jingjing liu

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

Period 2 forms a trimeric complex with p53 and Mdm2. As a result, p53’s transcriptional activity and stability are modulated in unstressed cells, ensuring that basal levels are present if a p53-mediated response is needed. These data provide evidence of cross-talk between circadian and checkpoint components, adding a level of regulation to the checkpoint.

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

Significance Cells sense changes in environmental conditions and translate them into physiological responses that are mediated by a plethora of molecular intermediaries that converge at critical nodes. This is particularly relevant to multiple levels of biological organization in which the circadian clock and cell division are interlocked. Unexpectedly, the circadian rhythms of two critical components of the interlocked system, Period 2 (Per2) and p53, were in antiphase. This finding was analyzed using a systematic modeling approach, and predictions of the model were validated by experimental studies that revealed a distinct and complex scenario. Accordingly, we show that the spatiotemporal organization of Per2:p53 interactions and the nature of their chemical modifications are critical for their time-dependent subcellular redistribution and potential biological functions. 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.

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

Association of the circadian Per2 factor to p53 results in cytosol–nuclear shuttling of the complex and further association to Mdm2. The trimeric complex remains in the nucleus until a genotoxic signal frees p53, allowing for a transcriptional checkpoint response.

Ligand binding reveals a role for heme in translationally-controlled tumor protein dimerization.

The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a “buffer protein” controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His76His77) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTPs cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTPs proposed buffer function, ligand binding occurs at high concentration, allowing the “buffer” condition to be dissociated from TCTPs role as a component of signal transduction mechanisms.

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

The circadian clock relies on post-translational modifications to set the timing for degradation of core regulatory components and, thus, sets clock progression. Ubiquitin-modifying enzymes targeting clock components for degradation are known to mostly recognize phosphorylated substrates. A case in point is the circadian factor PERIOD 2 (PER2) whose phospho-specific turnover involves its recognition by β-transducin repeat containing proteins (β-TrCPs). Yet, the existence of this unique mode of regulation of PER2’s stability falls short of explaining persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. In this study, we challenge the phosphorylation-centric view that PER2 degradation enhances circadian rhythm robustness by i) identifying the PER2:MDM2 endogenous complex, ii) establishing PER2 as a previously uncharacterized substrate for MDM2, iii) revealing an alternative phosphorylation-independent mechanism for PER2 ubiquitin-mediated degradation, iv) pinpointing residues for ubiquitin modification, and v) establishing the importance of MDM2-mediated PER2 turnover for defining the circadian period length. Our results not only expand MDM2’s suite of specific substrates beyond the cell cycle to include circadian components but also uncover novel regulatory players that likely impact our view of how other mechanisms crosstalk and modulate the clock itself.

Abstract 53: Interplay among E3 ubiquitin ligases regulate timely degradation of the circadian factor Period 2

Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our body to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Oscillatory rhythms are generated as result of the activation of positive and negative transcriptional feedback loops in which post-translational modifications, shuttling, and degradation are common themes. In mammals, the positive feedback loop involves regulation of bmal1 transcription. BMAL1/CLOCK selectively binds to E-box enhancers and drives the expression of per, cry and rev-erbα genes. REV-ERBa protein then represses bmal1 transcription through Rev-Erba/ROR response elements in its promoter. Then, the level of bmal1 RNA falls, as per and cry RNA levels peak. Period proteins (i.e., PER1, 2, and 3) accumulate in the cytoplasm, becomes phosphorylated by CK1e/d, ubiquitylated and degraded. Later in the day, cryptochrome accumulates (i.e., CRY2), associates with PER2/CK1e/d and this complex translocates to the nucleus where CRY disrupts the CLOCK/BMAL1-associated transcriptional complex, resulting in the inhibition of cry, per and rev-erba and de-repression of bmal1 transcription. In addition to the transcriptional loops, ubiquitin E3 ligases-mediated protein degradation is critical to sustain physiological rhythms. The F-box proteins b-TrCP1/2 target PER1 and PER2 for degradation via ubiquitination. Whereas knocking down β-trcp results in PER stabilization and period lengthening in synchronized culture cells; knock-out b-trcp1 mice do not display an abnormal circadian rhythmicity. Our findings indicate that PER2 ubiquitination engages two different types of E3 ligases, a process that directly influences the timely control of PER2 accumulation in the nucleus. Our data show that the mouse double-minute 2 homolog (Mdm2) RING finger E3 ligase binds to PER2 in multiple regions including an overlapping site with b-TrCP. We determined that PER2 is a specific substrate for Mdm2 and that PER29s polyubiquitination occurs both in vitro and in cells. Accordingly, overexpression Mdm2 results in the decrease of PER2 half-life, whereas, down-regulation of Mdm2 by siRNA increases PER2 stability. Furthermore, we found Mdm2-dependent post-translational modification of PER2 directly influences the amplitude of the circadian oscillatory response in synchronized cells and that both, b-TrCP and Mdm2 target PER2 at different circadian times. Overall, our findings support a model in which, the rate of nuclear degradation of PER2 leads to slow accumulation of the protein in the nucleus and, later in the day, to a temporal switch in which b-TrCP takes control over PER2 degradation. Thus, the interplaying between these two E3 ligases is indispensible for sustaining robust circadian oscillation. Citation Format: Carla V. Finkielstein, jingjing liu. Interplay among E3 ubiquitin ligases regulate timely degradation of the circadian factor Period 2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2015-53

