A Systems Biology Approach Identifies Hidden Regulatory Connections Between the Circadian and Cell-Cycle Checkpoints.

Circadian rhythms form a self-sustaining, endogenous, time-keeping system that allows organisms to anticipate daily environmental changes. The core of the clock network consists of interlocking transcriptional-translational feedback loops that ensures that metabolic, behavioral, and physiological processes run on a 24 h timescale. The hierarchical nature of the clock manifests itself in multiple points of control on the daily cell division cycle, which relies on synthesis, degradation, and post-translational modification for progression. This relationship is particularly important for understanding the role of clock components in sensing stress conditions and triggering checkpoint signals that stop cell cycle progression. A case in point is the interplay among the circadian factor PERIOD2 (PER2), the tumor suppressor p53, and the oncogenic mouse double minute-2 homolog protein (MDM2), which is the p53's negative regulator. Under unstressed conditions, PER2 and p53 form a stable complex in the cytosol and, along with MDM2, a trimeric complex in the nucleus. Association of PER2 to the C-terminus end of p53 prevents MDM2-mediated ubiquitylation and degradation of p53 as well as p53's transcriptional activation. Remarkably, when not bound to p53, PER2 acts as substrate for the E3-ligase activity of MDM2; thus, PER2 is degraded in a phosphorylation-independent fashion. Unexpectedly, the phase relationship between PER2 and p53 are opposite; however, a systematic modeling approach, inferred from the oscillatory time course data of PER2 and p53, aided in identifying additional regulatory scenarios that explained, a priori, seemingly conflicting experimental data. Therefore, we advocate for a combined experimental/mathematical approach to elucidating multilevel regulatory cellular processes.

Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein and ubiquitin ligase MDM2.

The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by ß-transducin repeat-containing proteins (ß-TrCPs), which are F-box-containing proteins that function as substrate recognition subunits of the SCFß-TRCP ubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2.

The circadian clock and cell cycle networks are interlocked on the molecular level, with the core clock loop exerting a multilevel regulatory role over cell cycle components. This is particularly relevant to the circadian factor Period 2 (Per2), which modulates the stability of the tumor suppressor p53 in unstressed cells and transcriptional activity in response to genotoxic stress. Per2 binding prevents Mdm2-mediated ubiquitination of p53 and, therefore, its degradation, and oscillations in the peaks of Per2 and p53 were expected to correspond. However, our findings showed that Per2 and p53 rhythms were significantly out-of-phase relative to each other in cell lysates and in purified cytoplasmic fractions. These seemingly conflicting experimental data motivated the use of a combined theoretical and experimental approach focusing on the role played by Per2 in dictating the phase of p53 oscillations. Systematic modeling of all possible regulatory scenarios predicted that the observed phase relationship between Per2 and p53 could be simulated if (i) p53 was more stable in the nucleus than in the cytoplasm, (ii) Per2 associates to various ubiquitinated forms of p53, and (iii) Per2 mediated p53 nuclear import. These predictions were supported by a sevenfold increase in p53's half-life in the nucleus and by in vitro binding of Per2 to the various ubiquitinated forms of p53. Last, p53's nuclear shuttling was significantly favored by ectopic expression of Per2 and reduced because of Per2 down-regulation. Our combined theoretical/mathematical approach reveals how clock regulatory nodes can be inferred from oscillating time course data.

Chronotherapy: Intuitive, Sound, Founded But Not Broadly Applied.

Circadian rhythms are a collection of endogenously driven biochemical, physiological, and behavioral processes that oscillate in a 24-h cycle and can be entrained by external cues. Circadian clock molecules are responsible for the expression of regulatory components that modulate, among others, the cell's metabolism and energy consumption. In clinical practice, the regulation of clock mechanisms is relevant to biotransformation of therapeutics. Accordingly, xenobiotic metabolism and detoxification, the two processes that directly influence drug effectiveness and toxicity, are direct manifestations of the daily oscillations of the cellular and biochemical processes taking place within the gastrointestinal, hepatic/biliary, and renal/urologic systems. Consequently, the impact of circadian timing should be factored in when developing therapeutic regimens aimed at achieving maximum efficacy, minimum toxicity, and decreased adverse effects in a patient. However, and despite a strong mechanistic foundation, only 0.16 % of ongoing clinical trials worldwide exploit the concept of 'time-of-day' administration to develop safer and more effective therapies. In this article, we (1) emphasize points of control at which circadian biology intersects critical processes governing treatment interventions; (2) explore the extent to which chronotherapeutics are incorporated into clinical trials; (3) recognize roadblocks; and (4) recommend approaches to precipitate the integration of chronobiological concepts into clinical practice.

