Period (PER) protein phosphorylation is a critical regulator of circadian period, yet an integrated understanding of the role and interaction between phosphorylation sites that can both increase and decrease PER2 stability remains elusive. Here, we propose a phosphoswitch model, where two competing phosphorylation sites determine whether PER2 has a fast or slow degradation rate. This mathematical model accurately reproduces the three-stage degradation kinetics of endogenous PER2. We predict and demonstrate that the phosphoswitch is intrinsically temperature sensitive, slowing down PER2 degradation as a result of faster reactions at higher temperatures. The phosphoswitch provides a biochemical mechanism for circadian temperature compensation of circadian period. This phosphoswitch additionally explains the phenotype of Familial Advanced Sleep Phase (FASP) and CK1ε(tau) genetic circadian rhythm disorders, metabolic control of PER2 stability, and how drugs that inhibit CK1 alter period. The phosphoswitch provides a general mechanism to integrate diverse stimuli to regulate circadian period.

The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABA A -mediated synaptic input. We show that increasing daylength changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABA A signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation-inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.day-length encoding | repulsive coupling | SCN | GABA | chloride T he physiological and behavioral rhythms of all life on earth are bound to the Earth's rotational cycle of ∼24 h. This fundamental rhythm is also affected by the planet's slanted rotational axis, which causes seasonal variations in the length of the day. How life has adapted to anticipate this yearly rhythm is still debated.The suprachiasmatic nucleus (SCN), the central circadian (∼24 h) pacemaker in mammals, consists of ∼10,000 "clock" neurons. These single-cell clocks maintain endogenous rhythms by autoregulatory feedback loops of genes including period (Per) and brain and muscle Arnt-like 1 (Bmal1) (1). Although it was speculated that seasonal rhythms might be encoded in a single cell (2), single-cell oscillations remain similar regardless of the seasonal time that the SCN tissue encodes (3). Seasonal timing is thus proposed to be encoded in the network of the SCN (4-7) through spatial reorganization of the relative phases of clocks within individual cells (8-10). Subsets of SCN clocks form phase clusters (11) that map approximately to dorsal (shell, D-SCN) and ventral (core, V-SCN) regions of the SCN. When measured through a luciferase reporter monitoring oscillations in the Bmal1 gene, the D-SCN and V-SCN clusters show a phase gap, ...

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

Computational modeling and experimentation explain how intercellular coupling and intracellular noise can generate oscillations in a mammalian neuronal network even in the absence of cell-autonomous oscillators.