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Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.

CENP-A is the histone H3 variant necessary to specify the location of all eukaryotic centromeres via its CENP-A targeting domain and either one of its terminal regions. In humans, several post-translational modifications occur on CENP-A, but their role in centromere function remains controversial. One of these modifications of CENP-A, phosphorylation on serine 7, has been proposed to control centromere assembly and function. Here, using gene targeting at both endogenous CENP-A alleles and gene replacement in human cells, we demonstrate that a CENP-A variant that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability. Thus, we conclude that phosphorylation of CENP-A on serine 7 is dispensable to maintain correct centromere dynamics and function., Phosphorylation of CENP-A on serine 7 has been proposed to control centromere assembly and function. Here, the authors use gene targeting at both endogenous CENP-A alleles and gene replacement in human cells to demonstrate that CENP-A that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability.

Shotgun proteomics generates valuable information from large-scale and target protein characterizations, including protein expression, protein quantification, protein post-translational modifications (PTMs), protein localization, and protein–protein interactions. Typically, peptides derived from proteolytic digestion, rather than intact proteins, are analyzed by mass spectrometers because peptides are more readily separated, ionized and fragmented. The amino acid sequences of peptides can be interpreted by matching the observed tandem mass spectra to theoretical spectra derived from a protein sequence database. Identified peptides serve as surrogates for their proteins and are often used to establish what proteins were present in the original mixture and to quantify protein abundance. Two major issues exist for assigning peptides to their originating protein. The first issue is maintaining a desired false discovery rate (FDR) when comparing or combining multiple large datasets generated by shotgun analysis and the second issue is properly assigning peptides to proteins when homologous proteins are present in the database. Herein we demonstrate a new computational tool, ProteinInferencer, which can be used for protein inference with both small- or large-scale data sets to produce a well-controlled protein FDR. In addition, ProteinInferencer introduces confidence scoring for individual proteins, which makes protein identifications evaluable.This article is part of a Special Issue entitled: Computational Proteomics.

SummaryHistidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.

Transcription by RNA polymerase III (Pol III) is an essential cellular process, and mutations in Pol III can cause neurodegenerative disease in humans. However, in contrast to Pol II transcription, which has been extensively studied, the knowledge of how Pol III is regulated is very limited. We report here that in budding yeast,Saccharomyces cerevisiae, Pol III is negatively regulated by theSmallUbiquitin-likeMOdifier (SUMO), an essential post-translational modification pathway. Besides sumoylation, Pol III is also targeted by ubiquitylation and the Cdc48/p97 segregase, the three of which likely act in a sequential manner and eventually lead to proteasomal degradation of Pol III subunits, thereby repressing Pol III transcription. This study not only uncovered a regulatory mechanism for Pol III, but also suggests that the SUMO and ubiquitin modification pathways and the Cdc48/p97 segregase can be potential therapeutic targets for Pol III-related human diseases.

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.

SummaryStem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the corepressor C-terminal binding protein (CtBP), which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo.

Summary The rich functional diversity of the nervous system is founded in the specific connectivity of the underlying neural circuitry. Neurons are often preprogrammed to respond to multiple axon guidance signals because they use sequential guideposts along their pathways, but this necessitates a strict spatiotemporal regulation of intracellular signaling to ensure the cues are detected in the correct order. We performed a mouse mutagenesis screen and identified the Rho GTPase antagonist p190RhoGAP as a critical regulator of motor axon guidance. Rather than acting as a compulsory signal relay, p190RhoGAP uses a non-conventional GAP-independent mode to transiently suppress attraction to Netrin-1 while motor axons exit the spinal cord. Once in the periphery, a subset of axons requires p190RhoGAP-mediated inhibition of Rho signaling to target specific muscles. Thus, the multifunctional activity of p190RhoGAP emerges from its modular design. Our findings reveal a cell-intrinsic gate that filters conflicting signals, establishing temporal windows of signal detection.

