Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock

Abstract

Interlocked feedback loops may represent a common feature among the regulatory systems controlling circadian rhythms. The Neurospora circadian feedback loops involve white collar-1 (wc-1), wc-2, and frequency (frq) genes. We show that WC-1 and WC-2 proteins activate the transcription of frq gene, whereas FRQ protein plays dual roles: repressing its own transcription, probably by interacting with the WC-1/WC-2 complex, and activating the expression of both WC proteins. Thus, they form two interlocked feedback loops: one negative and one positive. We establish the physiological significance of the interlocked positive feedback loops by showing that the levels of WC-1 and WC-2 determine the robustness and stability of the clock. Our data demonstrate that with WC-1 being the limiting factor in the WC-1/WC-2 complex, the greater the levels of WC-1 and WC-2, the higher the level of the FRQ oscillation and the more robust the overt rhythms. Our data also show that, despite considerable changes in the levels of WC-1, WC-2, and FRQ, the period of the clock has been limited to a small range, suggesting that the interlocked circadian feedback loops are also important for determining the circadian period length of the clock.

Figure 1

Both WC-1 and WC-2 are positively regulated by FRQ. The WC-2 level is low in the frq null strains in constant light (LL) (A) and constant darkness (DD) (B). Total protein extracts were prepared from the wild-type and the frq null strains (frq⁹ and frq¹⁰) grown in LL or in DD (at different times). Western blot analysis was performed by using WC-1, WC-2, or FRQ antisera (22). Representative results from three independent experiments are shown. (C) Western blot analysis shows that FRQ positively regulated the levels of WC-1 and WC-2 in the dark. Liquid cultures were first grown in LL in media with or without 1 × 10⁻² M QA for several hours before being transferred into constant darkness and were harvested at DD24. (D) Northern blot analysis reveals that frq differentially regulates wc-1 and wc-2. Cultures were harvested at DD14. Similar results were obtained for cultures harvested in LL. (E) Western blot analysis shows that the WC-2 level does not fluctuate much in constant darkness in the wild-type strain.

Figure 2

WC-1 and WC-2 activate the expression of frq. wc-1⁻, qa-WC-1 (A) and wc-2⁻, qa-WC-2 (B) strains were used in these experiments. At DD14, 1 × 10⁻² M QA was added to half of the cultures. Northern blot and Western blot analyses were performed. The representative results from several independent experiments are shown. The weak and high molecular weight FRQ signals in (A) are the extensively phosphorylated large FRQ forms. (C) Western blot analyses show that there is a low level of FRQ expression in wc-1⁻ strain. (D) Western blot analyses show that the level of WC-1 does not affect the abundance of WC-2 in a frq¹⁰, qa-WC-1 strain.

Figure 3

WC-1 and WC-2 are required for circadian rhythmicity, and their levels determine the robustness and stability of the clock. Race tube assays show the conidiation rhythms of the wild-type (A), wc-1⁻, qa-WC-1 (B, Upper), and wc-2⁻, qa-WC-2 (C, Upper) strains in the presence of different concentrations of QA (labeled at left). The race tubes shown are representative samples from six replicate tubes. The period lengths of the rhythm (Ave. + SEM) are labeled at right. The lower panels of B and C are Western blot analysis results showing the levels of WC-1, WC-2, and FRQ in the wild-type, wc-1⁻, qa-WC-1 or wc-2⁻, qa-WC-2 strain. Liquid cultures were grown in media with different concentrations (indicated above) of QA and were harvested at DD24. No QA was added to the wild-type culture. In C, two different exposures of the WC-2 blot are shown.

Figure 4

Western blot analysis results show that a higher level of WC-1 or WC-2 leads to a higher level of FRQ oscillation. (A and B) wc-1⁻, qa-WC-1 cultures were grown in media with or without QA (1 × 10⁻⁵ or 1 × 10⁻² M) in DD. The left lane in B shows the protein levels of the wild-type culture grown in LL. (C) A higher level of WC-2 resulted in a higher level of FRQ oscillation. wc-2⁻, qa-WC-2 cultures were grown in media with or without 1 × 10⁻² M QA in DD. (D and E) Densitometric analyses of the Western blots shown in B and C, respectively.

Figure 5

WC-1 is the rate-limiting factor in the WC-1/WC-2 complex. (A) Immunoprecipitation analysis shows that WC-1 is the rate-limiting factor in the WC-1/WC-2 complex. The same protein extracts (1 × 10⁻⁵, 1 × 10⁻⁴, and 1 × 10⁻⁵ M QA samples) shown in Fig. 3B were used, and they were immunoprecipitated with the WC-2 antiserum. Both the total extracts and the pellets of immunoprecipitation were subjected to Western blot analysis. (B) Western blot analysis shows that a higher level of WC-1 leads to a higher level of FRQ oscillation in a wild-type strain. wc-1⁺, qa-WC-1 liquid cultures were grown in media with or without 1 × 10⁻² M QA in DD. (Right) The densitometric analysis of the Western blot shown left. (C) Race tube assay results show the conidiation rhythms of the wc-1⁺, qa-WC-1 strain at different concentrations of QA (labeled at left). (D) Western blot analysis shows that the increase of WC-2 level in a wild-type strain (wc-2⁺, qa-WC-2) did not result in the increase of FRQ expression.

Figure 6

(A) Model for gene regulation within the Neurospora circadian oscillator. WC-1 and WC-2 form heterodimers to activate the transcription of frq. FRQ proteins interact with the WC-1/WC-2 complex to inhibit their transcriptional activation, forming the negative feedback loop. FRQ also positively regulates the levels of both WC-1 and WC-2, forming the positive feedback loops. (B) The levels of WC proteins determine the robustness of FRQ oscillation. When the levels of WCs are low, there is either no FRQ oscillation or it oscillates at a low level, whereas high levels of WCs lead to a robust and a high-level oscillation of FRQ. The parallel changes in WCs and FRQ limit the period of clock to a small range.