UNLABELLED: Theproto-oncogene regulates cellular proliferation, and its aberrant expression drives a range of human cancers. It also has a bidirectional regulatory relationship with the mammalian core circadian clock, with emerging evidence suggesting that MYC overexpression leads to clock disruption and loss of rhythms. While prior studies have probed MYC's role in clock disruption by overexpressing or mutating thegene, our understanding of the endogenous nature ofis limited. A major gap in knowledge is whether MYC itself is expressed rhythmically and if so, how its timing relates to that of core clock components. To address these shortcomings, we generated areporter and assessed its circadian nature, comparing it toand, and developed a computational model based on these and previous findings to evaluate its role(s). We developed lentiviral constructs for and established a U2OS (common circadian model) reporter cell line expressing luciferase () driven by a human-derivedpromoter sequence. To facilitate comparisons, as part of this work, we also developed a human-sequence derivedpromoter reporter to more readily recapitulate its behaviors. Using luminometry studies and subsequent data analyses, we demonstrated that thepromoter oscillated rhythmically in U2OS cells, which possess inherently low levels of. Furthermore, we found thatoscillates out-of-phase relative toand. Using this information, we built a mathematical model to better understand how's oscillations at both basal and over-expressed levels affect the clock and vice versa. The model reproduced expected alterations to the core clock resulting fromoverexpression and showed that MYC's role is as a disruptor, although the timing of MYC regulation can minimize its negative impact(s) on circadian timekeeping. This work is the first to assess's phase relationships relative to the core clock and to provide evidence for its circadian nature. c-MYC c-MYC c-MYC c-MYC BMAL1PER2luc c-MYC BMAL1c-MYC c-MYC c-MYC BMAL1PER2c-MYC c-MYC c-MYC
AUTHOR SUMMARY: is a transcription factor that is highly regulated and plays an important role in cellular proliferation. In cancers, deregulation ofcauses its overexpression, resulting in tumorigenesis. There have been multiple connections demonstrated between MYC and the circadian clock, including the clock's role in MYC expression and that its overexpression can lead to disruptions to the core circadian clock. However, knowledge of the expression patterns of MYC are limited, including whether they occur in a circadian manner. To address this, we developed aluciferase reporter in a human circadian cell model (U2OS). For the first time, we were able to directly assess the rhythmic nature ofusing this tool. Subsequently, we developed a mathematical model to gain insights into the disruptive role of MYC in clock regulation under disease-like conditions and, in turn, the effects of the circadian clock on MYC. We found thatoscillated in a circadian manner in U2OS cells and that the MYC protein's role is as a disruptor, but its timing can minimize its negative impact(s) on circadian rhythms. c-MYC c-MYC c-MYC-c-MYC c-MYC