Faithful transmission of genetic material is essential for cell viability and organism health. The occurrence of DNA damage, due to either spontaneous events or environmental agents, threatens the integrity of the genome. The consequences of these insults, if allowed to perpetuate and accumulate over time, are mutations that can lead to the development of diseases such as cancer. Alkylation is a relevant DNA lesion produced endogenously as well as by exogenous agents including certain chemotherapeutics. We sought to better understand the cellular response to this form of DNA damage using mass spectrometry-based proteomics. For this purpose, we performed sub-cellular fractionation to monitor the effect of methyl methanesulfonate (MMS) treatment on protein localization to chromatin. The levels of over 500 proteins were increased in the chromatin-enriched nuclear lysate including histone chaperones. Levels of ubiquitin and subunits of the proteasome were also increased within this fraction, suggesting that ubiquitin-mediated degradation by the proteasome has an important role in the chromatin response to MMS treatment. Finally, the levels of some proteins were decreased within the chromatin-enriched lysate including components of the nuclear pore complex. Our spatial proteomics data demonstrate that many proteins that influence chromatin organization are regulated in response to MMS treatment, presumably to open the DNA to allow access by other DNA damage response proteins. To gain further insight into the cellular response to MMS-induced DNA damage, we also performed phosphorylation enrichment on total cell lysates to identify proteins regulated via post-translational modification. Phosphoproteomic analysis demonstrated that many nuclear phosphorylation events were decreased in response to MMS treatment. This reflected changes in protein kinase and/or phosphatase activity in response to DNA damage rather than changes in total protein abundance. Using these two mass spectrometry-based approaches, we have identified a novel set of MMS-responsive proteins that will expand our understanding of DNA damage signaling.

Oncogene-induced senescence is an important tumor-suppressing defense mechanism. However, relatively little is known about the signaling pathway mediating the senescence response. Here, we demonstrate that a multifunctional acetyltransferase Tip60 plays an essential role in oncogenic ras-induced senescence. Further investigation reveals a novel cascade of posttranslational modifications involving p38, Tip60 and PRAK, three proteins that are essential for ras-induced senescence. Upon activation by ras, p38 induces the acetyltransferase activity of Tip60 through phosphorylation of Thr158; activated Tip60 in turn directly interacts with and induces the protein kinase activity of PRAK through acetylation of K364 in a manner that depends on phosphorylation of both Tip60 and PRAK by p38. These posttranslational modifications are critical for the pro-senescent function of Tip60 and PRAK, respectively. These results have defined a novel signaling pathway that mediate oncogene-induced senescence, and identified novel posttranslational modifications that regulate the enzymatic activity and biological functions of Tip60 and PRAK.

Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states.

MBF and SBF transcription factors regulate a large family of coordinately expressed G1/S genes required for early cell-cycle functions including DNA replication and repair. SBF is inactivated upon S-phase entry by Clb/CDK whereas MBF targets are repressed by the co-repressor, Nrm1. Using genome-wide expression analysis of cells treated with methyl methane sulfonate (MMS), hydroxyurea (HU) or camptothecin (CPT), we show that genotoxic stress during S phase specifically induces MBF-regulated genes. This occurs via direct phosphorylation of Nrm1 by Rad53, the effector checkpoint kinase, which prevents its binding to MBF target promoters. We conclude that MBF-regulated genes are distinguished from SBF-regulated genes by their sensitivity to activation by the S-phase checkpoint, thereby, providing an effective mechanism for enhancing DNA replication and repair and promoting genome stability.

The retinoblastoma tumor suppressor (RB) is a central cell cycle regulator and tumor suppressor. RB cellular functions are known to be regulated by a diversity of post-translational modifications such as phosphorylation and acetylation, raising the possibility that RB may also be methylated in cells. Here we demonstrate that RB can be methylated by SMYD2 at lysine 860, a highly conserved and novel site of modification. This methylation event occurs in vitro and in cells, and it is regulated during cell cycle progression, cellular differentiation, and in response to DNA damage. Furthermore, we show that RB monomethylation at lysine 860 provides a direct binding site for the methyl-binding domain of the transcriptional repressor L3MBTL1. These results support the idea that a code of post-translational modifications exists for RB and helps guide its functions in mammalian cells.

