SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation

The eukaryotic cell cycle is regulated by multiple ubiquitin-mediated events, such as the timely destruction of cyclins and replication licensing factors. The histone H4 methyltransferase SET8 (Pr-Set7) is required for chromosome compaction in mitosis and for maintenance of genome integrity. In this study, we show that SET8 is targeted for degradation during S phase by the CRL4(CDT2) ubiquitin ligase in a proliferating cell nuclear antigen (PCNA)-dependent manner. SET8 degradation requires a conserved degron responsible for its interaction with PCNA and recruitment to chromatin where ubiquitylation occurs. Efficient degradation of SET8 at the onset of S phase is required for the regulation of chromatin compaction status and cell cycle progression. Moreover, the turnover of SET8 is accelerated after ultraviolet irradiation dependent on the CRL4(CDT2) ubiquitin ligase and PCNA. Removal of SET8 supports the modulation of chromatin structure after DNA damage. These results demonstrate a novel regulatory mechanism, linking for the first time the ubiquitin-proteasome system with rapid degradation of a histone methyltransferase to control cell proliferation.     

CRL4CDT2 Targets CHK1 for PCNA-Independent Destruction

CDT2 targets proteins involved in replication licensing (CDT1), cell cycle control (p21), and chromatin modification (SET8) for destruction by the CUL4-based E3 ligase (CRL4). CRL4^CDT2 recruits these substrates through interactions with chromatin-bound PCNA and ubiquitinates them exclusively on chromatin. Rereplication and G₂ cell cycle arrest are observed in CDT2-depleted cells. The rereplication phenotype has been attributed to an inability to destroy CDT1, but the molecular target important for G₂ cell cycle arrest in CDT2-depleted cells has not been identified. Here we identify CHK1 as a novel CRL4^CDT2 substrate and demonstrate that CHK1 activity is required for maintaining G₂ arrest in CDT2-depleted cells. We demonstrate that CRL4^CDT2 targets the activated form of CHK1 for destruction in the nucleoplasm rather than on chromatin and that this occurs in a PCNA-independent manner. Although both CRL1 and CRL4 ubiquitinate CHK1, we report that they bind CHK1 in distinct cellular compartments. Our study provides insight into how elevated CDT2 expression levels may provide tumors with a proliferative advantage.     

Opposing effects of glutamine and asparagine govern prion formation by intrinsically disordered proteins

Several proteins, including the replication licensing factor CDT1 and the histone methyltransferase SET8, are targeted for proteolysis during DNA replication and repair by the E3 ubiquitin ligase CRL4(CDT2). CRL4(CDT2) function is coupled to replication and repair because it only ubiquitinates substrates that associate with chromatin-bound PCNA. Here, we report a genome-wide siRNA screen that identifies multiple factors necessary for CDT1 destruction after UV irradiation. Among these, nucleotide excision repair factors promote CDT1 destruction due to a role in recruiting PCNA to damaged DNA. The COP9/Signalosome regulates CDT2 stability through CUL4 deneddylation. Finally, the p97 AAA(+)-ATPase and its cofactor UFD1 are required for proteasome-dependent removal of ubiquitinated CDT1 and SET8 from chromatin and their subsequent degradation both in vivo and in a Xenopus egg extract system in vitro. This study provides insight into and a resource for the further exploration of pathways that promote timely degradation of chromatin-associated CRL4(CDT2) substrates.     

Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. Here we describe a novel function of Geminin. We demonstrate that human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2.     

PCNA-coupled p21 degradation after DNA damage: The exception that confirms the rule?

While many are the examples of DNA damaging treatments that induce p21 accumulation, the conception of p21 upregulation as the universal response to genotoxic stress has come to an end. Compelling evidences have demonstrated the existence of converging signals that negatively regulate p21 bellow basal levels when replication forks are blocked. Moreover, conclusive reports identified the E3-ligase CRL4^CDT2 (CUL4–DDB1–CDT2) as the enzymatic complex that promotes p21 proteolysis when treatments such as UV irradiation trigger replication fork stress. A pre-requisite for CRL4^CDT2-driven proteolysis is the interaction of p21 with PCNA. Interestingly as well, CRL4^CDT2-dependent proteolysis is not limited to p21 and affects other PCNA partners, including the specialized DNA polymerase η (pol eta). These recent discoveries are particularly intriguing since the UV-induced degradation of p21 has been shown to be required for efficient pol η recruitment to DNA lesions. Herein we review the findings that lead to the identification of the molecular mechanism that triggers damage-induced PCNA-coupled protein proteolysis. We propose a novel model in which CRL4^CDT2-dependent protein degradation facilitates a sequential and dynamic exchange between PIP box bearing proteins at stall forks during Translesion DNA synthesis (TLS). Moreover, given the tight spatiotemporal control that CRL4^CDT2-driven proteolysis is able to confer to PCNA-regulated processes, we discuss the impact that this degradation mechanism might have in other molecular switches associated with the repair of damaged DNA.     

