Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells.

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

A robust ultrasensitive transcriptional switch in noisy cellular environments.

Ultrasensitive transcriptional switches enable sharp transitions between transcriptional on and off states and are essential for cells to respond to environmental cues with high fidelity. However, conventional switches, which rely on direct repressor-DNA binding, are extremely noise-sensitive, leading to unintended changes in gene expression. Here, through model simulations and analysis, we discovered that an alternative design combining three indirect transcriptional repression mechanisms, sequestration, blocking, and displacement, can generate a noise-resilient ultrasensitive switch. Although sequestration alone can generate an ultrasensitive switch, it remains sensitive to noise because the unintended transcriptional state induced by noise persists for long periods. However, by jointly utilizing blocking and displacement, these noise-induced transitions can be rapidly restored to the original transcriptional state. Because this transcriptional switch is effective in noisy cellular contexts, it goes beyond previous synthetic transcriptional switches, making it particularly valuable for robust synthetic system design. Our findings also provide insights into the evolution of robust ultrasensitive switches in cells. Specifically, the concurrent use of seemingly redundant indirect repression mechanisms in diverse biological systems appears to be a strategy to achieve noise-resilience of ultrasensitive switches.

Combined multiple transcriptional repression mechanisms generate ultrasensitivity and oscillations.

Transcriptional repression can occur via various mechanisms, such as blocking, sequestration and displacement. For instance, the repressors can hold the activators to prevent binding with DNA or can bind to the DNA-bound activators to block their transcriptional activity. Although the transcription can be completely suppressed with a single mechanism, multiple repression mechanisms are used together to inhibit transcriptional activators in many systems, such as circadian clocks and NF-κB oscillators. This raises the question of what advantages arise if seemingly redundant repression mechanisms are combined. Here, by deriving equations describing the multiple repression mechanisms, we find that their combination can synergistically generate a sharply ultrasensitive transcription response and thus strong oscillations. This rationalizes why the multiple repression mechanisms are used together in various biological oscillators. The critical role of such combined transcriptional repression for strong oscillations is further supported by our analysis of formerly identified mutations disrupting the transcriptional repression of the mammalian circadian clock. The hitherto unrecognized source of the ultrasensitivity, the combined transcriptional repressions, can lead to robust synthetic oscillators with a previously unachievable simple design.

Systematic modeling-driven experiments identify distinct molecular clockworks underlying hierarchically organized pacemaker neurons.

In metazoan organisms, circadian (∼24 h) rhythms are regulated by pacemaker neurons organized in a master-slave hierarchy. Although it is widely accepted that master pacemakers and slave oscillators generate rhythms via an identical negative feedback loop of transcription factor CLOCK (CLK) and repressor PERIOD (PER), their different roles imply heterogeneity in their molecular clockworks. Indeed, in Drosophila, defective binding between CLK and PER disrupts molecular rhythms in the master pacemakers, small ventral lateral neurons (sLNvs), but not in the slave oscillator, posterior dorsal neuron 1s (DN1ps). Here, we develop a systematic and expandable approach that unbiasedly searches the source of the heterogeneity in molecular clockworks from time-series data. In combination with in vivo experiments, we find that sLNvs exhibit higher synthesis and turnover of PER and lower CLK levels than DN1ps. Importantly, light shift analysis reveals that due to such a distinct molecular clockwork, sLNvs can obtain paradoxical characteristics as the master pacemaker, generating strong rhythms that are also flexibly adjustable to environmental changes. Our results identify the different characteristics of molecular clockworks of pacemaker neurons that underlie hierarchical multi-oscillator structure to ensure the rhythmic fitness of the organism.