Central to the replication checkpoint are two protein kinases, ATR, and its downstream target kinase, Chk1. Signaling pathways leading to activation of ATR-Chk1 have been extensively investigated; however, events that mediate checkpoint termination and replication fork restart are less well understood. Here, we define a coupled activation-destruction mechanism of Chk1 that regulates checkpoint termination and cellular sensitivity to replicative stress. DNA damage-induced phosphorylation or mutation of a conserved motif of Chk1 both activates Chk1 and exposes a degron-like region at the carboxyl-terminus of Chk1 to a Fbx6-containing SCF (Skp1-Cul1-F-box) E3 ligase, which mediates the ubiquitination and degradation of Chk1, and, in turn, terminates the checkpoint. The expression levels of Chk1 and Fbx6 proteins showed an inverse correlation in both cultured cancer cell lines and in a small cohort of human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to killing by the chemotherapeutic agent, camptothecin (CPT). We propose that Fbx6-dependent Chk1 degradation contributes to S-phase checkpoint termination, and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.

Mass spectrometry has been widely used to analyze biological samples and has evolved into an indispensable tool for proteomics research. Our desire to understand the proteome has led to new technologies that push the boundary of mass spectrometry capabilities, which in return has allowed mass spectrometry to address an ever-increasing array of biological questions. The recent development of a novel mass spectrometer (Orbitrap) and new dissociation methods such as electron-transfer dissociation has made possible the exciting new areas of proteomic application. Although bottom-up proteomics (analysis of proteolytic peptide mixtures) remains the workhorse for proteomic analysis, middle-down and top-down strategies (analysis of longer peptides and intact proteins, respectively) should allow more complete characterization of protein isoforms and post-translational modifications. Finally, stable isotope labeling strategies have transformed mass spectrometry from merely descriptive to a tool for measuring dynamic changes in protein expression, interaction, and modification.

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ- Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Eμ- Myc transgenic mice lacking one or both E2f1 alleles exhibited a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27 Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27 Kip1 .

Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mixtures of intact proteins on a proteomic scale. Here we applied capillary electrophoresis (CE) through a sheathless capillary electrophoresis-electrospray ionization (CESI) interface coupled to an Orbitrap Elite mass spectrometer to profile the proteome from Pyrococcus furiosus. CESI-top-down MS analysis of Pyrococcus furiosus cell lysate identified 134 proteins and 291 proteoforms with a total sample consumption of 270 ng in 120 min of total analysis time. Truncations and various PTMs were detected, including acetylation, disulfide bonds, oxidation, glycosylation, and hypusine. This is the largest scale analysis of intact proteins by CE-top-down MS to date.

Post-translational modification by SUMO is a highly conserved pathway in eukaryotes that plays very important regulatory roles in many cellular processes. Deregulation of the SUMO pathway contributes to the development and progression of many diseases including cancer. Therefore, identifying additional SUMO substrates and studying how their cellular and biological functions are regulated by sumoylation should provide new insights. Our studies showed that sumoylation activity was significant in Xenopus egg extracts, and that a high level of sumoylation was associated with sperm chromatin when SUMO was incubated with Xenopus egg extracts. By isolating SUMO-conjugated substrates using His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified 346 proteins by mass spectrometry analysis that were not present in control pull-downs. Among them, 167 proteins were identified from interphase egg extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and 228 proteins by both. We validated the sumoylation of five candidates, CKB, ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in this study were involved in diverse biological processes. Additionally, SUMO substrates identified from different cell cycle stages or pulled down by different SUMO homologs were enriched for distinct cellular components and functional categories. Our results comprehensively profile the sumoylation occurring in the Xenopus egg extract system.

Motivation: We introduce Census 2, an update of a mass spectrometry data analysis tool for peptide/protein quantification. New features for analysis of isobaric labeling, such as Tandem Mass Tag (TMT) or Isobaric Tags for Relative and Absolute Quantification (iTRAQ), have been added in this version, including a reporter ion impurity correction, a reporter ion intensity threshold filter and an option for weighted normalization to correct mixing errors. TMT/iTRAQ analysis can be performed on experiments using HCD (High Energy Collision Dissociation) only, CID (Collision Induced Dissociation)/HCD (High Energy Collision Dissociation) dual scans or HCD triple-stage mass spectrometry data. To improve measurement accuracy, we implemented weighted normalization, multiple tandem spectral approach, impurity correction and dynamic intensity threshold features. Availability and implementation: Census 2 supports multiple input file formats including MS1/MS2, DTASelect, mzXML and pepXML. It requires JAVA version 6 or later to run. Free download of Census 2 for academic users is available at http://fields.scripps.edu/census/index.php . Contact: jyates@scripps.edu Supplementary information: Supplementary data are available at Bioinformatics online.