Abstract 5173: The circadian factor period 2 modulates p53 stability and function in downstream signaling.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The human Period 2 (hPer2) factor is a transcriptional regulator placed at the core of the circadian clock mechanism responsible for generating the negative feedback loop that sustains the clock. Its relevance to human diseases is underlined by alterations in its function that impacts many biochemical and physiological processes and, when absent, results in the development of various cancers. The tumor suppressor role of Per2 is speculated to involve transcriptional activation of p53, altered expression of cell cycle components, and regulation of the DNA-damage response pathway. However, it is entirely unclear how Per2 operates mechanistically. First, we identified hPer2 binds the C-terminus half of human p53 (hp53) and forms a stable trimeric complex with hp53’s negative regulator, the oncogenic protein Mdm2. Second, we determined that hPer2 binding to hp53 prevents Mdm2 from ubiquitinating and targeting hp53 by the proteasome. Accordingly, downregulation of hPer2 expression directly impacts hp53 levels whereas its overexpression influences both hp53 protein stability and transcription. Furthermore, we spatially define the distribution of the trimeric complex and determine the site for processing to be located in the nucleus. Third, we establish that hp53-mediated gene transcription is influenced by the presence of hPer2. Target genes such as 14-3-3σ, hp21WAF1/CIP1, and gadd45α show a synergistic increase in expression when hPer2 and hp53 are co-expressed in a hp53-deficient background. This result is the direct consequence of hPer2 dissociation of hp53 as result of checkpoint activation as shown by studies performed using a constitutively bound form of the hPer2/hp53 complex. Overall, our findings directly place hPer2 at the heart of the hp53-mediated response by modulating its stability and controlling its function. Citation Format: Carla V. Finkielstein, Tetsuya Gotoh, Marian Vila-Caballer, carlo santos, jingjing liu, jianhua yang. The circadian factor period 2 modulates p53 stability and function in downstream signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2013-5173 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract P6-06-05: Down-regulation of the circadian factor Period 2 by the oncogenic E3 ligase Mdm2: Relevance of circadian components for cell cycle progression

Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our body to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Circadian proteins also regulate genes involved in either cell division or death; and a perturbation of the balance among these processes leads to cancer development and progression. A key aspect of cancer research is identifying new regulatory pathways involved in proliferation and differentiation of cell. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression, nor which, if any, of its components is either the ultimate effector that influences the fate of the cell. Initial experiments were devoted to identifying new interacting partners for Per2 using a two-hybrid system. Interestingly, among the positive clones analyzed was the oncogenic protein Mdm2. This result was validated by immunoprecipitation of recombinant and endogenous Per2/Mdm2 complexes from unstressed cells. Pull-down assays using tagged-expressed proteins fragments and labeled proteins were later used to map the interacting regions between Per2 and Mdm2. Our results show Mdm2 binds to the central flexible region of Per2 known to interact with various protein partners. Thus, we hypothesized that binding of Mdm2 to Per2 might act by mediating its ubiquitination and therefore altering Per2 stability. We next examined the formation of the Mdm2/p53/Per2 complex by immunoprecipitation. Our data show anti-p53 antibody is able to co-immunoprecipitate Per2 and Mdm2. Moreover, in vitro and in vivo ubiquitination assays show that binding of Per2 to p53 prevented ubiquitination of p53 by Mdm2 without altering their binding. Immunofluorescence studies using H1299 cells (p53-) confirmed Per2 role in p53 stabilization and for localization. Overall our results suggest that Mdm2 modulates the stability of Per2 and p53 in unstressed cells, and might be responsible for the oscillatory levels of these proteins observed in a 24 h cycle. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-05.