InteractiveFly: GeneBrief

cul-4: Biological Overview | References

BIOLOGICAL OVERVIEW

The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate replication and transcription. To examine the roles of CUL4 in cell cycle regulation, the effect of inactivation of CUL4 was examined in both Drosophila and human cells. Loss of CUL4 in Drosophila cells causes G1 cell cycle arrest and an increased protein level of the CDK inhibitor Dacapo. Coelimination of Dacapo with CUL4 abolishes the G1 cell cycle arrest. In human cells, inactivation of CUL4A induces CDK inhibitor p27Kip1 stabilization and G1 cell cycle arrest which is dependent on the presence of p27, suggesting that this regulatory pathway is evolutionarily conserved. In addition, it was found that the Drosophila CUL4 also regulates the protein level of cyclin E independent of Dacapo. Evidence is provided that human CUL4B, a paralogue of human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causes the accumulation of cyclin E without a concomitant increase of p27. The human CUL4B and cyclin E proteins also interact with each other and the CUL4B complexes can polyubiquitinate the CUL4B-associated cyclin E. These studies suggest that the CUL4-containing ubiquitin E3 ligases play a critical role in regulating G1 cell cycle progression in both Drosophila and human cells (Higa, 2006a).

The CUL1 (cullin 1; see Drosophila Cul1) containing SCF (SKP1, CUL1/CDC53, F-box proteins) ubiquitin E3 ligases are key regulators of cell cycle progression from yeast to human. The SCF E3 ligases use different F-box proteins to bind and target various cell cycle regulators for ubiquitin-dependent proteolysis. In mammalian cells, it has been shown that SKP2, an F-box protein, primarily binds and targets phosphorylated CDK inhibitors p27Kip1 and p21Cip1 for ubiquitin-dependent proteolysis, while another F-box protein, human CDC4/AGO/FBXW7 regulates the proteolysis of phosphorylated cyclin E protein. In mammalian cells, the G1 cell cycle is regulated by the relative abundance of G1 cyclin/CDKs and CDK inhibitors such as p27 and p21. Similarly, the Drosophila G1 cell cycle is regulated by the balance between the CDK inhibitor Dacapo, which shares substantial homology to p27, and cyclin E. While cyclin E is regulated by the conserved Drosophila SCFAgo E3 ligase, it is not clear how the level of Dacapo is regulated in the cell cycle (Higa, 2006a).

Like other cullin family members, CUL1 is regulated by the covalent linkage of an ubiquitin like protein, NEDD8, through the neddylation activating enzyme E1 (APPBP1 and UBA3) and the E2 enzyme, UBC12. Neddylation of CUL1 dissociates CAND1, an inhibitor of SCF, from CUL1, and consequently promotes the binding of SKP1 and F-box proteins such as SKP2 to CUL1 and the assembly of the SCF E3 ligase complex. The neddylation of CUL1 is removed (deneddylated) by the peptidase activity of the COP9-signalosome complex (CSN; see Drosophila COP9 complex homolog subunit 5). Many lines of evidence suggest that the activity of cullins is regulated by the elegant balance between the neddylation and deneddylation activities (Higa, 2006a).

Cullin 4 (CUL4) is a conserved core component of a new class of ubiquitin E3 ligase that also contains the UV-damaged DNA-binding protein 1 (DDB1) and Ring finger protein ROC1 (also called RBX1 or HRT1). Unlike Drosophila or other metazoans, mammals encode two paralogues of CUL4, CUL4A and CUL4B (Kipreos, 1996; Higa, 2003). CUL4A and CUL4B are coexpressed in many cells but the functional differences between them remain unclear (Higa, 2003). Like other cullin E3 ligases, the CUL4 proteins also bind to CAND1 and CSN, and are regulated by neddylation and deneddylation processes (Zheng, 2002; Groisman, 2003). Previous studies suggest that CUL4-containing E3 ligase complexes and CSN regulate the proteolysis of replication licensing protein CDT1 (see Drosophila Cdt/Double parked) in response to UV or gamma-irradiation (Higa, 2003). Additional studies suggest that DDB1, a potential SKP1-like adaptor for CUL4 E3 ligase (McCall, 2005) is also involved in UV-induced CDT1 proteolysis (Hu, 2004). The CUL4ADDB1 complex also regulates the proteolysis of c-Jun and DDB2 (Wertz, 2004; Nag, 2001). However, the roles of CUL4-containing ubiquitin E3 ligases in cell cycle regulation remain uncharacterized. This study has investigated the regulation of cell cycle regulators by neddylation and CAND1 and reports the unexpected finding that CUL4 E3 ligase plays a critical role in regulating G1 cell cycle progression (Higa, 2006a).