The reversible nature of protein phosphorylation dictates that any protein kinase activity must be counteracted by protein phosphatase activity. How phosphatases target specific phosphoprotein substrates and reverse the action of kinases, however, is poorly understood in a biological context. We address this question by elucidating a novel function of the conserved PP4 family phosphatase Pph3-Psy2, the yeast counterpart of the mammalian PP4c-R3 complex, in the glucose-signaling pathway. Our studies show that Pph3-Psy2 specifically targets the glucose signal transducer protein Mth1 via direct binding of the EVH1 domain of the Psy2 regulatory subunit to the polyproline motif of Mth1. This activity is required for the timely dephosphorylation of the downstream transcriptional repressor Rgt1 upon glucose withdrawal, a critical event in the repression of HXT genes, which encode glucose transporters. Pph3-Psy2 dephosphorylates Mth1, an Rgt1 associated corepressor, but does not dephosphorylate Rgt1 at sites associated with inactivation, in vitro. We show that Pph3-Psy2 phosphatase antagonizes Mth1 phosphorylation by protein kinase A (PKA), the major protein kinase activated in response to glucose, in vitro and regulates Mth1 function via putative PKA phosphorylation sites in vivo. We conclude that the Pph3-Psy2 phosphatase modulates Mth1 activity to facilitate precise regulation of HXT gene expression by glucose.

Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.

Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3′-halves beginning with a 5′-hydroxyl, and 5′-halves ending in a 2′,3′-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3′-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2′,3′-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3′-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn 2+ binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3′-P RNA ligase.

Mms1 and Mms22 are subunits of an Rtt101-based E3 ubiquitin ligase required for replication of damaged DNA templates in Saccharomyces cerevisiae. The function and evolutionary conservation of this DNA repair module are unknown. Here we report the characterization of an Mms1 ortholog in Schizosaccharomyces pombe. Fission yeast Mms1 was discovered through its physical association with S. pombe Mms22 (also known as Mus7). Loss of S. pombe Mms1 results in the accumulation of spontaneous DNA damage, mitotic delay, and hypersensitivity to genotoxins such as camptothecin that perturb replisome progression. Homologous recombination repair proteins Rhp51 and Rad22 (Rad51 and Rad52 orthologs, respectively) are critical for survival in the absence of Mms1; however, there is no such requirement for Mus81-Eme1 Holliday junction resolvase that is essential for recovery from broken replication forks. Mms1 and Mms22 mutants share similar phenotypes and are genetically epistatic under unperturbed growth conditions and following exposure to genotoxins. From these data we conclude that an evolutionary conserved Mms1-Mms22 complex is required for replication of damaged DNA in fission yeast.

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G 1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication.