Thymine DNA Glycosylase Is a CRL4Cdt2 Substrate

The E3 ubiquitin ligase CRL4^Cdt2 targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4^Cdt2 while binding to PCNA. In vertebrates, CRL4^Cdt2 promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4^Cdt2 targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4^Cdt2. Our results expand the group of vertebrate CRL4^Cdt2 substrates to include a bona fide DNA repair enzyme.     

Ubiquitin E3 Ligase CRL4CDT2/DCAF2 as a Potential Chemotherapeutic Target for Ovarian Surface Epithelial Cancer

Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4^CDT2 repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4^CDT2 is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.     

Cell Type–dependent Requirement for PIP Box–regulated Cdt1 Destruction During S Phase

DNA synthesis–coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4^Cdt2 E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4^Cdt2-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (Dup^ΔPIP) and which cannot be regulated by CRL4^Cdt2. Dup^ΔPIP is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. Dup^ΔPIP restores DNA synthesis to dup null mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, Dup^ΔPIP accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of Dup^ΔPIP expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4^Cdt2 components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box–mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup^ΔPIP activity in some endocycling cell types.     

Spy1 Enhances Phosphorylation and Degradation of the Cell Cycle Inhibitor p27

The cyclin dependent kinase inhibitor (CKI) p27Kip1 binds to cyclin E/CDK2 complexes and prevents premature S-phase entry. During late G₁ and throughout S phase, p27 phosphorylation at T187 leads to its subsequent degradation, which relieves CDK2 inhibition to promote cell cycle progression. However, critical events that trigger CDK2 complexes to phosphorylate p27 remain unclear. Utilizing recombinant proteins, we demonstrate that human Speedy (Spy1) activates CDK2 to phosphorylate p27 at T187 in vitro. Addition of Spy1 or Spy1/CDK2 to a preformed, inhibited cyclin E/CDK2/p27 complex also promoted this phosphorylation. Furthermore, Spy1 protected cyclin E/CDK2 from p27 inhibition toward histone H1, in vitro. Inducible Spy1 expression in U2OS cells reduced levels of endogenous p27 and exogenous p27WT, but not a p27T187A mutant. Additionally, Spy1 expression in synchronized HeLa cells enhanced T187 phosphorylation and degradation of endogenous p27 in late G₁ and throughout S phase. Our studies provide evidence that Spy1 expression enhances CDK2-dependent p27 degradation during late G₁ and throughout S phase.     

Stress-stimulated mitogen-activated protein kinases control the stability and activity of the Cdt1 DNA replication licensing factor

DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G(1) phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G(2) phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4(Cdt2) targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G(2) promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.     

CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase

The proper coordination between DNA replication and mitosis during cell-cycle progression is crucial for genomic stability. During G2 and mitosis, Set8 catalyzes monomethylation of histone H4 on lysine 20 (H4K20me1), which promotes chromatin compaction. Set8 levels decline in S phase, but why and how this occurs is unclear. Here, we show that Set8 is targeted for proteolysis in S phase and in response to DNA damage by the E3 ubiquitin ligase, CRL4(Cdt2). Set8 ubiquitylation occurs on chromatin and is coupled to DNA replication via a specific degron in Set8 that binds PCNA. Inactivation of CRL4(Cdt2) leads to Set8 stabilization and aberrant H4K20me1 accumulation in replicating cells. Transient S phase expression of a Set8 mutant lacking the degron promotes premature H4K20me1 accumulation and chromatin compaction, and triggers a checkpoint-mediated G2 arrest. Thus, CRL4(Cdt2)-dependent destruction of Set8 in S phase preserves genome stability by preventing aberrant chromatin compaction during DNA synthesis.     

14-3-3 Proteins Play a Role in the Cell Cycle by Shielding Cdt2 from Ubiquitin-Mediated Degradation

Cdt2 is the substrate recognition adaptor of CRL4^Cdt2 E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCF^FbxO11-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G₂/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4^Cdt2 substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression.     

