The Neurospora crassa circadian clock

The filamentous fungus Neurospora crassa represents another model organism for the analysis of the cellular and molecular bases of circadian rhythms in eukaryotes, because of its relative simplicity and its easily assayed circadian rhythmicity. N.crassa is a haploid fungus that can grow and propagate asexually (production of conidia) or via a sexual cycle (formation of ascospores), both of these developments being regulated in a circadian fashion (Figure 7) (Bobrowicz et al., 2002; Dunlap and Loros, 2004). Classical genetic screens allowed the characterization and cloning of core clock and clock controlled genes (Aronson et al., 1994a; Loros et al., 1989). The N.crassa circadian system is based on interlocked negative and positive feedback loops. The positive core components include the transcription factors WC-1 and WC-2 that heterodimerize and drive the transcription of the negative element, frq, and of other clock-controlled genes (CCGs). WC-1 and WC-2 are able to bind DNA via their zinc finger domains and are held together via the interaction of protein dimerization domains called PAS domains (from PER-ARNT-SIM that were the first proteins shown to have this domain) (Aronson et al., 1994b; Crosthwaite et al., 1997; Froehlich et al., 2003). The presence of PAS domains in positive regulatory proteins was shown to be a characteristic

Figure 7. In N.crassa, the endogenous clock controls conidia formation. Correlation between circadian conidial bands formation (left, upper track) and bioluminescence expression from frq core clock promoter fused to luciferase (frq-luc-l) under free running condition in DD. Rhythms are most intense at the growth front, although there is a clear, but dampening, rhythm in older conidial bands (left, lower track). The trace on the right represents quantification of the luminescence of the entire race tube. These Figures are issued from (Hastings et al., 2008) and (Gooch et al., 2008).

of other eukaryotic circadian systems, such as Drosophila (Allada et al., 1998; Rutila et al., 1998) and mouse (King et al., 1997). Wc-1 and wc-2 transcription are not rhythmic. WC-2 proteins level is high and constant throughout the circadian period, while WC-1 proteins display an abundance rhythm almost antiphasic to frq. The FRQ proteins participate in separate actions during the clock cycle, each being essential to the daily rhythm. First, the FRQ proteins homodimerize and interfere with WC complex (WCC) by modulating the phosphorylation status of both subunits WC-1 and WC-2 (Schafmeier et al., 2005). FRQ thus feeds back on its own synthesis and regulates at the same time other genes controlled by WCC. This feedback regulation results in high amplitude circadian rhythms of frq RNA and protein levels. A second role of FRQ occurs in the cytoplasm, where FRQ is phosphorylated by several kinases and becomes an activator. In this state, it may promote the translation of wc-1 RNA, generating the cyclic accumulation of WC-1 proteins and facilitating the formation of WCC on a post-translational level. Finally, hyperphosphorylated FRQ is targeted to degradation. WCC is thus progressively dephosphorylated and becomes again able to activate frq transcription, thereby initiating a new circadian cycle (Figure 8) (Lee et al., 2000;

Schafmeier et al., 2006), for review, see (Brunner and Schafmeier, 2006; Dunlap and Loros,

2004).

The circadian oscillator regulates many biological processes, including the well studied conidia formation, but also CO2 production, a number of enzymatic activities, lipid and diacylglycerol metabolism, and growth rate. Attempts to identify CCGs in N.crassa have led to the estimation that approximately 10% of its transcripts are cycling. Some of them contain a C box identical to the one found in the frq promoter and bound by the WCC, thus suggesting a direct regulation by WCC. Some harbor ACEs (activating circadian elements) and/or LREs (light responsive elements) within their promoters. Still others have neither element, suggesting additional clock-controlled cis-acting elements that are perhaps the targets of transcriptional regulation by yet unknown CCGs. Interestingly, some genes were discovered to continue to cycle in a frq-null strain, albeit with a somewhat altered

Figure 8. Model for interconnected negative feedback loop and positive loop of N.crassa circadian system. (Morning) Newly synthesized FRQ is active as a repressor by promoting phosphorylation and inactivation of WCC. (Evening) High levels of hyperphosphorylated cytosolic FRQ are required to support WC-1 accumulation.

Cytosolic FRQ may also act on the level of WCC assembly. Newly assembled WCC accumulates in the nucleus and is still inactivated via FRQ-dependent phosphorylation. frq transcription remains repressed. (Night) FRQ is progressively phosphorylated and degraded. As the FRQ concentration decreases, WCC is progressively dephosphorylated (Schafmeier et al., 2005). Dephosphorylated WCC binds to the frq promoter and activates transcription. This Figure is taken from (Schafmeier et al., 2006)

phase (Correa et al., 2003). This is in accordance with the fact that frq-null strain have long been known to occasionally express a rhythm in conidial banding, although one that has lost most typical circadian features (Aronson et al., 1994a). Thus, these residual oscillations appeared to be irregular, damping, not resettable by input pathways, and, not temperature compensated. Nonetheless, they were observed under certain conditions, and their period length was dependent on temperature (for conidiation) and nutrition (for nitrate reductase (NR) activity) period length (Christensen et al., 2004; Merrow et al., 1999). Such non-circadian oscillators, which can exist in the absence of the FRQ/WC loop, were named FRQ-less oscillators (FLOs). These are possible slave oscillators that can be coupled to the FRQ/WCC circadian feedback loop, but can run on their own in a non-circadian manner in absence of the FRQ/WCC loop (Figure 9).