Loss of CUL4 E3 ligases causes a G1 cell cycle arrest that is dependent on CDK inhibitors Dacapo in Drosophila and p27 in human cells. The regulation of Dacapo and p27 by CUL4 E3 ligases occurs at the post-transcriptional levels of protein stability. Although it has not been demonstrated that p27 can be directly polyubiquitinated by the CUL4 E3 ligase complexes in vitro due to technical difficulties, this study raises the possibility that CUL4 E3 ligases may regulate Dacapo or p27 by directly targeting them for ubiquitin-dependent proteolysis. Several lines of evidence support this hypothesis. Dacapo protein is regulated by CUL4 but not by CUL1 in Drosophila cells. Although in human cells, SCFSKP2 regulates p27, there is no structural and functional evidence that SKP2 is conserved in Drosophila cells. In addition, although Dacapo shares substantial homology to p27 or p21 in the core region that mediates cyclin or CDK binding, it diverges greatly at the carboxy terminal end with p27 in which the critical threonine 187 is located for the SCFSKP2- dependent proteolysis of p27 (this threonine is absent in Dacapo). Furthermore, it was found that there are no significant differences in the SCF-dependent p27 degradation between extracts derived from the control and DDB1 or CUL4A siRNA treated cells, suggesting that reduced levels of DDB1 and CUL4A proteins does not significantly affect SCFSKP2 activity. However, these experiments do not completely rule out the possibility that CUL4A/DDB1 are catalytically involved in SCFSKP2-mediated p27 degradation since small amounts of DDB1 and CUL4A proteins remain in the siRNA treated cells. Moreover, although SKP2 represents a major proteolysis pathway for regulating p27 degradation in S phase of human cells, substantial evidence suggests there are additional pathways that regulate the stability of CDK inhibitors. For example, it was found that the Xenopus p27 homologue p27Xic1 is polyubiquitinated on chromatin only when DNA replication starts in the Xenopus egg extracts. Replication licensing protein CDT1 is proteolyzed by CUL4/ROC1 E3 ligase in response to UV or gamma-irradiation. CDT1 is also degraded in S phase in mammalian cells and such an event can be reproduced in Xenopus egg extracts in which CDT1 was found to undergo ubiquitin-dependent proteolysis once DNA replication starts. In C. elegans, loss of CUL4 stabilizes CDT1 in S phase and causes the accumulation of polyploid nuclei containing 100C DNA content. It is possible that CUL4 may also regulate the proteolysis of Dacapo or p27 in similar processes in Drosophila or human cells (Higa, 2006a).

Cyclin E protein accumulates in CUL4 silenced Drosophila and human cells often in the absence of CDK inhibitors Dacapo or p27. Although this effect is more pronounced in Drosophila cells, the CUL4 E3 ligase may represent one of several pathways that regulate cyclin E in response to certain signals in mammalian cells (Strohmaier, 2001). It has been shown that CUL1- and CUL3-containing E3 ligases regulate cyclin E stability in mammalian cells. Cyclin E expression and its protein stability are also regulated by an E2F/DP-1 dependent process. This study found that cyclin E directly interacts with Drosophila CUL4 and human CUL4B and the isolated CUL4A or CUL4B immunocomplexes can polyubiquitinate the associated cyclin E in vitro. These observations raise the possibility that cyclin E may also be a direct ubiquitination target of CUL4 E3 ligases in vivo. These studies indicated that loss of CAND1, APPBP1, or CSN has differential effects on Armadillo/β-catenin and cyclin E. This effect could be partly explained by the observation that while Armadillo is regulated by CUL1-containing SCF ligase, cyclin E is controlled by both CUL1 and CUL4 E3 ligases. Evidence is also provided that the effects of CAND1, APPBP1 or CSN deficiency on the substrates of various cullin E3 ligases may be different. Further analysis is required to investigate the mechanisms for these observations (Higa, 2006a).

These data reveal that CUL4 E3 ligase represents a novel and conserved pathway from Drosophila to human cells in regulating CDK inhibitors and cyclin E. In the G1 cell cycle, the CDK inhibitors Dacapo and p27 appear to be the primary targets of CUL4 E3 ligases, since loss of CUL4 in Drosophila or CUL4A in human leads to the G1 cell cycle arrest rather than enhanced S phase entry. Since the gene encoding CUL4A is amplified in many breast cancers and hepatocellular carcinomas (Chen, 1998; Yasui, 2002) and since low or absent expression of p27 is often associated with malignant cancers, these studies also highlight how altered regulation of CUL4 E3 ligase may contribute to the genesis and progression of human cancers (Higa, 2006a).

L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage

The CUL4 proteins are the core components of a new class of ubiquitin E3 ligases that regulate cell cycle, DNA replication and DNA damage response. To determine the composition of CUL4 ubiquitin E3 ligase complex, anti-CUL4 antibody affinity chromatography was used to isolate the proteins that associated with human CUL4 complexes, and they were identified by mass-spectrometry. A novel and conserved WD40 domain-containing protein, the human homologue of Drosophila Lethal(2) denticleless protein (L2DTL) or fission yeast CDT2, was found to associate with CUL4 and DDB1. L2DTL also interacts with replication licensing protein CDT1 in vivo. Loss of L2DTL in Drosophila S2 and human cells suppresses proteolysis of CDT1 in response to DNA damage. The human L2DTL complexes weere isolated by anti-L2DTL immuno-affinity chromatography from HeLa cells, and it was found to associate with DDB1, components of the COP9-signalosome complex (CSN) and PCNA (see Drosophila PCNA). PCNA interacts with CDT1 and loss of PCNA suppresses CDT1 proteolysis after DNA damage. These data also revealed that in vivo, inactivation of L2DTL causes the dissociation of DDB1 from the CUL4 complex. These studies suggest that L2DTL and PCNA interact with CUL4/DDB1 complexes and are involved in CDT1 degradation after DNA damage (Higa, 2006b).

CUL4/ROC1 E3 ligase targets CDT1 for ubiquitin-dependent proteolysis in response to UV or gamma-irradiation (Higa, 2003). Since the CUL4 complex represents a new type of cullin-containing E3 ligases that is very different from the SCF, CUL2 and CUL3 ubiquitin E3 ligase complexes, attempts were made to determine the protein composition of the CUL4 complex to understand the mechanism of substrate recognition and its regulation. To identify these proteins, anti-CUL4 antibody affinity chromatography was used to isolate CUL4 complexes from human cells. Mass-spectrometry analysis revealed that the isolated human CUL4B complex from HeLa cells contained DDB1, components of the CSN complex, and the human homologue of Drosophila lethal(2) denticleless protein (Kurzik-Dumke, 1996). L2DTL is a conserved WD40 repeat-containing protein which is essential for Drosophila embryogenesis but without any known molecular function. L2DTL also shares homology with the S. pombe CDT2. To further determine the interaction, rabbit polyclonal antibodies were raised against human L2DTL protein (Higa, 2006b).