Association of the circadian factor Period 2 to p53 influences p53's function in DNA-damage signaling.

Circadian period proteins influence cell division and death by associating with checkpoint components, although their mode of regulation has not been firmly established. hPer2 forms a trimeric complex with hp53 and its negative regulator Mdm2. In unstressed cells, this association leads to increased hp53 stability by blocking Mdm2-dependent ubiquitination and transcription of hp53 target genes. Because of the relevance of hp53 in checkpoint signaling, we hypothesize that hPer2 association with hp53 acts as a regulatory module that influences hp53's downstream response to genotoxic stress. Unlike the trimeric complex, whose distribution was confined to the nuclear compartment, hPer2/hp53 was identified in both cytosol and nucleus. At the transcriptional level, a reporter containing the hp21(WAF1/CIP1) promoter, a target of hp53, remained inactive in cells expressing a stable form of the hPer2/hp53 complex even when treated with γ-radiation. Finally, we established that hPer2 directly acts on the hp53 node, as checkpoint components upstream of hp53 remained active in response to DNA damage. Quantitative transcriptional analyses of hp53 target genes demonstrated that unbound hp53 was absolutely required for activation of the DNA-damage response. Our results provide evidence of the mode by which the circadian tumor suppressor hPer2 modulates hp53 signaling in response to genotoxic stress.

The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells.

Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanism that is responsible for generating the negative feedback loop that sustains the clock. Its relevance to human disease is underlined by alterations in its function that affect numerous biochemical and physiological processes. When absent, it results in the development of various cancers and an increase in the cell's susceptibility to genotoxic stress. Thus we sought to define a yet-uncharacterized checkpoint node in which circadian components integrate environmental stress signals to the DNA-damage response. We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53's negative regulator, Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from being ubiquitinated and targeting hp53 by the proteasome. Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes. Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

Regulatory pathways coordinating cell cycle progression in early Xenopus development.

The African clawed frog, Xenopus laevis, is used extensively as a model organism for studying both cell development and cell cycle regulation. For over 20 years now, this model organism has contributed to answering fundamental questions concerning the mechanisms that underlie cell cycle transitions--the cellular components that synthesize, modify, repair, and degrade nucleic acids and proteins, the signaling pathways that allow cells to communicate, and the regulatory pathways that lead to selective expression of subsets of genes. In addition, the remarkable simplicity of the Xenopus early cell cycle allows for tractable manipulation and dissection of the basic components driving each transition. In this organism, early cell divisions are characterized by rapid cycles alternating phases of DNA synthesis and division. The post-blastula stages incorporate gap phases, lengthening progression, and allowing more time for DNA repair. Various cyclin/Cdk complexes are differentially expressed during the early cycles with orderly progression being driven by both the combined action of cyclin synthesis and degradation and the appropriate selection of specific substrates by their Cdk components. Like other multicellular organisms, chief developmental events in early Xenopus embryogenesis coincide with profound remodeling of the cell cycle, suggesting that cell proliferation and differentiation events are linked and coordinated through crosstalk mechanisms acting on signaling pathways involving the expression of cell cycle control genes.

Phosphorylation of Claspin is triggered by the nucleocytoplasmic ratio at the Xenopus laevis midblastula transition.

At the Xenopus midblastula transition (MBT), cell cycles lengthen, and checkpoints that respond to damaged or unreplicated DNA are established. The MBT is triggered by a critical nucleocytoplasmic (N/C) ratio; however, the molecular basis for its initiation remains unknown. In egg extracts, activation of Chk1 checkpoint kinase requires the adaptor protein Claspin, which recruits Chk1 for phosphorylation by ATR. At the MBT in embryos, Chk1 is transiently activated to lengthen the cell cycle. We show that Xenopus Claspin is phosphorylated at the MBT at both DNA replication checkpoint-dependent and -independent sites. Further, in egg extracts, Claspin phosphorylation depends on a threshold N/C ratio, but occurs even when ATR is inhibited. Not all phosphorylation that occurs at the MBT is reproduced in egg extracts. Our results identify Claspin as the most upstream molecule in the signaling pathway that responds to the N/C ratio and indicate that Claspin may also respond to an independent timer to trigger the MBT and activation of cell cycle checkpoints.

Near-infrared laser delivery of nanoparticles to developing embryos: a study of efficacy and viability.

