The repetitive activity pattern of all life on earth follows the daily cycle of sunrise and sunset and is controlled by an endogenous biological clock composed of a network of interactive molecules. For the biological clock to exert its control over the fundamental activities of life, the molecules in the clock network must interact with molecules of other functional networks, such as signaling networks that control feeding, sleep-awake cycle, growth and metabolic regulation, though these interactions remain to be elucidated. Our recent investigations have clearly established direct interactions between PER2, a key regulatory molecule in the clock network, and promyelocytic leukemia protein (PML), which was initially characterized as a tumor suppressor protein responsible for acute promyelocytic leukemia (APL). Currently PML is known as the central component of a large nuclear protein complex, PML nuclear bodies (PML NBs), where PML is suggested to act as a "recruiter and organizer" for nuclear proteins from various functional networks. At the core of the circadian clock mechanism, nuclear proteins BMAL1 and CLOCK form the heterodimers that drive the transcriptional expressions of the key clock regulators, including Per and Cry genes, as well as many clock output genes. We found PML not only interacts directly with PER2 in the nucleus, it also facilitates the process of PER2 nuclear localization. Recently, BMAL1 was also found to colocalize with PML NBs. These lines of evidence, led us to the hypothesis that PML plays the role of "recruiter and organizer" for the nuclear regulatory network of the circadian clock mechanism. Here we propose to investigate the "recruiter and organizer" function of PML in the circadian clock mechanism by constructing a profile of subcellular distributions and interactions of various clock regulators and PML. We propose to carry out in vitro and in vivo experiments using a combination of tools including immunofluorescence, reciprocal coimmunoprecipitation, recombinant expression constructs, genetically engineered cell lines and animal models. Our recent studies have revealed that the acetylation status of PML affects its interaction with PER2. We have undertaken a small molecule screen and identified a compound, which we have renamed PINEF, that is capable of facilitating PER2 nuclear entry in the absence of PML. We also propose to study how the status of PML acetylation and the actions of PINEF in the absence of PML may alter the profile of subcellular distributions and interactions of various clock regulators. These proposed studies will result in a more comprehensive spatiotemporal profile of the distribution and interactions of key clock proteins, including BMAL1, CLOCK, PERs, CRYs, and PML. The findings will contribute to a better understanding of the mammalian circadian clock mechanism. In addition, as dozens of nuclear proteins involved in many different cellular pathways have been localized in PML NBs, this study will help to further understand if and how PML contributes to the connections among different functional networks, including the circadian clock regulatory network.
The biological clock within our body controls our daily physiological rhythms including our sleep-wake cycle, hormonal release and many other biological processes that are critical to our health. The proposed study will help us to further th understanding of how the biological clock works and how the clock mechanism is connected to functioning of other life processes, such as normal and abnormal cell growth, including tumorigenesis.