Coimmunoprecipitation performed with L2DTL antibodies confirmed that human L2DTL indeed interacts with endogenous CUL4A, CUL4B and DDB1 in human cells. While the anti-L2DTL antibodies consistently coimmunoprecipitated DDB1, the interaction between L2DTL and CUL4A or CUL4B varies between experiments, suggesting L2DTL may preferentially interact with DDB1. However, the lack of a DDB1 antibody that can immunoprecipitate the CUL4/DDB1/L2DTL complexes prevented further exploration of this possibility. Antibody was also raised against the Drosophila L2DTL protein. The data indicate that the Drosophila L2DTL protein can be detected in the anti-CUL4 immuno-complex in Drosophila Schneider D2 (S2) cells (Higa, 2006b).

Structurally, the L2DTL protein contains a conserved WD40 repeat region at its amino terminus and a less conserved carboxy half. To determine the region in L2DTL that mediates its interaction with CUL4 and DDB1, deletion mutants were made that lack either the WD40 repeats or the carboxy terminal region, and their binding to CUL4 or DDB1 was analyzed. While wild-type L2DTL binds both CUL4 and DDB1, loss of either the WD40 repeats or the carboxy terminal region severely impairs the association between L2DTL and CUL4 or DDB1, suggesting that both domains are required for L2DTL interaction with DDB1-CUL4 complexes (Higa, 2006b).

One of the best characterized CUL4 substrates is replication licensing protein CDT1, To determine L2DTL function, the expression of Drosophila L2DTL was silenced in S2 cells by RNA interference (RNAi) because of highly efficient gene silencing in these cells. The effect of L2DTL inactivation on CDT1 proteolysis induced by gamma-irradiation was examined. While CDT1 is rapidly degraded in response to gamma-irradiation in S2 cells treated with control dsRNA, inactivation of L2DTL by RNAi completely blocked CDT1 proteolysis after radiation. This effect is similar to inactivation of CUL4 on CDT1 in parallel experiments, indicating that Drosophila L2DTL is required for CDT1 proteolysis in response to DNA damage (Higa, 2006b).

Replication licensing protein CDT1 serves as a substrate of CUL4 ubiquitin E3 ligase complex. The inactivation of Drosophila L2DTL suppresses CDT1 proteolysis in response to DNA damage. Therefore whether L2DTL can interact with CDT1 was tested. Since Drosophila anti-L2DTL antibodies do not immunoprecipitate L2DTL complex, the interaction between L2DTL and CDT1 was characterized in human cells. L2DTL can be detected in the anti-CDT1 immunocomplexes while CDT1 is also present in anti-L2DTL complexes, albeit the signal is relatively weak under the immunoprecipitation conditions used. These data indicate that there is an interaction between the endogenous L2DTL and CDT1 proteins. These studies are consistent with observations CUL4 and its associated DDB1 interact with CDT1 in vivo (Higa, 2006b).

Because of the relative weak interaction between L2DTL and CDT1 under various conditions, the possibility was considered that there may be additional subunits in the CUL4 complexes that regulates CDT1 stability. To further identify the proteins that associate with L2DTL and CUL4 complexes, L2DTL complexes were affinity purified using anti-L2DTL antibodies as the affinity resins for chromatography from HeLa cell lysates. Mass-spectrometry was used to analyze proteins that are specifically associated with L2DTL complexes. Peptides corresponding to DDB1 and subunits of the CSN complex were obtained from the isolated protein bands in L2DTL complexes. One of the peptides in L2DTL complexes corresponds to PCNA. To confirm these interactions, immunoprecipitation of cell lysates prepared from various human cells was performend, followed by western blotting using anti-L2DTL, CSN5 and PCNA antibodies. The data confirmed that endogenous CSN5 and PCNA proteins are indeed present in L2DTL complexes isolated from various mammalian cells (Higa, 2006b).

To determine whether L2DTL functions to regulate CDT1 stability in human cells, the expression of human L2DTL was silenced in HeLa, U2OS, or other human cell lines by small interfering RNA (siRNA). Loss of human L2DTL in these cells also suppresses CDT1 degradation in response to gamma-irradiation. These studies demonstrate that L2DTL is a novel protein that associates with CUL4 and DDB1 and is required for CDT1 degradation in response to DNA damage in both Drosophila and human cells. Sometimes, it was observed that CDT1 is stabilized in L2DTL silenced cells in the abscence of irradiation. This effect was also sometimes observed in DDB1 or CUL4 silenced cells. Since the protein stability of CDT1 is regulated in S phase it is possible that L2DTL and DDB1/CUL4 complexes regulate CDT1 proteolysis in S phase cells (Higa, 2006b).

Since inactivation of L2DTL prevents CDT1 degradation in response to DNA damage, the expression of PCNA was also silenced in human cells. Similar to L2DTL or DDB1 silenced cells, inactivation of PCNA by siRNA prevented CDT1 proteolysis in response to gamma irradiation. Whether CDT1 interacts with PCNA was also tested. The recombinant CDT1 and PCNA directly interact in insect SF9 cells infected with baculoviruses encoding CDT1 and PCNA cDNAs. These studies suggest that PCNA is involved in regulating DNA-damage induced proteolysis of CDT1. These data suggest that CDT1 proteolysis after DNA damage requires the presence of L2DTL, PCNA and the DDB1/CUL4 E3 ligase complexes (Higa, 2006b).

Since L2DTL binds to DDB1 and CUL4, the mechanism for the requirement of L2DTL by the CUL4 complex was explored. It was found that loss of L2DTL by siRNA sometimes reduces the binding of DDB1 to CUL4 complexes. These observations suggest that one function of L2DTL may be to facilitate the interaction between DDB1 and CUL4A complexes in vivo (Higa, 2006b).

