Cofactors UFD-1 Research Articles

Cdc48 and its co-factor Ufd1 extract CENP-A from centromeric chromatin and can induce chromosome elimination in the fission yeast Schizosaccharomyces pombe.

CENP-A determines the identity of the centromere. Because the position and size of the centromere and its number per chromosome must be maintained, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A (Cnp1SP) in fission yeast. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to Cnp1 expressed at a high level and allows mislocalization of Cnp1. The level of Cnp1 in centromeric chromatin is increased in the ufd1-73 mutant even when Cnp1 is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins interact physically with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of Cnp1 from Ch16, leading to an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 remove excess Cnp1 from chromatin, likely in a direct manner. This mechanism may play a role in centromere disassembly, a process to eliminate Cnp1 to inactivate the kinetochore function during development, differentiation, and stress response.

The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation

Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its invivo implications.

The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA.

Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

The CDC48 complex mediates ubiquitin-dependent degradation of intra-chloroplast proteins in plants.

Chloroplasts are the site of numerous biochemical reactions including photosynthesis, but they also produce reactive oxygen species (ROS) that negatively affect chloroplast integrity. The chaperone-like CDC48 complex plays critical roles in ubiquitin-dependent protein degradation in yeast and mammals, but its function in plants is largely unknown. Here, we show that defects in CDC48A and its cofactors UFD1 and NPL4 lead to the accumulation of ubiquitinated chloroplast proteins in Arabidopsis thaliana. We reveal that two plastid genome-encoded proteins, RbcL and AtpB, associate with the CDC48 complex. Strikingly, RbcL and AtpB are ubiquitinated and degraded by the 26S proteasome pathway upon ROS stress, and these processes are impaired by defects of the CDC48 complex. Functional analysis demonstrates that the CDC48 complex is required for plant tolerance to ROS. This study reveals a role for the plant CDC48 complex in modulating ubiquitin-dependent degradation of intra-chloroplast proteins in response to oxidative stress.

Cdc48Ufd1/Npl4 segregase removes mislocalized centromeric histone H3 variant CENP-A from non-centromeric chromatin.

Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 incdc48,ufd1andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed incdc48-3, ufd1-2andnpl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork.

SummaryThe AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.

Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.ImportanceJEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

Involvement of VCP/UFD1/Nucleolin in the viral entry of Enterovirus A species

Valosin-containing protein (VCP) plays roles in various cellular activities. Recently, Enterovirus A71 (EVA71) infection was found to hijack the VCP protein. However, the mechanism by which VCP participates in the EVA71 life cycle remains unclear. Using chemical inhibitor, RNA interference and dominant negative mutant, we confirmed that the VCP and its ATPase activity were critical for EVA71 infection. To identify the factors downstream of VCP in enterovirus infection, 31 known VCP-cofactors were screened in the siRNA knockdown experiments. The results showed that UFD1 (ubiquitin recognition factor in ER associated degradation 1), but not NPL4 (NPL4 homolog, ubiquitin recognition factor), played critical roles in infections by EVA71. UFD1 knockdown suppressed the activity of EVA71 pseudovirus (causing single round infection) while it did not affect the viral replication in replicon RNA transfection assays. In addition, knockdown of VCP and UFD1 reduced viral infections by multiple human Enterovirus A serotypes. Mechanistically, we found that knockdown of UFD1 significantly decreased the binding and the subsequent entry of EVA71 to host cells through modulating the levels of nucleolin protein, a coreceptor of EVA71. Together, these data reveal novel roles of VCP and its cofactor UFD1 in the virus entry by EVA71.

PINK1 Interacts with VCP/p97 and Activates PKA to Promote NSFL1C/p47 Phosphorylation and Dendritic Arborization in Neurons.

While PTEN-induced kinase 1 (PINK1) is well characterized for its role in mitochondrial homeostasis, much less is known concerning its ability to prevent synaptodendritic degeneration. Using unbiased proteomic methods, we identified valosin-containing protein (VCP) as a major PINK1-interacting protein. RNAi studies demonstrate that both VCP and its cofactor NSFL1C/p47 are necessary for the ability of PINK1 to increase dendritic complexity. Moreover, PINK1 regulates phosphorylation of p47, but not the VCP co-factor UFD1. Although neither VCP nor p47 interact directly with PKA, we found that PINK1 binds and phosphorylates the catalytic subunit of PKA at T197 [PKAcat(pT197)], a site known to activate the PKA holoenzyme. PKA in turn phosphorylates p47 at a novel site (S176) to regulate dendritic complexity. Given that PINK1 physically interacts with both the PKA holoenzyme and the VCP-p47 complex to promote dendritic arborization, we propose that PINK1 scaffolds a novel PINK1-VCP-PKA-p47 signaling pathway to orchestrate dendritogenesis in neurons. These findings highlight an important mechanism by which proteins genetically implicated in Parkinson's disease (PD; PINK1) and frontotemporal dementia (FTD; VCP) interact to support the health and maintenance of neuronal arbors.

Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1-Npl4.

