Analysis of circadian clock PERIOD complexes in the cytoplasm

• Applicant: Dr. Michael Gebert
• Subject Area: Cell Biology
• Term: from 2013 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 235474185

Molecular clocks are found in virtually any cell of mice or humans and govern daily rhythms of physiology, metabolism and behavior. At least ten percent of all genes in a mammalian cell are circadianly regulated, which is even more impressive considering that the set of cycling genes can be distinct in different cell types. Essential cellular processes like the cell cycle, DNA damage response or cellular senescence are coordinated by the circadian clock and disruptions can have fatal consequences. Cancer, cardiovascular abnormalities, immune defects, and obesity are only a few examples.The underlying basic clock mechanism is the same in all cell types and comprises a transcription translation feedback loop generating self-sustaining rhythms with an intrinsic period close to 24 hours. In this cycle the heterodimeric transcription factor BMAL1-CLOCK is responsible for the activation of the circadian transcriptome. Among the regulated genes are the Per and Cry genes encoding for the negative elements in the loop. After translation PER and CRY proteins reside in the cytoplasm for several hours before they transfer into the nucleus and repress the activity of BMAL1-CLOCK. The cycle can restart after the turnover of the nuclear PER and CRY proteins.The cytoplasmic retention of PER and CRY proteins is an essential feature of the mammalian circadian clock. The delayed repression enables oscillations in the system and is a major determinant of the period length. How this cytoplasmic accumulation is achieved is still a fundamental open question.Clearly other proteins are involved this process and consequently the goal of my proposal is to identify and characterize these as-yet unknown factors involved in the extra-nuclear phase of PER proteins. In a first step I will isolate cytoplasmic PER-containing protein complexes from mouse liver tissue at the appropriate time in the cycle by immunoaffinity purification. For this purpose I can use mouse strains in the lab in which the endogenous copy of a PER protein was replaced by a fusion protein with FLAG-Hemagglutinin. The eluates derived from the livers containing fusion proteins will be compared to the corresponding wildtype control by SDS-PAGE. Bands exclusively visible in the fusion protein eluates will be analyzed by mass spectrometry. The role of candidate proteins for the clock will be assessed by loss-of-function studies in circadian reporter cell lines carrying luciferase transgenes. By measuring real-time bioluminescence traces I will be able to obtain information about the period length and oscillation behavior of the clock in the absence of a protein of interest. The further characterization of newly identified PER interactors will depend on the nature of the identified proteins.Altogether the proposed project is focused on an elementary characteristic of the mammalian clock enabling important findings for a better understanding of the basic molecular mechanism of circadian rhythms.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Charles J. Weitz