Therefore a novel WD40 repeat-containing protein, L2DTL, binds to DDB1, CUL4, PCNA and CSN. These studies further indicate that PCNA associates with L2DTL and CDT1. Similar to the effect of loss of CUL4 and DDB1, inactivation of either L2DTL or PCNA prevented CDT1 proteolysis in response to DNA damage. In contrast, inactivation of CSN5 or CSN2, components of CSN complex, by siRNAs did not alter CDT1 proteolysis after DNA damage, even though the protein levels of CSN2 or CSN5 were substantially reduced. This differs from previous data, which demonstrated loss of CSN complex abolished CDT1 degradation in Drosophila S2 cells after gamma-irradiation. It is possible that the siRNAs used against human CSN2 or CSN5 were still not sufficient to silence the expression and the activity of CSN to the level that can impact CDT1 proteolysis. Alternatively, because cullin deneddylation can be mediated by CSN and DEN1 it is possible that the function of CSN and DEN1 may overlap. Additional studies also showed that CUL4A, DDB1, L2DTL and PCNA also interact with p53 and MDM2 in human cells and are required for the CUL4-mediated p53 polyubiquitination activity. These studies suggest that L2DTL and PCNA may be part of the CUL4 complexes that regulate the protein stability of CUL4 substrates such as CDT1 and p53. In this regard, it was found that loss of L2DTL often leads to the dissociation of DDB1 from CUL4 complex. It is possible that L2DTL may play a role in promoting and/or stabilizing the interaction between DDB1 and CUL4 complex. Consistently, the fission yeast CDT2 was recently isolated as a CUL4 binding protein. Since L2DTL and PCNA interact with CDT1 and p53/MDM2, these studies suggest that L2DTL and PCNA also contribute to substrate recognition of the DDB1/CUL4 E3 ligase complex (Higa, 2006b).

Cul4 and DDB1 regulate Orc2 localization, BrdU incorporation and Dup stability during gene amplification in Drosophila follicle cells

In higher eukaryotes, the pre-replication complex (pre-RC) component Cdt1 is the major regulator in licensing control for DNA replication. The Cul4-DDB1-based ubiquitin ligase mediates Cdt1 ubiquitylation for subsequent proteolysis. During the initiation of chorion gene amplification, Double-parked (Dup), the Drosophila ortholog of Cdt1, is restricted to chorion gene foci. This study found that Dup accumulated in nuclei in Cul4 mutant follicle cells, and the accumulation was less prominent in DDB1 (piccolo) mutant cells. Loss of Cul4 or DDB1 activity in follicle cells also compromised chorion gene amplification and induced ectopic genomic DNA replication. The focal localization of Orc2, a subunit of the origin recognition complex, is frequently absent in Cul4 mutant follicle cells. Therefore, Cul4 and DDB1 have differential functions during chorion gene amplification (Lin, 2009).

In this study, Cul4 and DDB1 mutants were isolated which were larval lethal with growth arrest in the second instar stage, similarly to previous results (Hu, 2008). It was further shown that Cul4 mutant clones in developing wing discs were defective in proliferation and had a reduced number of S-phase cells. To focus on the role of Cul4 during DNA replication and bypass the requirement of Cul4 in G1-S transition, mutant follicle cells were analyzed during gene amplification stages in follicle cells. It was shown that the Dup protein level and Orc2 focal localization are regulated by Cul4 and, differentially, by DDB1. In addition, BrdU focal patterns are defective in Cul4 and DDB1 mutant follicle cells (Lin, 2009).

Previous studies have shown the replication-dependent degradation of human and Xenopus Cdt1 by the Cul4-DDB1 E3 ligase, and the replication-coupled recruitment of the DDB1 to chromatin in Xenopus cells. This study has shown the requirement of Cul4 for the suppression of Dup protein levels during gene amplification in Drosophila follicle cells. Comparison of Dup nuclear accumulation in Cul4 and DDB1 mutant follicle cells, however, reveals substantial differences. Almost all Cul4 mutant cells, except those undergoing apoptosis, accumulated Dup in the nucleoplasm in stage 10B, consistent with Cul4 being a dedicated component of the E3 ligase in promoting Cdt1 degradation. By contrast, accumulation of Dup levels was observed in much smaller fractions of two DDB1 alleles analyzed. These analyses have not excluded the involvement of DDB1 in downregulating Dup protein levels. Compensatory or parallel Cul4-mediated Dup degradation pathways might be present in addition to the Cul4/DDB1-mediated Dup degradation. In agreement with this speculation, a recent study has shown that nuclear accumulation of cyclin D1 during the S phase promotes human Cdt1 stabilization and triggers DNA re-replication. This Cdt1 stabilization could be suppressed by the overexpression of Cul4A and Cul4B but not DDB1 or Cdt2, also an adaptor for the Cul4 ligases, implying the involvement of other adaptors in mediating the Cul4-dependent degradation of Cdt1 (Lin, 2009).

This study found that BrdU incorporation at chorion gene amplification foci was reduced or even absent in about half of Cul4^G1-3 mutant follicle cells, if apoptotic cells that cannot be scored for their capability in BrdU incorporation were excluded. Similarly, 40% of Cul4^G1-3 mutant cells displayed ectopic BrdU incorporation (the abnormal genomic replication group). These cells were not scored for their BrdU incorporation at focal sites because of overall strong nuclear signals. Therefore, the effect of Cul4 on the chorion gene amplification might be underestimated in this Cul4-null allele. Using the Cul4 hypomorphic allele KG02900 in which both the fractions of apoptotic cells and abnormally BrdU-incorporated cells were reduced, the combined percentages for reduced and absent BrdU incorporation combined at chorion gene amplification foci reached more than 70%. DDB1^5-1 mutant follicle cells also displayed a severe phenotype in the BrdU incorporation assay. When apoptotic cells were disregarded, cells with a reduction or absence of BrdU incorporation accounted for more than 60% of DDB1^5-1 mutant cells. Therefore, these BrdU incorporation analyses lend support to the notion that certain processes in chorion gene amplification require both Cul4 and DDB1 (Lin, 2009).