Many poly-ubiquitinated proteins are extracted from membranes or complexes by a conserved ATPase, called Cdc48 in yeast and p97/VCP in mammals, before proteasomal degradation1. Each Cdc48 hexamer contains two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains2. Cdc48 binds various cofactors, including a heterodimer of Ufd1 and Npl43. Here, we report structures of the Cdc48-Ufd1-Npl4 ATPase complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and uses two Zn2+-finger domains to anchor an enzymatically inactive Mpr1/Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring. The MPN domain of Npl4 is located above Cdc48’s central pore, similarly to the MPN of the de-ubiquitinase Rpn11 in the proteasome4. Our results indicate that Npl4 is unique among Cdc48 cofactors, and suggest a mechanism for how poly-ubiquitinated substrates bind to and translocate into the ATPase.

Cdc48/p97 segregase is modulated by cyclin‐dependent kinase to determine cyclin fate during G1 progression

Cells sense myriad signals during G1, and a rapid response to prevent cell cycle entry is of crucial importance for proper development and adaptation. Cln3, the most upstream G1 cyclin in budding yeast, is an extremely short-lived protein subject to ubiquitination and proteasomal degradation. On the other hand, nuclear accumulation of Cln3 depends on chaperones that are also important for its degradation. However, how these processes are intertwined to control G1-cyclin fate is not well understood. Here, we show that Cln3 undergoes a challenging ubiquitination step required for both degradation and full activation. Segregase Cdc48/p97 prevents degradation of ubiquitinated Cln3, and concurrently stimulates its ER release and nuclear accumulation to trigger Start. Cdc48/p97 phosphorylation at conserved Cdk-target sites is important for recruitment of specific cofactors and, in both yeast and mammalian cells, to attain proper G1-cyclin levels and activity. Cdk-dependent modulation of Cdc48 would subjugate G1 cyclins to fast and reversible state switching, thus arresting cells promptly in G1 at developmental or environmental checkpoints, but also resuming G1 progression immediately after proliferative signals reappear.

Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast

Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA+) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe, generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe.

CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination andmitosis.

Replisome disassembly is the final step of DNA replication in eukaryotes, involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase (Cdc45-MCM-GINS). Using Caenorhabditis elegans early embryos and Xenopus egg extracts, we show that the E3 ligase CUL-2LRR-1 associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 co-factors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2LRR1 as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2LRR-1, but is then removed by a mitotic pathway that requires the CDC-48 co-factor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future approaches by which a deeper understanding of CMG disassembly in metazoa could be exploited therapeutically.

UV-induced proteolysis of RNA polymerase II is mediated by VCP/p97 segregase and timely orchestration by Cockayne syndrome B protein.

RNA polymerase II (RNAPII) acts as a damage sensor for transcription-coupled nucleotide excision repair (TC-NER) and undergoes proteolytic clearance from damaged chromatin by the ubiquitin-proteasome system (UPS). Here, we report that Valosin-containing protein (VCP)/p97, a druggable oncotarget, is essential for RNAPII's proteolytic clearance in mammalian cells. We show that inhibition of VCP/p97, or siRNA-mediated ablation of VCP/p97 and its cofactors UFD1 and UBXD7 severely impairs ultraviolet radiation (UVR)-induced RNAPII degradation. VCP/p97 interacts with RNAPII, and the interaction is enhanced by Cockayne syndrome B protein (CSB). However, the VCP/p97-mediated RNAPII proteolysis occurs independent of CSB. Surprisingly, CSB enhances UVR-induced RNAPII ubiquitination but delays its turnover. Additionally, VCP/p97-mediated RNAPII turnover occurs with and without Von Hippel-Lindau tumor suppressor protein (pVHL), a known substrate receptor of Elongin E3 ubiquitin ligase for RNAPII. Moreover, pVHL re-expression improves cell viability following UVR. Whereas, VCP/p97 inhibition decreases cell viability and enhances a low-dose UVR killing in presence of pVHL. These findings reveal a function of VCP/p97 segregase in UVR-induced RNAPII degradation in mammalian cells, and suggest a role of CSB in coordinating VCP/p97-mediated extraction of ubiquitinated RNAPII and CSB itself from chromatin.

Valosin-containing Protein (VCP)/p97 Segregase Mediates Proteolytic Processing of Cockayne Syndrome Group B (CSB) in Damaged Chromatin

Cockayne syndrome group A and B (CSB) proteins act in transcription-coupled repair, a subpathway of nucleotide excision repair. Here we demonstrate that valosin-containing protein (VCP)/p97 segregase functions in ultraviolet radiation (UVR)-induced ubiquitin-mediated CSB degradation. We show that VCP/p97 inhibition and siRNA-mediated ablation of VCP/p97 and its cofactors UFD1 and UBXD7 impair CSB degradation. VCP/p97 inhibition also results in the accumulation of CSB in chromatin. Moreover, VCP/p97 interacts with both native and ubiquitin-conjugated forms of CSB. The localized cellular UVR exposures lead to VCP/p97 accumulation at DNA damage spots, forming distinct UVR-induced foci. However, manifestation of VCP/p97 foci is independent of CSB and UBXD7. Furthermore, VCP/p97 and UBXD7 associate with the Cockayne syndrome group A-DDB1-Cul4A complex, an E3 ligase responsible for CSB ubiquitination. Compromising proteasome and VCP/p97 function allows accumulation of both native and ubiquitinated CSB and results in an increase of UBXD7, proteasomal RPN2, and Sug1 in the chromatin compartment. Surprisingly, both biochemical inhibition and genetic defect of VCP/p97 enhance the recovery of RNA synthesis following UVR, whereas both VCP/p97 and proteasome inhibitions decrease cell viability. Our findings reveal a new role of VCP/p97 segregase in the timely processing of ubiquitinated CSB from damaged chromatin.