DNA Damage Triggers p21WAF1-dependent Emi1 Down-Regulation That Maintains G2 Arrest

Several regulatory proteins control cell cycle progression. These include Emi1, an anaphase-promoting complex (APC) inhibitor whose destruction controls progression through mitosis to G1, and p21^WAF1, a cyclin-dependent kinase (CDK) inhibitor activated by DNA damage. We have analyzed the role of p21^WAF1 in G2-M phase checkpoint control and in prevention of polyploidy after DNA damage. After DNA damage, p21^+/+ cells stably arrest in G2, whereas p21^−/− cells ultimately progress into mitosis. We report that p21 down-regulates Emi1 in cells arrested in G2 by DNA damage. This down-regulation contributes to APC activation and results in the degradation of key mitotic proteins including cyclins A2 and B1 in p21^+/+ cells. Inactivation of APC in irradiated p21^+/+ cells can overcome the G2 arrest. siRNA-mediated Emi1 down-regulation prevents irradiated p21^−/− cells from entering mitosis, whereas concomitant down-regulation of APC activity counteracts this effect. Our results demonstrate that Emi1 down-regulation and APC activation leads to stable p21-dependent G2 arrest after DNA damage. This is the first demonstration that Emi1 regulation plays a role in the G2 DNA damage checkpoint. Further, our work identifies a new p21-dependent mechanism to maintain G2 arrest after DNA damage.     

Regulation of TGF-β signaling,exit from the cell cycle,and cellular migration through cullin cross-regulation: SCF-FBXO11 turns off CRL4-Cdt2

Deregulation of the cell cycle and genome instability are common features of cancer cells and various mechanisms exist to preserve the integrity of the genome and guard against cancer. The cullin 4-RING ubiquitin ligase (CRL4) with the substrate receptor Cdt2 (CRL4^Cdt2) promotes cell cycle progression and prevents genome instability through ubiquitylation and degradation of Cdt1, p21, and Set8 during S phase of the cell cycle and following DNA damage. Two recently published studies report the ubiquitin-dependent degradation of Cdt2 via the cullin 1-RING ubiquitin ligase (CRL1) in association with the substrate specificity factor and tumor suppressor FBXO11 (CRL1^FBXO11). The newly identified pathway restrains the activity of CRL4^Cdt2 on p21 and Set8 and regulates cellular response to TGF-β, exit from the cell cycle and cellular migration. Here, we show that the CRL1^FBXO11 also promotes the degradation of Cdt2 during an unperturbed cell cycle to promote efficient progression through S and G₂/M phases of the cell cycle. We discuss how this new method of regulating the abundance of Cdt2 participates in various cellular activities.     

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

Multiple mechanisms determine the order of APC/C substrate degradation in mitosis

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C^Cdc20 substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1–Cks1 complex and the presence of a Cdc20-binding “ABBA motif” in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C^Cdc20 substrate destruction.     

Human cyclin A is required for mitosis until mid prophase.

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21(Cip1/Waf1/Sdi1) (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.     

Two different replication factor C proteins, Ctf18 and RFC1, separately control PCNA-CRL4Cdt2-mediated Cdt1 proteolysis during S phase and following UV irradiation

Recent work identified the E3 ubiquitin ligase CRL4(Cdt2) as mediating the timely degradation of Cdt1 during DNA replication and following DNA damage. In both cases, proliferating cell nuclear antigen (PCNA) loaded on chromatin mediates the CRL4(Cdt2)-dependent proteolysis of Cdt1. Here, we demonstrate that while replication factor C subunit 1 (RFC1)-RFC is required for Cdt1 degradation after UV irradiation during the nucleotide excision repair process, another RFC complex, Ctf18-RFC, which is known to be involved in the establishment of cohesion, has a key role in Cdt1 degradation in S phase. Cdt1 segments having only the degron, a specific sequence element in target protein for ubiquitination, for CRL4(Cdt2) were stabilized during S phase in Ctf18-depleted cells. Additionally, endogenous Cdt1 was stabilized when both Skp2 and Ctf18 were depleted. Since a substantial amount of PCNA was detected on chromatin in Ctf18-depleted cells, Ctf18 is required in addition to loaded PCNA for Cdt1 degradation in S phase. Our data suggest that Ctf18 is involved in recruiting CRL4(Cdt2) to PCNA foci during S phase. Ctf18-mediated Cdt1 proteolysis occurs independent of cohesion establishment, and depletion of Ctf18 potentiates rereplication. Our findings indicate that individual RFC complexes differentially control CRL4(Cdt2)-dependent proteolysis of Cdt1 during DNA replication and repair.