These BrdU foci represent DNA amplification of chorion genes within 100 kb of origins, and the phenotype of absence or reduction in BrdU incorporation could reflect a failure in the initiation of DNA replication, reduced processivity in DNA synthesis or fewer rounds of gene amplification. To further support the idea that Cul4 is involved in gene amplification, advantage was taken of the dominant-negative Cul4KR mutant in which the neddylation site has been mutated. Acute expression of Cul4KR suppressed BrdU incorporation in almost all follicle cells. When assayed by quantification PCR, gene amplification at chorion foci was strongly suppressed, supporting a role of Cul4 in the chorion gene amplification process (Lin, 2009).

Abnormal genomic replication, as inferred from ectopic BrdU incorporation, was observed in Cul4 mutant follicle cells in both Cul4 mutant alleles tested. The percentage of cells with such a defect was reduced in the cells with the hypomorphic mutant allele, indicating that the low level of Cul4 activity partially suppresses this defect. Abnormal genomic replication was also detected in DDB1 mutant follicle cells with a lower frequency than in Cul4-null mutants. The phenotype of ectopic genomic replication is less likely to be a retarded developmental process in the previous endocycle stage, because these cells with ectopic BrdU signals show normal DNA contents as estimated by Hoechst staining. Ectopic genomic replication might require some prerequisite steps in DNA replication, such as Orc2 localization at replication origins, which is defective in Cul4 mutant cells, thus blocking abnormal DNA replication throughout the genome (Lin, 2009).

The localization of Orc2, a component of the pre-RC, at chorion gene foci was examined during gene amplification. Mutations in Cul4 caused reduced or no Orc2 localization at gene amplification foci, a prominent phenotype in both G1-3 (59%) and KG02900 alleles (60%) when apoptotic cells were discounted. Failure of proper Orc2 focal localization might represent defects in the initial loading of Orc2 or the maintenance of Orc2 localization at amplification foci. Such Orc2 localization defects were not prominent in DDB1 mutant follicle cells (Lin, 2009).

Consequently, defective Orc2 localization at regular gene amplification foci might evoke ectopic genomic replication in Cul4 mutant follicle cells. Upon co-labeling for Orc2 and BrdU in Cul4 mutant cells, the absence of or reduction in Orc2 signals was found in conjunction with a reduction in BrdU incorporation at gene amplification loci or with abnormal BrdU incorporation throughout the genome. In some cells, normal Orc2 loading was accompanied with abnormal BrdU incorporation. The decoupling of both phenotypes therefore suggests that Cul4 functions distinctively in Orc2 localization and in suppression of abnormal BrdU incorporation during chorion gene amplification (Lin, 2009).

Many interesting questions remain to be answered regarding how genetic loci are selected for amplification, how the pre-RC is assembled only in specific loci and how other genomic regions are kept silent. Previous evidence suggests that high transcriptional activity of specific loci controls replication origin firing during the gene amplification stage. Mutants for transcription factors such as E2f2, Dp, Rbf, Myb and Mip130 show increased mRNA and protein levels of replication factors, such as components of the Orc and MCM complexes, and ectopic genomic replication during the gene amplification stage. Mutant follicle cells for Cul4 displayed both Orc2 localization defects and abnormal genomic replication, implying that Cul4 is probably involved in both processes by modulating the transcriptional activity in DNA replication. Interestingly, a recent study suggests that Cul4 targets degradation of the transcriptional activator E2F1 during S phase. However, Drosophila E2F1 proteins became abundant in the nucleus and rich at ACE3 origin DNA during gene amplification. How this developmental regulation of E2F is involved in Cul4 activity needs further investigation. Some studies suggest that Cul4 functions in histone modification and heterochromatin maintenance. It is speculated that the Cul4 E3 complex also functions in regulating Orc2 origin localization through a local remodeling of the chromatin structure on ACE3 and Ori-β (Lin, 2009).

Control of Drosophila endocycles by E2F and CRL4^CDT2

Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world's biomass. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. This study used genetic tests in parallel with computational modelling to show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the PCNA/replication fork-associated E3 ubiquitin ligase CRL4^CDT2 (Cul-4), and this in turn mediates the destruction of E2F1 (Shibutani, 2008). It is proposed that the transient loss of E2F1 during S phases creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, it was found that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants, indicating that elements of this mechanism act in most growth-dependent cell cycles (Zielke, 2011).

Altogether these results indicate that periodic E2F1 degradation is necessary for endocycling for three reasons: (1) it creates a window of low CYCE/CDK2 activity; (2) it promotes high APC^Fzr/Cdh1 activity and thereby suppresses geminin accumulation; and (3) it allows E2F2 to maintain repression of CDK1 and its cyclins. Each of these conditions is required for preRC assembly and endocycle progression. This cell cycle mechanism is fundamentally different from that used in mitotic cycles, wherein destruction of the M-phase cyclins by APC^Cdc20/Fzy, rather than of E2F1 by the CRL4^CDT2, throws the switch that allows preRC assembly. Indeed it is noteworthy that the periodic degradation of E2F1 and depletion of CYCE are not required for mitotic cell cycles in Drosophila. CRL4^CDT2 is required for endocycling in plants, indicating that this element of the endocycle oscillator is conserved (Zielke, 2011).

