Browsing by Author "Zou, Xianlin"
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- Interlocking mechanisms regulating the circadian clock response to DNA damageZou, Xianlin (Virginia Tech, 2021-06-15)Almost all organisms have an endogenously generated and self-sustained time-keeping system that oscillates with a periodicity of about 24 h, namely the circadian clock, that help them adapt to daily environmental changes. Mammalian circadian rhythms are generated and maintained by transcription-translation feedback loops (TTFLs) and include post-translational modifications to help fine-tune the oscillation. Circadian rhythms control a broad range of cellular signaling pathways including those mechanisms involved in cell division and DNA damage response (DDR). We have previously established that the core clock component PERIOD2 (PER2) binds to the tumor suppressor protein p53, a key regulatory checkpoint component that modulates cell cycle progression and the cellular response to genotoxic stress. PER2 binding to p53 modulates p53's stability, cellular localization, and transcriptional activity. As described in Chapter 2, we now identified PER2 as a previously uncharacterized substrate for the ubiquitin E3 ligase mouse double minute 2 homolog (MDM2), an oncoprotein and negative regulator of p53. Our findings showed that the association between PER2 and MDM2 is independent of the presence of p53. In addition, MDM2 targets PER2 for ubiquitylation and degradation in a phosphorylation-independent fashion. Lastly, our studies showed that MDM2 collaborates with β-transducin repeat-containing proteins (β-TrCPs), an E3 ligase that targets PER2 for ubiquitylation in a phosphorylation-dependent manner, to control PER2 degradation and thus the length of circadian period. Because the p53:MDM2 pathway plays a critical role in the cellular response to genotoxic stress, the project described in Chapter 3 is based on the hypothesis that DNA damage caused by radiation shifts the circadian clock phase via the p53:PER2:MDM2 complex. Firstly, we generated Trp53KO (Trp53 gene encodes mouse p53) cell lines in NIH 3T3 Per2:dLuc reporter cells expressing luciferase driven by the Per2 promoter. Phase-response curves (PRCs) for Trp53WT and Trp53KO reporter cells were obtained in response to ionizing radiation (IR) treatments. Results indicated that Trp53 knockout did not affect radiation-induced circadian phase shifts, whereas increased p53 levels induced by transient inhibitor treatments prevented phase shifts when IR was performed at the trough of PER2 abundance. Additional mechanisms were unveiled that kinases ATM (Ataxia Telangiectasia Mutated), ATR (ATM- and Rad3-related) and CHK2 (Checkpoint Kinase 2) regulate radiation-induced phase shifts. Lastly, we found that CLOCK (Circadian Locomotor Output Cycles Kaput) and CRY1 (CRYPTOCHROME 1) were phosphorylated in response to radiation. Taken together, these results indicate that radiation-induced clock phase shifts involve the activity of kinases ATM, ATR and CHK2, and the modification in CLOCK and CRY1. Chapter 4 is a review of current findings about the interaction between circadian rhythms and the cell division cycle regulation pathway. The article highlights a multidisciplinary approach that combines mathematical modeling and experimental data to reveal how p53:PER2:MDM2 acts as a node controlling timely cell cycle progression. In summary, our work provided evidence that MDM2 targets PER2 for ubiquitylation and degradation in a phosphorylation-independent manner, and this influences circadian oscillation. Furthermore, the exploration of p53:PER2:MDM2 association shed light on how radiation-induced DNA damage shifts clock phase. These findings expose a crosstalk mechanism that senses DNA damage and shifts the clock system.
- A Systems Biology Approach Identifies Hidden Regulatory Connections Between the Circadian and Cell-Cycle CheckpointsZou, Xianlin; Kim, Dae Wook; Gotoh, Tetsuya; Liu, Jingjing; Kim, Jae Kyoung; Finkielstein, Carla V. (Frontiers Media, 2020-04-16)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.