A circadian clock in Neurospora: how genes and proteins cooperate to produce a sustained, entrainable, and compensated biological oscillator with a period of about a day

Abstract

Neurospora has proven to be a tractable model system for understanding the molecular bases of circadian rhythms in eukaryotes. At the core of the circadian oscillatory system is a negative feedback loop in which two transcription factors, WC-1 and WC-2, act together to drive expression of the frq gene. WC-2 enters the promoter region of frq coincident with increases in frq expression and then exits when the cycle of transcription is over, whereas WC-1 can always be found there. FRQ promotes the phosphorylation of the WCs, thereby decreasing their activity, and phosphorylation of FRQ then leads to its turnover, allowing the cycle to reinitiate. By understanding the action of light and temperature on frq and FRQ expression, the molecular basis of circadian entrainment to environmental light and temperature cues can be understood, and recently a specific role for casein kinase 2 has been found in the mechanism underlying circadian temperature-compensation. These data promise molecular explanations for all of the canonical circadian properties of this model system, providing biochemical answers and regulatory logic that may be extended to more complex eukaryotes including humans.

Figure 1

Similar regulatory relationships constitute the circadian oscillators in model organisms. The shaded region in the center of each loop covers the core transcription-translation feedback loop that underlies circadian rhythms. Regulatory relationships outside this core are helpful but not essential for circadian rhythmicity. (Adapted, with permission, from Bell-Pedersen et al. 2005 [© Nature Publishing Group].)

Figure 2

Complex regulation and splicing involved in the expression of frq. The Clock Box and PLRE are regulatory elements upstream of P_U and P_D, the upstream and downstream promoters, respectively. (Green) Parts of the primary transcript that are retained after splicing; (asterisks) upstream ORFs that influence the amount of FRQ made; (purple) FRQ ORF. Temperature-regulated splicing of intron 2 governs whether AUG_L or AUG_S is used to initiate FRQ. (Red) qrf, the antisense transcript (see text for details). (Adapted, with permission, from Dunlap 2006 [© ASBMB].)

Figure 3

Molecular details within the Neurospora circadian mechanism. (Top) Cycles in the levels of clock-pertinent RNAs and proteins. (Bottom) Locations of RNAs and proteins important for the circadian oscillator are shown as a function of time, from left to right, beginning at subjective dawn (CT 0). The trash can indicates the proteasome. (Adapted, with permission, from Dunlap 2006 [© ASBMB].)

Figure 4

How light resets the clock. (Top) Cycle in frq RNA abundance and effect of brief exposure to light at any time. (Middle) Blackrepresents the cycle of frq transcription; colored lines show the effect of transient light induction of frq on the steady-state rhythm. (Bottom) The cycle in frq transcript levels juxtaposed with response of the clock to light, showing how light seen while frq is rising leads to advances, and light seen while frq levels are falling leads to delays (see text for details). (Adapted, with permission, from Dunlap 1999 [© Elsevier].)

Figure 5

A possible mechanism for how temperature resets the clock. (Green) Cycles in FRQ levels at different temperatures. (Red arrows) Step up at any time leaves the clock with too little FRQ to close the feedback loop, so the clock is reset to the time corresponding to low FRQ, subjective dawn. (Blue arrows) Step down at any time leaves the clock with enough FRQ to close the feedback loop, so the clock is reset to the time corresponding to high FRQ, around subjective evening (see text for details). (Adapted, with permission, from Dunlap 1999 [© Elsevier].)

Figure 6

Strains expressing only long FRQ are not deficient in temperature-compensation. Period length is plotted as a function of temperature from a wild-type strain and from two independently engineered strains that express only long FRQ (HVC16 from Colot et al. 2005; I-6^mut from Diernfellner et al. 2005). The error bars indicate +/− one standard deviation; n = 6. Both strains clearly show compensation. See text for details.

Figure 7

A special role for casein kinase 2 (CK2) in temperature-compensation. Strains bearing inducible copies of the gene encoding the β subunit of CK2 (qa-ckb-1) or CK1 (qa-ck-1a) were grown at different levels of inducer and at different temperatures. (Left) As levels of ckb-1 decrease leading to less CK2 activity, the period length increases and compensation moves from the slight undercompensation typical of wild type to distinct overcompensation. (Right) As CK1 levels drop, the period gets longer, but in contrast to the behavior seen with CK2, the mode of compensation remains like that of wild type. These illustrations depict unpublished data from A. Mehra et al. (in prep.).