Targeted delivery of materials to individual cells remains a challenge in nanoscience and nanomedicine. Near infrared (NIR) laser injection may be a promising alternative to manual injection (where the micropipet diameter limits targeting to small cells) or other laser techniques (such as picosecond green and UV lasers, which can be damaging to cells). However, the efficiency with which NIR pulses can deliver nanoparticles and any adverse effects on living cells needs thorough testing. Toward this end, we have determined the efficacy and toxicity of delivering quantum dots (QDs) into cells of Xenopus laevis embryos by NIR laser injection. Because this model system provides not only living cells but also a developing organism, we were able to assess relatively long-term effects of NIR pulses on embryonic development (through the tadpole stage). We developed parameters for NIR pulses that did not affect embryonic viability or morphology and delivered QDs as effectively as manual injection. Higher intensities of NIR pulses caused permanent damage to the targeted cells, and thus NIR pulses may also prove useful for ablation of specific cells within tissues.

Cyclin E2 is required for embryogenesis in Xenopus laevis.

In mammalian cells, E-type cyclins (E1 and E2) are generally believed to be required for entry into S phase. However, in mice, cyclin E is largely dispensable for normal embryogenesis. Moreover, Drosophila cyclin E plays a critical role in cell fate determination in neural lineages independently of proliferation. Thus, the functions of cyclin E, particularly during early development, remain elusive. Here, we investigated the requirement for E-type cyclins during Xenopus embryogenesis. Although cyclin E1 has been reported as a maternal cyclin, inhibition of its translation in the embryo caused no serious defects. We isolated a Xenopus homologue of human cyclin E2, which was zygotically expressed. Sufficient inhibition of its expression led to death at late gastrula, while partial inhibition allowed survival. These observations indicate distinct roles for Xenopus cyclins E1 and E2, and an absolute requirement of cyclin E2 for Xenopus embryogenesis.

NESH (Abi-3) is present in the Abi/WAVE complex but does not promote c-Abl-mediated phosphorylation.

Abl interactor (Abi) was identified as an Abl tyrosine kinase-binding protein and subsequently shown to be a component of the macromolecular Abi/WAVE complex, which is a key regulator of Rac-dependent actin polymerization. Previous studies showed that Abi-1 promotes c-Abl-mediated phosphorylation of Mammalian Enabled (Mena) and WAVE2. In addition to Abi-1, mammals possess Abi-2 and NESH (Abi-3). In this study, we compared the three Abi proteins in terms of the promotion of c-Abl-mediated phosphorylation and the formation of Abi/WAVE complex. Although Abi-2, like Abi-1, promoted the c-Abl-mediated phosphorylation of Mena and WAVE2, NESH (Abi-3) had no such effect. This difference was likely due to their binding abilities as to c-Abl. Immunoprecipitation revealed that NESH (Abi-3) is present in the Abi/WAVE complex. Our results suggest that NESH (Abi-3), like Abi-1 and Abi-2, is a component of the Abi/WAVE complex, but likely plays a different role in the regulation of c-Abl.

Emi1-mediated M-phase arrest in Xenopus eggs is distinct from cytostatic factor arrest.

Oocytes of most vertebrates arrest at metaphase of the second meiosis (meta-II) to await fertilization, thus preventing parthenogenetic activation. This arrest is caused by a cytoplasmic activity called cytostatic factor (CSF), which was first identified in the frog Rana pipiens oocyte >30 years ago. CSF arrest is executed by maintaining the activity of cyclin B-Cdc2 at elevated levels largely through prevention of cyclin B destruction. Although CSF arrest is established by the Mos-mitogen-activated protein kinase pathway and is released by the Ca-calmodulin kinase II pathway, it remains unclear precisely how cyclin B destruction is regulated. Recently, an early mitotic inhibitor, Emi1, was reported to be a critical component of CSF. This report has been expected to provide a final resolution to the CSF problem because Emi1 inhibits the anaphase-promoting complex/cyclosome, a ubiquitin ligase for cyclin B destruction, through sequestration of Cdc20, an activator for the anaphase-promoting complex/cyclosome. In mitotic cycles, however, Emi1 is destroyed in every pro-metaphase, and accordingly, it is unclear why Emi1 should be required for CSF activity, which is seen only in meta-II. Here, we show that Emi1 is absent in unfertilized mature Xenopus eggs and that exogenous Emi1 is destroyed in meta-II and mitotic metaphase. The expression of Emi1 in oocytes hinders meiotic progression. Although both Emi1 and Mos can inhibit progression through M phase, the Emi1-mediated arrest does not require mitogen-activated protein kinase activity and is not released by Ca. Together, our results indicate that Emi1 is unlikely to be a component of CSF.