Finally, it was asked what factors control E2F production to regulate endocycle rates. Endocycle speed and number can be manipulated by altering cell growth through changes in dietary protein or growth-regulatory genes including Myc and insulin/PI3K/TOR signalling components. Hence larvae were starved of protein to suppress insulin/TOR signalling, reduce protein synthesis, and block cell growth. Starvation arrested the salivary endocycles within 24h and strongly depleted E2F1. E2f1 and Dp mRNA levels were not affected, but the E2F targets CycE, pcna and rnrS were reduced. To test whether this was responsible for starvation-induced endocycle arrest E2F1 was overexpressed in the salivary glands of starved animals. Although these glands failed to grow their nuclei incorporated BrdU and accrued approximately sevenfold more DNA than controls. Overexpression of RHEB, which activates the Target of rapamycin (TOR) kinase and increases ribosome biogenesis and cap-dependent translation, also restored cell growth, E2F1 protein, and endocycle progression in starved animals. Thus E2F1 appears to act as a 'growth sensor' that couples rates of endocycle progression to rates of cell growth. A likely mechanism for this, corroborated by modelling, involves increased translation of E2F1 in rapidly growing cells. Indeed, it was found that the association of E2F1 mRNA with polyribosomes was greatly reduced in protein-starved animals. Translational control of E2F is an attractive mechanism for coupling growth to G1/S progression not only in endocycling cells, but also in growth-dependent mitotic cells with extended G1 periods (Zielke, 2011).

Rbf1 degron dysfunction enhances cellular DNA replication

The E2F family of transcription factors contributes to oncogenesis through activation of multiple genes involved in cellular proliferation, a process that is opposed by the Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by inhibiting its proteasome-mediated degradation, but the consequences of this post-translational regulation of E2F1 remain unknown. To better understand the mechanism of E2F stabilization and its physiological relevance, this study examined the streamlined Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that modulates E3 ubiquitin ligase activity. This study report that the COP9 signalosome also protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels, enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1. It was further shown that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1 cell cycle-target genes are distinct properties. Removal of an evolutionarily conserved Rbf1 C terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This mutant form of Rbf1 also enhanced G₁-to-S phase progression when expressed in Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate gain-of-function properties relevant to cellular transformation. Consistent with this idea, several studies have identified mutations in the homologous C terminal domains of RB and p130 in human cancer (Raj, 2012).

The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes

Cell competition, the elimination of cells surrounded by more fit neighbors, is proposed to suppress tumorigenesis. Mahjong (Mahj), a ubiquitin E3 ligase substrate receptor, has been thought to mediate competition of cells mutated for lethal giant larvae (lgl), a neoplastic tumor suppressor that defines apical-basal polarity of epithelial cells. This study shows that Drosophila cells mutated for mahjong, but not for lgl [l(2)gl], are competed because they express the bZip-domain transcription factor Xrp1, already known to eliminate cells heterozygous for ribosomal protein gene mutations (Rp/+ cells). Xrp1 expression in mahj mutant cells results in activation of JNK signaling, autophagosome accumulation, eIF2α phosphorylation and lower translation, just as in Rp/+ cells. Cells mutated for damage DNA binding-protein 1 (ddb1; pic) or cullin 4 (cul4), which encode E3 ligase partners of Mahj, also display Xrp1-dependent phenotypes, as does knockdown of proteasome subunits. These data suggest a new model of mahj-mediated cell competition that is independent of apical-basal polarity and couples Xrp1 to protein turnover (Kumar, 2022).

Cell competition, which is the elimination of, in most cases, slower-growing cells by faster-growing cells in mosaics, is important for precise development, regeneration and physiological maintenance. Cell competition was first recognized in Drosophila in the case of cells lacking one copy of ribosomal protein genes (Rp/+). These mutants, which are known as 'Minutes' because of their thin body bristles, also display slow growth. Minute mutant cells are eliminated from mosaics with wild-type cells by caspase-dependent cell death. Super-competition, the name given to the process of eliminating wild-type cells, happens in mosaics with faster-growing Myc- or Yorkie (Yki)-expressing cells. Considered together, cell competition and super-competition suggest that comparison of cellular fitness leads to cell competition. Because the mammalian homologs of Myc and Yki are proto-oncogenes, it has been suggested that super-competition might contribute to tumor expansion, as several recent studies have confirmed (Kumar, 2022).

Cell competition may also be tumor suppressive. Global loss of apico-basal polarity genes such as lgl [l(2)gl)] or scribble (scrib) leads to polarity-deficient neoplasia of Drosophila imaginal discs, but clones of lgl or scrib cells are eliminated from mosaics. These lgl or scrib mutant clones do not form tumors unless cell competition is blocked and mutant cells remain in the epithelium (Kumar, 2022).

Competition of Rp/+ mutant cells might also serve a tumor-surveillance role. Rp genes are spread throughout the genome, and it has been shown they can serve as sensors for aneuploidy, leading to elimination of aneuploid cells containing monosomies that affect Rp gene dose. Mutants with disrupted cell competition accumulate aneuploid cells. These would be expected to be tumorigenic in mammals, where aneuploidy is associated with tumorigenesis. Competition of Rp/+ cells depends on the Drosophila bZip AT-hook domain transcription factor Xrp1. The Xrp1 expression induced in Rp/+ imaginal discs is also responsible for most of their altered gene expression, their slow growth and their reduced translation, in addition to their propensity to be eliminated by cell competition. Xrp1 is also expressed in the DNA damage response, where its transcription is p53 dependent. Xrp1 induction in Rp/+ cells is independent of p53 but dependent on a particular Rp protein, RpS12, which is thought to play a role in signaling the defect in ribosome biogenesis (Kumar, 2022).

The tumor-suppressive cell competition of polarity gene mutant cells has been proposed to go through Mahjong (Mahj) (Tamori, 2010), a CRL4 E3 ubiquitin ligase (Ly, 2019). mahj physically interacts with Lgl, and its overexpression in lgl mutant clones suppresses their elimination from mosaic tissues (Tamori, 2010). Interestingly, mahj knockdown in MDCK cell cultures also leads to their elimination by co-cultured normal MDCK cells, suggesting a cell competition mechanism that is conserved between Drosophila and mammalian cells (Tamori, 2010). The mammalian homolog of mahj, known as DDB1-Cul4-associated factor 1 (DCAF1) or human immunodeficiency virus type 1 accessory protein Vpr-binding protein (VprBP), is important for G2 cell cycle arrest and virus replication after HIV1 infection. Dcaf1 is required for mouse embryogenesis and its knockdown affects cell proliferation, cell cycle and cell survival in multiple cell types. Dcaf1 interacts with the Hippo pathway and its knockdown also stabilizes p53, but there has been no report of Dcaf1/VprBP affecting epithelial cell polarity in mammals (Kumar, 2022).

