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.     

Cell Cycle-dependent Subcellular Translocation of the Human DNA Licensing Inhibitor Geminin

Once per cell cycle replication is crucial for maintaining genome integrity. Geminin interacts with the licensing factor Cdt1 to prevent untimely replication and is controlled by APC/C-dependent cell cycle specific proteolysis during mitosis and in G₁. We show here that human geminin, when expressed in human cells in culture under a constitutive promoter, is excluded from the nucleus during part of the G₁ phase and at the transition from G₀ to G₁. The N-terminal 30 amino acids of geminin, which contain its destruction box, are essential for nuclear exclusion. In addition, 30 amino acids within the central domain of geminin are required for both nuclear exclusion and nuclear accumulation. Cdt1 overexpression targets geminin to the nucleus, while reducing Cdt1 levels by RNAi leads to the appearance of endogenous geminin in the cytoplasm. Our data propose a novel means of regulating the balance of Cdt1/geminin in human cells, at the level of the subcellular localization of geminin.     

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2–7 complex onto chromatin during G₁ phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G₁ phase. The immobile fraction of MCM2 and MCM4 increases during G₁ phase, suggestive of reiterative licensing. In late G₁ phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G₁ phase progresses and do not colocalize with sites of DNA synthesis in S phase.     

Geminin-Cdt1 balance is critical for genetic stability

A cell limits its DNA replication activity to once per cell division cycle to maintain its genomic integrity. Studies in a variety of organisms are elucidating how these controls are exercised. Key amongst these is the regulation of replication initiator proteins such as Cdt1. Cdt1 is present in cells in G1 phase where it is required for initiation of replication. Once origins have fired, Cdt1 is either exported out of the nucleus or degraded, thereby preventing another round of replication. Higher eukaryotes have evolved another redundant mechanism, an inhibitor called geminin, to restrain Cdt1 activity. Studies in multiple organisms have shown that unregulated Cdt1 activity stimulates overreplication of the genome. Interestingly, the same seems to be true when geminin is depleted. The imbalance in the activities of these proteins causes the activation of key checkpoint proteins, the ATM/ATR kinases and the tumor suppressor, p53. This review proposes that a balance between Cdt1 and geminin is important for maintaining genomic stability.     

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G₁ phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G₁ phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G₁ phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC.     

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.     

Acetylation/Deacetylation Modulates the Stability of DNA Replication Licensing Factor Cdt1

Proper expression of the replication licensing factor Cdt1 is primarily regulated post-translationally by ubiquitylation and proteasome degradation. In a screen to identify novel non-histone targets of histone deacetylases (HDACs), we found Cdt1 as a binding partner for HDAC11. Cdt1 associates specifically and directly with HDAC11. We show that Cdt1 undergoes acetylation and is reversibly deacetylated by HDAC11. In vitro, Cdt1 can be acetylated at its N terminus by the lysine acetyltransferases KAT2B and KAT3B. Acetylation protects Cdt1 from ubiquitylation and subsequent proteasomal degradation. These results extend the list of non-histone acetylated proteins to include a critical DNA replication factor and provide an additional level of complexity to the regulation of Cdt1.To maintain genomic integrity, DNA replication must be tightly controlled to ensure that each portion of the genome replicates once and only once per cell cycle (reviewed in Ref. 1). Replication licensing begins by the formation of the prereplication complex at multiple potential origins of replication. This is established sequentially, with the origin recognition complex (ORC)² proteins binding first, followed by the recruitment of Cdc6 and Cdt1, which in turn recruit the MCM2–7 proteins. MCM proteins act as the replicative helicase. The licensed replication origins are activated by cyclin-dependent kinases at the start of S phase. Licensing occurs throughout the cell cycle once S phase is complete.Cdt1 levels fluctuate throughout the cell cycle. It is destabilized at G₁/S transition, and then levels begin to climb again upon S phase completion. To prevent licensing at inappropriate times, two separate processes regulate the inactivation or destruction of Cdt1. First, geminin negatively regulates Cdt1 function by prevention of the association of Cdt1 with MCM2–7 via steric hindrance (2). Interestingly, geminin also positively regulates Cdt1 by preventing its ubiquitylation, perhaps by prevention of its interaction with an E3 ligase. This allows Cdt1 to accumulate in G₂ and M phases, to ensure adequate pools of Cdt1 to license the next cycle of replication (3). The ratio of geminin to Cdt1 likely determines whether geminin positively or negatively regulates Cdt1 (4). Second, Cdt1 is targeted for proteolysis by two distinct ubiquitin E3 ligases: the SCF-Skp2 complex and the DDB1-Cul4 complex (5). Phosphorylation by cyclin A/Cdk2 promotes interaction of Cdt1 with Skp2, leading to Cdt1 degradation during S phase (6–8). In addition, DDB1-Cul4 utilizes proliferating cell nuclear antigen as a binding platform to contact Cdt1, targeting the destruction of Cdt1 in S phase or following DNA damage (9, 10). Ubiquitylation by either of these E3 ligases promotes degradation of Cdt1 by the proteasome.Ubiquitylation occurs primarily (but not exclusively) on the ε-amino group of lysine residues. Another prominent post-translational modification that occurs on that residue is acetylation. Acetylation and, correspondingly, deacetylation can modulate the function and activity of a variety of proteins (see Ref. 11 for review). Here, we report that Cdt1 physically interacts with HDAC11, a class IV histone deacetylase (12, 13), as well as with several lysine acetyltransferases (KATs). We show that Cdt1 is an acetylated protein and further show that acetylation protects Cdt1 from ubiquitylation and subsequent proteasomal degradation. This study uncovers yet another layer of complexity to the regulation of the critical licensing factor Cdt1.     

