Assessing the contribution of transcriptional and posttranslational mechanisms in the clockworks: understanding circadian period and phase via CK1 dissections, and transcriptional rewiring.

In the fungus Neurospora crassa, the circadian core-oscillator is composed of the positive element (White Collar Complex) that commands the expression of the negative element frequency (frq). As FRQ is made, it inhibits the WCC thus shutting down its own expression. CK1, as in other circadian systems, plays a pivotal role by progressively phosphorylating the negative element and setting the pace of the clock. To obtain new insights into how period is determined, we have systematically analyzed the effect of diverse mutations in FRQ and CK1: this has allowed us to register a wide range of periods, as long as ~60, and as short as 11 hours!

Casein Kinase 1 (CK1) is an essential enzyme participating in a plethora of cellular processes. Importantly, CK1 also plays a pivotal role in all eukaryotic circadian systems described to date. The enzyme can modulate the circadian period and is involved in temperature compensation, making of this protein a crucial regulatory hotspot for clock-related inputs. It is hypothesized that regulatory aspects of CK1's general activity rely on the interaction of its unstructured C-terminal tail with the active domain of the kinase. However, such mechanisms in the context of circadian clocks remain unclear. We’ve explored the regulatory motives of CK1 by dissecting the enzyme structure and analyzing circadian parameters. Altogether, our results imply that CK1's C-terminal tail encodes for an essential autoregulatory structure and may provide yet uncharacterized mechanism to actively modulate period upon certain input stimuli.

In addition, we have found -through transcriptional rewiring strategies- that altering transcriptional aspects of the core-oscillator circuitry has rather limited impact on period, yet has major effects on phase determination. Indeed, we have obtained semi-synthetic oscillators that while maintaining period and temperature compensation, they exhibit a revert phase-logic, as they work as “lights-on” instead of a lights-off timer.

Moreover, while evaluating the topology of alternative core-clock circuitries, we observed a fascinating phenomenon: the emergence of ultradian rhythms that appear as subharmonics of a WT period. Our analyses uncover that these ultradian rhythms have a period highly dependent on kinase activity, particularly CK1, while the molecular characteristics of FRQ only appear to have a partial effect. Thus, these results highlight the relevance of CK1 in period determination, while our evidence also indicates CK1’s role in ultradian rhythms, which are controlled by a, yet, unknown molecular oscillator.