In Drosophila, the overall transcriptional signature of mahj mutant wing discs is unexpectedly similar to that of Rp/+ mutants, including upregulation of Xrp1 mRNA. Because mahj and Rp/+ cells were thought to represent distinct mechanisms of cell competition, this finding suggested a gene expression signature common to cells targeted by cell competition. Besides transcription, other similarities have been reported between mahj and Rp/+ mutant cells, including autophagosome accumulation and evidence of proteotoxic stress (Kumar, 2022).

This study shows that mahj mutant cells trigger cell competition through an Xrp1-dependent pathway like that in Rp/+ cells, and distinct from cell competition of lgl or scrib clones, which do not express or depend on Xrp1 function for elimination. Xrp1 expression also makes mahj mutant cells phenotypically like Rp/+ cells, that is, results in 'Minute-like' thin thoracic bristles, slow growth, reduced translation, altered autophagy and increased JNK signaling. Regulation of Xrp1 by mahj likely requires its E3 ligase activity, depending on DNA Damage Binding Protein 1 (Ddb1) and Cullin 4 (Cul4). These results show that mahj mutant cells suffer cell competition because of a transcriptional response to altered ubiquitinylation mediated by Xrp1 and therefore resembling Rp/+ mutant cells. This seems unrelated to elimination of scrib or lgl mutant cells - the polarity-defective cells. Thus, loss of mahj function is an additional genotype triggering elimination by the Xrp1-dependent pathway that also removes Minute cells, not the mechanism for eliminating tumorigenic polarity-deficient cells (Kumar, 2022).

This research explored the cell competition mechanisms of Mahj, a CRL4 ubiquitin ligase (Ly, 2019), the mutation of which triggers similar cellular effects to Rp/+ mutations, including similar changes in gene expression, global translation rates, JNK activity and autophagy, leading mahj cells to be eliminated by competition with wild-type cells, as Rp/+ cells are. The basis of the similarity is that mahj and Rp loss of function both activate expression of Xrp1, the transcription factor that coordinates these effects. Unlike Rp/+ genotypes, which activate Xrp1 through a rpS12-dependent mechanism, mahj regulates Xrp1 most likely through its ubiquitin ligase activity, which depends on DDB1, Cul4 and Roc1a, although the specific ubiquitylated target has not yet been identified. It is suggested that Xrp1 is likely to be activated by a protein, or proteins, that are normally degraded by Mahj-dependent ubiquitylation, because Xrp1 is also activated by inhibition of the proteasome, which is expected to affect the degradation of ubiquitylated proteins, but not other functional consequences of ubiquitylation. The relevant target does not seem to be Warts, despite the fact that levels of Warts and Hippo pathway activity also control cellular growth and global translation levels, and can stimulate cell competition. These studies support the notion that Xrp1 is a sensor of multiple cellular defects that cause cell competition, rather than that of a 'loser signature' common to distinct cell competition mechanisms (Kumar, 2022).

Mahj was previously thought to be responsible for the cell competition of cells mutated for lgl (Tamori, 2010; Baker, 2011; Levayer, 2013), a gene that controls apical basal cell polarity. mahj was originally linked to apical-basal polarity because of a physical interaction with Lgl, and because mahj overexpression can rescue lgl mutant clones for elimination, suggesting that mahj behaves as an intracellular signal transducer of lgl activity in cell competition (Tamori, 2010). As such, it was surprising when similar gene expression changes were observed in mahj mutant and Rp mutant wing discs, because these were assumed to reflect distinct cell competition pathways and suggested a common gene expression signature associated with competed cells. This study shows, however, that neither lgl nor scrib, another related cell polarity gene, affects cell competition by the same mechanism as mahj, because neither lgl nor scrib mutant cells express or require Xrp1. Interestingly, several distinct pathways have recently been described to mediate the elimination of scrib mutant cells in competition with wild-type cells, and none of these pathways are shown to be required for the elimination of Rp/+ mutant cells. In addition, mahj loss by itself does not result in apical-basal polarity defects (Tamori, 2010), and its mammalian homolog is implicated in cell cycle regulation, genome integrity and p53 activity (Hrecka, 2007; Cooper 2014; Lubow, 2020). Drosophila mahj, which is an essential gene, regulates neural stem cell reactivation (Ly, 2019) and may have other roles in non-neuronal tissues, as suggested by defects observed when mahj is depleted in posterior wing compartments. Accordingly, it is concluded that mahj mutants affect cellular growth and cell competition in a manner unrelated to lgl and scrib, and that the functional relationship of mahj to apical-basal polarity pathways, should any exist, is unclear. The functional importance of physical interaction between Mahj and Lgl remains to be explored. It is known that lgl clones are rescued by reduced Hippo signaling, although this study did not detect reduced Hippo signaling after mahj overexpression in the absence of lgl mutations (Kumar, 2022).

