Tumorigenesis is the result of uncontrolled cell growth due to the deregulation of cell cycle checkpoints 1. Period 2 (Per2) is a tumor suppressor that oscillate in expression in a 24-hour cycle 2, 3. Here, we show that Per2 interacts with the tumor suppressor protein p53. Both G1 and G2 checkpoint pathways involve a p53 dependent pathway which can trigger the cell to go through cell arrest or programmed cell death4. Understanding all the mitigating factors involved in regulating cell cycle progression under DNA damage can offer a better idea in how cells become immortal. Initially discovered through screening of a human liver cDNA library, the novel interaction between p53-Per2 was further documented using co-precipitation. Interestingly, under genotoxic stress conditions, p53 and Per2 were not found to bind which leads us to suspect that Per2 does not affect active p53 which may possibly be due to post translational modifications of its active state. Furthermore we investigated p53's ability to act as a transcription factor in the presence of Per2, showing that the Per2-p53 complex prevents p53 from binding to DNA. This implies that the tetramerization of p53 may also be another factor in Per2's ability to bind to p53. A truncated p53 lacking the last 30 amino acids that theoretically increase p53's ability to form a tetramer showed a drastic reduction in binding to Per2 5, 6. On the other hand, p53 lacking the tetramerization domain showed binding similar to wildtype. Consequently we speculate that the ability of Per2 to modulate p53 and act as a tumor suppressor protein may be dependent on either the post translational modifications of p53 or its oligomeric state. / Master of Science

Circadian rhythms are self-sustained, 24h, biological oscillatory processes that are present in organisms ranging from bacteria to human. Circadian rhythms, which can be synchronized by external cues, are important for organisms to adjust their behavior, physiological activity, and metabolic reactions to changes in environmental conditions. Another well-established oscillatory mechanism that shares common organizational and regulatory features with the circadian system, is the cell division cycle. Recent findings reveal that some essential regulators are common to both the cell cycle and the circadian clock. The first half of my thesis (Chapter 2-3) focuses on the function of Period 2 (Per2), a key regulatory component of the negative feedback arm of the clock and tumor suppressor protein, as a modulator of cell cycle response. We found that Per2 binds the C-terminus end of the tumor suppressor p53 thus forming a trimeric complex with p53's negative regulator Mdm2 and preventing Mdm2-mediated p53's ubiquitination and degradation. Thus, Per2 stabilizes p53 under unstressed conditions allowing for basal levels of the protein to exist and be available for a rapid response to take place in case of any stressed signals. Our experiments prove that Per2 plays an indispensible role in p53 signaling pathway. The second half of my thesis (Chapter 4-5) focuses on how Mdm2 and Per2 interplay regulate Per2 availability and its impact on circadian clock function. My research found that Mdm2 targets Per2 for ubiquitination as Mdm2 depletion stabilizes Per2 and, conversely, Mdm2 ectopic expression shorten Per2's half-life. Accordingly, association of Per2 to Mdm2 maps C-terminus of the p53 binding region in Mdm2 and thus, the RING domain remains accessible. Next, we tested the hypothesis that Mdm2-dependent ubiquitination of Per2 directly impacts circadian clock period length. Accordingly, addition of sempervirine nitrate (SN), a specific molecular inhibitor of Mdm2, to MEF cells abrogated Per2 ubiquitination leading to the accumulation of a stable pool of Per2. By recording the oscillatory behavior of the Per2:Luc reporter system in MEF cells treated with SN at different circadian times, we found that inhibition of Mdm2 E3 ligase activity promoted phase advance only when treatment took place during the degradation period. This is in agreement with our findings that radiation, but not light pulses, causes the same phase behavior. Considering the established role of both Mdm2 and p53 in the response of cells to genotoxic stress and Per2 in modulating the clock, the existence of the Mdm2-Per2-p53 complex opens the possibility of various stimuli triggering regulatory mechanisms converging in a critical node. Overall, our work provides a holistic view of how signals are integrated at multiple levels to ensure that environmental signals are sense and responses triggered timely. / Ph. D.

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. / Doctor of Philosophy / Mammals have a time-keeping system that oscillates with a periodicity of about 24 h, namely the circadian clock, that allows physiological and behavioral adaptation to environmental changes. The circadian clock controls and coordinates processes as diverse as sleep/wake cycle, feeding cycle, daily changes in body temperature, blood pressure and hormone secretion. At the cellular level, the circadian clock exists in almost all cells and controls a broad range of cellular signaling pathways including mechanisms involved in cell division and DNA damage response (DDR) pathway. Circadian disruption, for example, by night shift work, results in accumulation of DNA damage in cells and increases risk of cancer. In my thesis, we found that MDM2, a protein that is involved in the DDR signaling pathway and has the potential to cause cancer, controls the degradation of the core clock protein PERIOD2 (PER2), and thus regulates the length of circadian period. Further work exposed the mechanism for how DNA damage shifts the circadian clock. Our findings will have significant impacts on health and biomedical science, especially shedding light on optimizing the time in a day to give chemo- and radiation therapies to cancer patients.

The circadian clock is a molecular mechanism that synchronizes physiological changes with environmental variations. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, and cancer). Period 2 (Per2), a circadian protein, is at the center of the clock's function. The loss or deregulation of per2 has been shown to be common in several types of cancer including breast and ovarian [1, 2]. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERα) mRNA oscillates in a 24-hour period and, unlike Per2, ERα peaks during the light phase of the day. Because up regulation of ERα relates to tumor development, defining the mechanisms of ERα expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of ERα transcription. Transcriptional activation of ERα was measured using a reporter system in Chinese hamster ovary (CHO) cell lines. Data show that Per2 influences ERα transcription through a non-canonical mechanism independent of its circadian counterparts. Breast cancer susceptibility protein 1 (BRCA1) was confirmed to be an interactor of Per2 via bacterial two-hybrid assays, in accordance with previous studies [2]. BRCA1 is a transcriptional activator of ERα promoter in the presence of octamer transcription factor-1 (OCT-1) [3]. Our results indicate that the DNA binding domain of OCT-1, POU, to directly interact with Per2 and BRCA1, in vitro. Pull-down assays were used to map direct interaction of various Per2 and BRCA1 recombinant proteins and POU. Chromatin immunoprecipitation assays confirmed the recruitment of PER2 and BRCA1 to the estrogen promoter by OCT-1 and the recruitment of Per2 to the ERα promoter decreases ERα mRNA expression levels in MCF-7 cells. Our work supports a circadian regulation of ERα through the repression of esr1 by Per2 in MCF-7 cells. / Ph. D.

Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism. The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled. The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior. Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus. / Ph. D.