The development of mammalian tumors is dependent upon the disruption of two key biological activities, the control of cellular proliferation and the apoptotic response (Hanahan and Weinberg 2000). Remarkably, the Ink4a/Arf locus encodes two distinct tumor-suppressor proteins, p16Ink4a and p19Arf (p14Arf in humans), that influence one or both of these processes (Chin et al. 1998; Sherr 2001). p16Ink4a is a core component of the cell cycle control machinery (Sherr and Roberts 1999). It controls the activity of the G1 kinase, cyclinD · cdk4/6, and consequently, the phosphorylation status of the pocket protein family. This family includes the retinoblastoma protein (pRB) tumor suppressor and its relatives, p107 and p130. In the unphosphorylated state, the pocket proteins bind to the E2F family of transcription factors and prevent the expression of genes that are essential for entry into, and passage through the cell cycle (Trimarchi and Lees 2002). This inhibition occurs through two distinct mechanisms. pRB binds to the activating E2Fs, E2F1, E2F2, and E2F3a, and blocks their transcriptional activity. At the same time, the repressive E2Fs, E2F4, and E2F5 recruit p107 or p130 and their associated histone deactylases to E2F-responsive promoters. Under these conditions, the cell is blocked in G0/G1. Mitogenic signaling activates cell cycle re-entry by allowing cyclinD · cdk4/6 to overcome the repression by p16Ink4a. The consequent phosphorylation of the pocket proteins causes them to dissociate from E2F, enabling activation of E2F-responsive genes. In normal cells, the p16Ink4a–cyclinD · cdk4/6–pRB–E2F pathway responds to both positive and negative growth regulatory signals to determine whether or not a cell will divide (Sherr and Roberts 1999). This pathway is disrupted in most, if not all, mammalian tumors through loss of p16Ink4a, up-regulation of cyclinD · cdk4/6 or loss of pRB (Sherr 1996). The resulting deregulated proliferation is due, at least in part, to the inappropriate activation of E2F (Pan et al. 1998; Tsai et al. 1998; Yamasaki et al. 1998; McCaffrey et al. 1999; Ziebold et al. 2001, 2003). The second product of the Ink4a/Arf locus, p19Arf,isa key component of the p53 tumor-surveillance network (Sherr 2001). p19Arf exists at low or undetectable levels in most normal cell and tissue types (Zindy et al. 2003). However, its expression is specifically activated by abnormal proliferative signals. These include the continued in vitro culturing of mouse embryonic fibroblasts (MEFs; Kamijo et al. 1997) and the inappropriate expression of proliferative oncogenes including activated ras, c-myc, E2F, E1A, and v-Abl (Serrano et al. 1997; de Stanchina et al. 1998; Palmero et al. 1998; Radfar et al. 1998; Zindy et al. 1998; Dimri et al. 2000). Once it is expressed, p19Arf inhibits the p53 ubiquitin ligase, mdm2, allowing activation of the p53 tumor suppressor (Pomerantz et al. 1998; Stott et al. 1998; Zhang et al. 1998; Honda and Yasuda 1999; Weber et al. 1999; Llanos et al. 2001). Depending on the cellular context, p53 triggers either cell cycle arrest (via induction of the cdk inhibitor, p21Cip1) or apoptosis (through activation of various apoptosis inducers). In either case, this counteracts the effect of the abnormal proliferative signals. Essentially, p19Arf acts as a defense to oncogenic signals. The recent analysis of a mouse strain that expresses GFP in place of p19Arf confirms that Arf is induced by the oncogenic signals present in incipient tumors (Zindy et al. 2003). This explains why inactivation of the p19Arf–p53 network is essential for the survival and proliferation of tumor cells in vivo (Sherr 2001). The ability of Arf to specifically respond to inappropriate, but not normal proliferative signals must require a careful balance of transcriptional signals. Understanding how this is achieved remains a major challenge. Numerous studies have implicated E2F in this process (Phillips and Vousden 2001). The Arf promoter contains consensus E2F-binding sites and the overexpression of E2F1 is sufficient to trigger its transcriptional activation (DeGregori et al. 1997; Bates et al. 1998). However, it is unclear whether this regulation is direct because the identified E2F sites are not required for E2F-dependent activation (Parisi et al. 2002; Berkovich et al. 2003). There is also considerable debate as to which E2F family members might activate Arf (Trimarchi and Lees 2002). Some groups conclude that this is an E2F1-specific activity, whereas others propose that this is a shared property of the activating E2Fs. Certainly, E2F1 is not required for Arf induction in numerous settings (Palmero et al. 2002; Baudino et al. 2003) and p19Arf itself is dispensable for E2F-dependent apoptosis (Russell et al. 2002; Tolbert et al. 2002; Tsai et al. 2002). These findings could reflect redundancy; perhaps multiple E2Fs can activate a large panel of apoptotic inducers that includes p19Arf. Alternatively, E2F may not contribute to Arf activation in vivo. Others have suggested that Arf is regulated by repressive E2F · pocket protein complexes (Rowland et al. 2002). However, unlike classic E2F-responsive genes, Arf is not appreciably induced during cell cycle entry. Thus, if Arf is a genuine E2F target, it must be regulated in a distinct manner from classic E2F-responsive genes. In this study, we use E2f3-deficient MEFs to probe the role of E2F in Arf regulation. This analysis shows that a single member of the E2F family, E2f3, is required to maintain the transcriptional repression of Arf under normal proliferative conditions.