLPS in vitro through both self-interactions and cross-interactions with a wild-type IDR sequence, it will be useful to determine whether such a construct (with or without the native CDK sequences) can support normal kinetics of chromatin association and MCM recruitment and/or cell viability. Such studies, along with deletion and more targeted mutagenesis efforts, will be important to define how the plasticity of IDR sequences can be fine-tuned to elicit specific timing and partnering responses (Parker, 2021).

Defining the molecular 'grammar' that encodes LLPS in metazoan initiators likewise has implications for understanding the impact of initiator phase separation in vivo. Admittedly, neither this nor a previous study provides unequivocal evidence for initiator phase separation in cells. It is also unclear what function such concentrated initiator assemblies might have, such as improving the kinetics or efficiency of helicase loading (as has suggested, or something else entirely, such as maintaining the appropriate nuclear levels of the licensing factors. It is noted that the system lacks the convenience of identifying initiator condensation morphologically (i.e., by the presence of round cellular foci), as mitotic chromosomes - the stage of initiator assembly - are relatively rigid structures built from a central proteinaceous scaffold through an energy-consuming reaction. Thus, it is predicted that initiators condense on the surface of mitotic chromosomes without altering their shape. Consistently, work in multiple model organisms has revealed a switch-like transition in initiator localization during anaphase, at which point initiators begin to coat chromosomes. It is predicted that phosphorylation-dependent control of initiator self-assembly underlies this behavior and produces a dense, replication-competent zone of chromatin-bound initiators poised to drive the precipitous, genome-wide loading of the Mcm2-7 complex in late mitosis. This moment in the cell cycle is an opportune time to prepare for replication, as conflicts with the transcriptional machinery are minimized and the chromatin substrate is, relative to interphase, uniformly compacted. The identification of Cdt1 mutants that selectively block phase separation, but not DNA binding, affords an opportunity to directly investigate the physical nature of these global initiator interactions in vivo (Parker, 2021).

Beyond helicase loading and licensing, initiator LLPS might also serve to prime replisome assembly. Following helicase loading, Mcm2-7 is phosphorylated by the Dbf4-dependent kinase, a heterodimer of Cdc7 and Dbf4. Intriguingly, the Drosophila homolog of Dbf4, Chiffon, contains multiple IDRs, including a large internal IDR (residues 903-1209) that possesses striking compositional similarity to initiator-type IDRs. The identification of an IDR in Chiffon with compositional similarity to the initiator-type IDRs suggests that such sequences may be present in chromatin-associated factors beyond replication licensing components. Indeed, the capacity to undergo DNA-dependent phase separation may have broad utility for chromatin-localized factors and their respective cellular pathways. Thus, future work will aim to develop algorithms that accurately identify proteins proteome wide that possess disordered domains with compositional homology to initiator IDRs and to understand how these sequences impact protein functional dynamics (Parker, 2021).

In summary, the present work provides a detailed view of the DNA-dependent LLPS mechanism for Drosophila Cdt1. Due to a high level of IDR compositional homology and an ability to form comingled phases, the mechanism describe for Cdt1 phase separation likely extends to the other replication initiation factors, ORC and Cdc6. These studies set the stage for investigating the physiological significance of initiator self-assembly in replication licensing and for identifying other sequences in the proteome that possess IDRs capable of associating with chromatin and initiation factors alike (Parker, 2021).

GENE STRUCTURE

cDNA clone length - 2761

Bases in 5' UTR - 211

Bases in 3' UTR - 318

PROTEIN STRUCTURE

Amino Acids - 743

Structural Domains

Blast searches with the Dup sequence reveal that it is a member of a conserved family of proteins. The carboxy-terminal end of Dup shares very high similarity to a fully sequenced human cDNA the function of which has not been reported. The human cDNA is 34% identical and has 52% conservative changes compared to the carboxy-terminal 446 amino acids of Dup. The human cDNA sequence has been reported to be identical to a sequence 3' to the human adenine phosphoribosal transferase gene, making it likely that human Dup maps to this region on chromosome 16. Dup also shows high homology to several partially sequenced mouse ESTs. One of these shows 42% identity and 63% positives when compared to the carboxy-terminal end of Dup. There are predicted protein orthologs to Dup protein in Arabidopsis thaliana and Caenorhabditis elegans (22% identity and 36% positives and 23% identity and 39% positives, respectively). Interestingly, Dup is also related to S. pombe Cdt1 (20% identity and 37% positives), a protein known to be required for replication initiation (Hofmann, 1994). Alignments of Dup, Cdt1, and the human, C. elegans and A. thaliana orthologs show that these proteins share several conserved motifs, making it likely that they represent members of the same family of proteins. Arginine 342, which is changed to a cysteine in the fs(2)PA77 allele is conserved in humans, mouse, and A. thaliana, consistent with it playing an essential role in the function of Dup. The amino acid sequence of Dup does not reveal predicted biochemical activities (Whittaker, 2000).

date revised: 22 April 2023

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