These studies provide further evidence for Xrp1 as an integrator of multiple seemingly independent cellular defects that each result in a common spectrum of cellular responses and predispose cells to competitive elimination by wild-type neighbors (Kiparaki, 2022). These functional roles for Xrp1 first became apparent through its role in the slow growth, reduced translation and competitive elimination of Rp/+ cells, in which Xrp1 expression is induced in an rpS12-dependent manner. In the case of mahj, Xrp1 protein expression is induced to confer a very similar spectrum of cellular effects, but independently of rpS12 and perhaps depending on stabilization of a protein normally targeted for proteasomal turnover by mahj-dependent ubiquitylation. Xrp1 expression was first found as a p53-regulated gene, perhaps part of the DNA damage response. Recently, Xrp1 induction has also been found as a response to ER stress, possibly through translational regulation downstream of eIF2α phosphorylation. It has been suggested that eIF2α phosphorylation, and Xrp1 expression, can also be triggered by a global, cytoplasmic proteotoxic stress, which is suggested to occur as a consequence of deficient ribosome assembly in Rp mutant cells. Xrp1 expression in response to proteasome inhibition is one piece of evidence for this model. This study shows, however, that Xrp1 is induced, and cell competition results after loss of mahj, a single E3-ligase adapter protein that probably targets only a moderate number of proteins for degradation. Thus, an alternative explanation of Xrp1 induction after proteasome inhibition is that this could reflect stabilization of one or a few specific proteins. Overall, a picture is emerging of Xrp1 as a stress-responsive transcription factor whose expression can be initiated by multiple distinct pathways, then leading to a common cellular response, including the elimination of the stressed cells by competition with nearby wild-type cells, when such cells are available (Kumar, 2022).

Importantly, cells depleted for DCAF1/VprBP, the mammalian homolog of Mahj, are eliminated by competition with wild-type cells in mammalian cell culture. Thus, cell competition of mahj mutant cells may be a conserved process. Conservation of cell competition has not yet been demonstrated for Rp/+ cells in mammals, although it may very well occur. In mammals, knockdown of either mahj or its binding partner ddb1 results in P53 activation, which is functionally required for the resulting phenotypes. Differences in p53 activity lead to cell competition in many mammalian systems. p53 is not required for mahj-mediated cell competition in Drosophila, but because Xrp1 is a target of Drosophila p53 in irradiated cells, it is possible Xrp1 is a p53 target that has replaced the cell competition role of p53 in Drosophila, as has already been suggested for the competition of Rp/+ cells, which is also p53 independent in Drosophila, although Rp mutations activate p53 in mammals. Thus, mahj-mediated cell competition may provide another example where Xrp1 mediates a process in Drosophila that is dependent on p53 in mammals (Kumar, 2022).

Search PubMed for articles about Drosophila cul-4

Search PubMed for articles about Cul4 in general

Chen, L. C., et al. (1998). The human homologue for the Caenorhabditis elegans cul-4 gene is amplified and overexpressed in primary breast cancers. Cancer Res. 58: 3677-83. PubMed ID: 9721878

Groisman, R., et al. (2003). The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell 113: 357-67. PubMed ID: 12732143

Higa, L. A., et al. (2003). Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat. Cell Biol. 5: 1008-15. PubMed ID: 14578910

Higa, L. A., et al. (2006a). Involvement of CUL4 ubiquitin E3 ligases in regulating CDK inhibitors Dacapo/p27Kip1 and cyclin E degradation. Cell Cycle 5(1): 71-7. PubMed ID: 16322693

Higa, L. A., et al. (2006b). L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage. Cell Cycle 5(15): 1675-80. PubMed ID: 16861906

Hu, J., McCall, C. M., Ohta, T. and Xiong, Y. (2004). Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage. Nat. Cell Biol. 6: 1003-9. PubMed ID: 15448697

Hu, J., Zacharek, S., He, Y. J., Lee, H., Shumway, S., Duronio, R. J. and Xiong, Y. (2008). WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase. Genes Dev. 22: 866-871. PubMed ID: 18381890

Kipreos, E. T., et al. (1996). cul-1 is required for cell cycle exit in C. elegans and identifies a novel gene family. Cell 85: 829-39. PubMed ID: 8681378

Kumar, A. and Baker, N. E. (2022). The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes. Development 149(22). PubMed ID: 36278853

Kurzik-Dumke, U., Neubauer, M. and Debes, A. (1996). Identification of a novel Drosophila melanogaster heat-shock gene, lethal(2)denticleless [l(2)dtl], coding for an 83-kDa protein. Gene 171: 163-70. PubMed ID: 8666267

Lin, H. C., Wu, J. T., Tan, B. C., and Chien, C. T. (2009). Cul4 and DDB1 regulate Orc2 localization, BrdU incorporation and Dup stability during gene amplification in Drosophila follicle cells. J. Cell Sci 122: 2393-2401. PubMed ID: 19531585

McCall, C. M., Hu, J. and Xiong, Y. (2005). Recruiting substrates to cullin 4-dependent ubiquitin ligases by DDB1. Cell Cycle 4: 27-9. PubMed ID: 15655366

Nag, A., Bondar, T., Shiv, S. and Raychaudhuri, P. (2001). The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells. Mol. Cell Biol. 21: 6738-47. PubMed ID: 11564859

Raj, N., Zhang, L., Wei, Y., Arnosti, D. N. and Henry, R. W. (2012). Rbf1 degron dysfunction enhances cellular DNA replication. Cell Cycle 11: 3731-3738. PubMed ID: 22895052

Shibutani, S. T. et al. (2008). Intrinsic negative cell cycle regulation provided by PIP box- and Cul4^Cdt2-mediated destruction of E2f1 during S phase. Dev. Cell 15: 890-900. PubMed ID: 19081076

Strohmaier, H., et al. (2001). Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413: 316-22. PubMed ID: 11565034

Wertz, I. E., et al. (2004). De-etiolated-1 regulates c-Jun by assembling a CUL4A ubiquitin ligase. Science 303: 1371-4. PubMed ID: 14739464

Yasui, K., et al. (2002). TFDP1, CUL4A, and CDC16 identified as targets for amplification at 13q34 in hepatocellular carcinomas. Hepatology 35: 1476-84. PubMed ID: 12029633

Zheng, J., et al. (2002). CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex. Mol. Cell 10: 1519-26. PubMed ID: 12504026

Zielke, N., et al. (2011). Control of Drosophila endocycles by E2F and CRL4^CDT2. Nature 480(7375): 123-7. PubMed ID: 22037307

date revised: 15 July 2012

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