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G₁ phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G₁ to homologous recombination in S/G₂.     

Chromatin unfolding by Cdt1 regulates MCM loading via opposing functions of HBO1 and HDAC11-geminin

The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G₁, is suppressed by Geminin and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G₁ relative to S phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G₁ and repressed by Geminin-HDAC11 association with Cdt1 in S phase and represents a novel form of replication licensing control.Key words: Cdt1, HBO1, HDAC11, chromatin, DNA replication     

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double strand break repair, and stress-induced cell-cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27, and show that Cul4A-DDB1^DDB2-Artemis regulates G₁ phase cell cycle progression in mammalian cells.     

TRIM16 inhibits neuroblastoma cell proliferation through cell cycle regulation and dynamic nuclear localization

Neuroblastoma is the most common solid tumor in childhood and represents 15% of all children’s cancer deaths. We have previously demonstrated that tripartite motif 16 (TRIM16), a member of the RING B-box coiled-coil (RBCC)/tripartite totif (TRIM) protein family, has significant effects on neuroblastoma proliferation and migration in vitro and tumorigenicity in vivo. However, the mechanism by which this putative tumor suppressor influences cell proliferation and tumorigenicity was undetermined. Here we show, for the first time, TRIM16’s striking pattern of expression and dynamic localization during cell cycle progression and neuroblastoma tumor development. In a tyrosine hydroxylase MYCN (TH-MYCN) neuroblastoma mouse model, immunohistochemical staining revealed strong nuclear TRIM16 expression in differentiating ganglia cells but not in the tumor-initiating cells. Furthermore in vitro studies clearly demonstrated that during G₁ cell cycle phase, TRIM16 protein expression is upregulated and shifts to the nucleus of cells. TRIM16 also plays a role in cell cycle progression through changes in Cyclin D1 and p27 expression. Importantly, using TRIM16 deletion mutants, an uncharacterized protein domain of TRIM16 was found to be required for both TRIM16’s growth inhibitory effects and its nuclear localization. Taken together, our data suggest that TRIM16 acts as a novel regulator of both neuroblastoma G₁/S progression and cell differentiation.     

Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G₀/G₁ phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G₂/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G₀/G₁ to S/G₂/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G₂, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G₂ phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of “decoy, trap and shoot” chemotherapy.     

Growth inhibitory effects of three miR-129 family members on gastric cancer

Reduced expression of microRNA-129 (miR-129) has been reported in several types of tumor cell lines as well as in primary tumor tissues. However, little is known about how miR-129 affects cell proliferation in gastric cancer. Here, we show that all miR-129 family members, miR-129-1-3p, miR-129-2-3p, and miR-129-5p, are down-regulated in gastric cancer cell lines compared with normal gastric epithelial cells. Furthermore, using the real-time cell analyzer assay to observe the growth effects of miR-129 on gastric cancer cells, we found that all three mature products of miR-129 showed tumor suppressor activities. To elucidate the molecular mechanisms underlying down-regulation of miR-129 in gastric cancer, we analyzed the effects of miR-129 mimics on the cell cycle. We found that increased miR-129 levels in gastric cancer cells resulted in significant G₀/G₁ phase arrest. Interestingly, we showed that cyclin dependent kinase 6 (CDK6), a cell cycle-associated protein involved in G₁-S transition, was a target of miR-129. We also found that expression of the sex determining region Y-box 4 (SOX4) was inversely associated with that of miR-129-2-3p and miR-129-5p but not of miR-129-1-3p. Together, our data indicate that all miR-129 family members, not only miR-129-5p, as previously thought, play an important role in regulating cell proliferation in gastric cancer.     

The Oncogenic MicroRNA-21 Inhibits the Tumor Suppressive Activity of FBXO11 to Promote Tumorigenesis

The microRNA miR-21 is overexpressed in most human cancers and accumulating evidence indicates that it functions as an oncogene. Since miRNAs suppress the expression of their target genes, we hypothesized that some miR-21 targets may act as tumor suppressors, and thus their expression would be anticipated to be reduced by the high miR-21 levels observed in various human cancers. By microarray analysis and quantitative PCR we identified and validated FBXO11 (a member of the F-box subfamily lacking a distinct unifying domain) as a miR-21 target gene. FBXO11 is a component of the SKP1-CUL1-F-box ubiquitin ligase complex that targets proteins for ubiquitination and proteosomal degradation. By loss of function and gain of function studies, we show that FBXO11 acts as a tumor suppressor, promotes apoptosis and mediates the degradation of the oncogenic protein BCL6. The critical role that FBXO11 plays in miR-21-mediated tumorigenesis was demonstrated by a rescue experiment, in which silencing FBXO11 in miR-21KD cancer cells restored their high tumorigenicity. Expression of miR-21 and FBXO11 are inversely correlated in tumor tissue, and their expression correlates with patient survival and tumor grade. High FBXO11 expression correlates with better patient survival and lower tumor grade consistent with its tumor suppressor activity. In contrast high miR-21 expression, which correlates with poor patient survival and higher tumor grade, is consistent with its oncogenic activity. Our results identify FBXO11 as a novel miR-21 target gene, and demonstrate that the oncogenic miRNA miR-21 decreases the expression of FBXO11, which normally acts as a tumor suppressor, and thereby promotes tumorigenesis.     

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G₁ phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G₁. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G₁ phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G₂/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G₁.     

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc⁻) DT40 cells arrest in G₂ phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc⁻ cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G₂ checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G₂ arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G₂ phase.     

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double-strand break repair and stress-induced cell cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27 and show that Cul4A-DDB1^DDB2-Artemis regulates G₁-phase cell cycle progression in mammalian cells.Key words: artemis, DDB2, p27, Cul4A-DDB1, ubiquitylation