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Re-evaluation of the circadian clock model in mammals

PADLOM, Apirada 名古屋大学

2023.01.31

概要

Circadian rhythms are biological oscillations of physical, mental, and behavioral activities with a period of approximately 24 hours driven by an endogenous cell - autonomous timing system called the circadian clock. The current molecular models of the mammalian circadian clocks are based on a molecular mechanism regulated by a transcriptional-translational negative feedback loop (TTFL) in which the translational products of clock genes repress transcription of their own mRNA. Consistent with this model, transcriptional and translational products of Period, Cryptochrome, and Bmal1 show circadian rhythm in their accumulation. However, several studies have revealed that constitutively expressed clock genes effectively restore circadian oscillations. To understand this point more quantitatively, I expressed Bmal1 from a doxycycline (DOX)-inducible promoter in Bmal1-disrupted U2OS cells containing a luciferase reporter under the control of the Bmal1 promoter (PBmal1 ), and followed the PBmal1 and PPer2 promoter activities. In the presence of 0.1 and 1 μg/mL of DOX, constitutively expressed BMAL1 restored circadian oscillation in PBmal1 and PPer 2 promoter activities as well as the antiphase relationship between PBmal1 and PPer 2 oscillations, although the level of BMAL1 and other clock proteins, REV-ERBα and CLOCK showed no clear rhythmicity. I applied a transient response analysis to P Bmal1 luminescence data in the presence of various concentrations of doxycycline and found that a slightly damped linear oscillator system can reproduce P Bmal1 promoter activity. The oscillation parameters were not dramatically impacted by the levels of Bmal1 expression, however, the behavior of the baseline of oscillations was greatly impacted. Based on the obtained transfer functions, this study suggests that BMAL1 is not directly invol ved in the oscillatory process but modulates the robustness of the oscillations by regulating the basal activity of the clock gene promoter.

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