In a rhythmic environment, the phase of the N.crassa clock is adjusted to light and temperature cycles. The light response is maximal at blue light and acts through WCC to reset the clock by a rapid frq induction. WC-1 is associated with a flavin adenine dinucleotide (FAD) chromophore via its flavin-binding LOV domain (He et al., 2002). Light perceived by this LOV domain may induce a conformational change in WCC, thereby promoting its binding to LREs within the frq promoter. At the same time, light exposure renders WCC susceptible to phosphorylation, which then leads, with a kinetic delay, to the inactivation of WCC by reducing its affinity for LREs (He and Liu, 2005). The VVD protein allows the

Figure 9. A model for oscillators and networks controlling and integrating light and temperature influences in the N.crassa circadian system. Solid loops, lines, and arrows represent known feedback loops or regulatory relationships, and dotted lines represent those predicted to exist.

This Figure is issued from (Dunlap and Loros, 2004)

circadian clock to modulate its sensitivity to light resetting cues, as a function of daytime. In response to light, VVD mediates a photoadaptation process that modulates the organism’s response to subsequent light signals. Vvd is an immediate early light-induced gene controlled by WCC (Heintzen et al., 2001). It appears to be a negative regulator of light responses, since vvd mutants exhibit prolonged transcription of immediate early light-induced genes and altered light-induced phase-shifting properties. VVD proteins are present 30 minutes after light exposure and may participate in the alteration of WCC activity (Brunner and Schafmeier, 2006). VVD may help to entrain the N.crassa circadian clock properly with natural photoperiods. After completion of the Neurospora genome project,two putative red-light photoreceptors (N. crassa phytochrome orthologs phy-1 and phy-2) and one additional blue-light photoreceptor (N. crassa cryptochrome orthologue cry) were identified. Cry transcript and protein levels are strongly induced by blue light in a wc-1 dependent manner.

However, due to the lack of a detectable phenotype and atypical light responses in the respective knockout strains, the biological functions of PHY-1, PHY-2 and CRY remain to be discovered (Chen and Loros, 2009) (Figure 9).

Resetting of the clock by temperature is mediated via the levels of FRQ. As mentioned previously (section 2.2, last paragraph), N.crassa frq ORF encodes two versions of FRQ proteins, a large (l-FRQ) and a small (s-FRQ) isoform. Thermosensitive splicing and translation regulate the ratio and abundance of these isoforms, respectively; which are crucial for high-amplitude, self-sustained rhythmicity and for temperature compensation of the circadian clock (Garceau et al., 1997; Liu et al., 1997). At low temperature, frq precursor mRNA is preferentially spliced to give rise to s-FRQ. There is thus more s-FRQ at low temperature and more l-FRQ at high temperature. In parallel, the global abundance of both isoforms is regulated at the level of translation initiation. In the high-temperature range, ribosomes are scanning through nonconsensus AUGs of the upstream ORFs (uORFs) present in the 5′-UTR of frq, but mostly initiate translation at the main frq ORF, leading to efficient

expression of FRQ. In contrast, at low temperatures, scanning ribosomes initiate translation more efficiently at the uORFs, leading to a reduced translation of the downstream frq ORF.

Accordingly, the levels of both isoforms are elevated at high temperature and diminishe while temperature is decreasing (Diernfellner et al., 2005). So after a temperature change, the relation among frq mRNAs and FRQ proteins are instantaneously changed allowing a rapid response accordingly to the new situation. In this way, temperature changes reset the circadian clock directly within the core loop, and it has been shown that physiologically natural temperature differences can be dominant over light/dark transitions (Liu et al., 1998).

One of the goals of studying the circadian clocks in multiple model organisms is to determine if the properties of the clock in one organism are conserved in other organisms. It is interesting to note that another member of the fungi kingdom, Saccharomyces cerevisiae, which represents the most powerful genetic experimental system, still remains absent from the repertoire of circadian model organisms. It seems indeed that yeast cells do not harbor a circadian clock and that they have no homologs of known clock genes. They have however developed mechanisms allowing them to partition time allocated to diverse aspects of their metabolism (Chen et al., 2007; Robertson et al., 2008; Tu et al., 2007). Interestingly, a recent study has shown that the application of temperature cycle protocols reveals a circadian timing mechanism in S.cerevisiae, using pH as readout. The metabolism in yeast shows systematic circadian entrainment, responding to temperature cycle length and Zeitgeber (stimulus) strength, and rapidly damps in free running constant conditions. The ammonium permease, MEP2, and the general amino acid permease, GAP1, are expressed several hours ahead of the acidification of the media under temperature entrainment (Eelderink-Chen et al., 2010).

Although the authors suggest that the temperature cycle could entrain a circadian system that actively regulates the timing of the observed oscillations, more experiments are required to investigate circadian behavior and to identify the underlying genes. Nevertheless, the preliminary observations on the putative yeast clock could open new approaches to study

circadian timing system in eukaryotes, since yeast is easily cultured in the laboratory, its genome is sequenced, and null-strains are available for every non-essential gene.