Setting the cellular clock

Many organisms live out their lives on schedules established by internal clock mechanisms, generated by the combined action of multiple regulatory networks that interlock like gears in a watch. The resulting circadian rhythms establish one’s internal perception of day and night, as well as numerous time-points in between.

In 2005, a team led by Hiroki Ueda of the RIKEN Center for Developmental Biology in Kobe made significant progress in identifying the core components of the complex circadian circuitry¹. They found several regulatory elements that specifically mark genes for activation or inhibition in the morning, daytime or night, as well as numerous genes that mediate regulation via these elements.

“Our team identified a natural transcriptional circuit for mammalian circadian clocks,” explain Maki Ukai-Tadenuma and Takeya Kasukawa, members of Ueda’s team. “However, no one has yet confirmed the mechanism that generates practically continuous phases from these three, discrete basic phases.”

However, the investigators had ideas about how such patterns might emerge, and were able to sketch a rough map of how the various time-specific regulatory loops may interact in vivoto produce a stable day–night cycle. To test their hypotheses, they constructed a series of synthetic circadian circuits within live cells based on their models, and examined the extent to which their activity replicated natural biological cycles².

In fact, these experimental scenarios provided strong support for their regulatory models. One of the synthetic circuits consisted of a bioluminescent indicator gene under the regulation of a morning-specific activator and a nighttime-specific repressor, and the resulting pattern of indicator activity was a cyclic oscillation that very closely matches the natural expression pattern of daytime-specific genes.

They were similarly able to replicate night-cycle activity with a daytime-specific activator and morning-specific repressor, and were even able to generate novel ‘late night’ or ‘dusk’ output peaks by further tinkering with the timing of activation and repression. Most importantly, their experimental findings were all consistent with the predictions of their previously developed theoretical models. “Both our simulation model and the derived design principles successfully recapitulated the natural transcriptional circuit in the circadian clocks,” say the researchers.

Although their modeling system has proven effective, the researchers have yet to fully reconstruct all the phases of the mammalian circadian cycle. “In our study, morning transcriptional regulation is still a ‘missing link’,” they point out. The team’s focus now is on successfully identifying the regulatory processes required to restore these final time-points to their reconstructed cellular clock.