Role of Cdc48/p97 as a SUMO-targeted segregase curbing Rad51–Rad52 interaction

Cdc48 (also known as p97), a conserved chaperone-like ATPase, plays a strategic role in the ubiquitin system. Empowered by ATP-driven conformational changes, Cdc48 acts as a segregase by dislodging ubiquitylated proteins from their environment. Ufd1, a known co-factor of Cdc48, also binds SUMO (ref. 6), but whether SUMOylated proteins are subject to the segregase activity of Cdc48 as well and what these substrates are remains unknown. Here we show that Cdc48 with its co-factor Ufd1 is SUMO-targeted to proteins involved in DNA double-strand break repair. Cdc48 associates with SUMOylated Rad52, a factor that assembles the Rad51 recombinase on chromatin. By acting on the Rad52-Rad51 complex, Cdc48 curbs their physical interaction and displaces the proteins from DNA. Genetically interfering with SUMO-targeting or segregase activity leads to an increase in spontaneous recombination rates, accompanied by aberrant in vivo Rad51 foci formation in yeast and mammalian cells. Our data thus suggest that SUMO-targeted Cdc48 restricts the recombinase Rad51 by counterbalancing the activity of Rad52. We propose that Cdc48, through its ability to associate with co-factors that have affinities for ubiquitin and SUMO, connects the two modification pathways for protein degradation or other regulatory purposes.

The Cdc48 Protein and Its Cofactor Vms1 Are Involved in Cdc13 Protein Degradation

Vms1 is a newly identified Cdc48-binding protein. The biological function of Vms1 remains obscure. Here, we show that both Cdc48 and Vms1, but not Cdc48 cofactors Ufd1 and Ufd2, are crucial for the degradation of Cdc13, a telomere regulator. Interestingly, both autophagy and the proteasome are involved in Cdc13 turnover. Toxicity associated with accumulation of large amounts of Cdc13 in vms1Δ or autophagy mutants underscores the significance of the proteolytic regulation of Cdc13. Because few ubiquitylated yeast proteins are known to be degraded by autophagy under non-stress conditions, the identification of Cdc13 as a target of autophagy provides a valuable tool to unravel the mechanism of autophagy-mediated selective protein degradation.

Expanding into new markets – VCP/p97 in endocytosis and autophagy

The AAA-ATPase p97 (also called VCP for Valosin-containing protein) is essential for a number of cellular processes as diverse as ER-associated degradation, DNA damage response, and cell cycle control. Mechanistically, p97 cooperates with its cofactor Ufd1-Npl4 in these processes to segregate polyubiquitinated misfolded or regulatory client proteins from intracellular structures for subsequent degradation by the proteasome. Recent work now connects p97, independently of Ufd1-Npl4, to endosomal trafficking and autophagy. Interestingly, these pathways also deliver proteins for degradation, albeit by the lysosome. While monoubiquitination and alternative p97-cofactors, including UBXD1, have been associated with these activities, the underlying molecular mechanism(s) are still unclear or controversial. In this review, we aim to summarize the available data and discuss mechanistic models.

The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks

The accumulation of the human tumor suppressor 53BP1 at DNA damage sites requires the ubiquitin ligases RNF8 and RNF168. As 53BP1 recognizes dimethylated Lys20 in histone H4 (H4K20me2), the requirement for RNF8- and RNF168-mediated ubiquitylation has been unclear. Here we show that RNF8-mediated ubiquitylation facilitates the recruitment of the AAA-ATPase valosin-containing protein (VCP, also known as p97) and its cofactor NPL4 to sites of double-strand breaks. RIDDLE cells, which lack functional RNF168, also show impaired recruitment of VCP to DNA damage. The ATPase activity of VCP promotes the release of the Polycomb protein L3MBTL1 from chromatin, which also binds the H4K20me2 histone mark, thereby facilitating 53BP1 recruitment. Consistent with this, nematodes lacking the VCP orthologs CDC-48.1 or CDC-48.2, or cofactors UFD-1 or NPL-4, are highly sensitive to ionizing radiation. Our data suggest that human RNF8 and RNF168 promote VCP-mediated displacement of L3MBTL1 to unmask 53BP1 chromatin binding sites.

A Genome-wide Screen Identifies p97 as an Essential Regulator of DNA Damage-Dependent CDT1 Destruction

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.