Rhythmic binding of a WHITE COLLAR-containing complex to the frequency promoter is inhibited by FREQUENCY

Abstract

The biological clock of Neurospora crassa includes interconnected transcriptional and translational feedback loops that cause both the transcript and protein encoded by the frequency gene (frq) to undergo the robust daily oscillations in abundance, which are essential for clock function. To understand better the mechanism generating rhythmic frq transcript, reporter constructs were used to show that the oscillation in frq message is transcriptionally regulated, and a single cis-acting element in the frq promoter, the Clock Box (C box), is both necessary and sufficient for this rhythmic transcription. Nuclear protein extracts used in binding assays revealed that a White Collar (WC)-1-and WC-2-containing complex (WCC) binds to the C box in a time-of-day-specific manner. Overexpression of an ectopic copy of FRQ or addition of in vitrogenerated FRQ resulted in reduced WCC binding to the C box. These data suggest that oscillations in frq transcript result from WCC binding to the frq promoter and activating transcription with subsequent changes in FRQ levels having an inverse effect on WCC binding. In this way rhythmic expression and turnover of FRQ drives the rhythm in its own transcription. Life on earth has evolved under the continual daily fluctuations in light and temperature. Many organisms have evolved the ability to anticipate these external changes in their environment by using endogenous ''biological clocks.'' In recent years, the molecular components that make up these intracellular clocks have begun to be identified, and similarities among a wide range of organisms have emerged (1-5). The clock in a fungus, fruit fly, or mammal contains a negative feedback loop in which two PAS domain-containing proteins [White Collar (WC)-2͞ WC-1, dCLOCK (dCLK)͞CYCLE (CYC), CLOCK͞BMAL1] heterodimerize and act as positive elements to activate the expression of a negative element [FREQUENCY (FRQ), PERIOD (PER)͞TIMELESS (TIM), CRYPTOCHROME 1 (CRY1)͞CRY2͞mPERs]. The negative element(s) in turn feeds back to repress the activity of the positive elements. These transcription͞translation-based negative feedback loops ultimately generate self-sustaining circadian (circa ϭ about; dies ϭ day) oscillations or rhythms in the level(s) of one or more of the elements within the loop. The robustness and stability of these oscillations is enhanced further by interlocking positive feedback loops (6-8) and multiple layers of regulation (2,3,9).In Neurospora, a negative feedback loop comprised of the products of the frq, wc-1, and wc-2 genes is central to clock function. WC-1 and WC-2 are predominately nuclear transcription factors containing trans-activation domains and Zn-finger DNA-binding domains (10, 11). They form a WC complex (WCC) by heterodimerizing via PAS domains (12) and act as positive elements in the expression of frq (13); in a wc-1 KO or wc-2 KO strain, very limited, unregulated transcription of frq occurs (14-16). During the course of a day, FRQ is progressively phosphorylated and degraded (17)(18)(19), but when present FRQ ...

Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation

“…This is consistent with the earlier result using in vitro translated WC proteins made from the rabbit reticulocyte lysate system (Froehlich et al 2002(Froehlich et al , 2003. Therefore, the L-WCC should be the result of multimerization of the WC proteins, a notion that is supported by the observation of WC-1 self-association in vivo (Cheng et al 2003b).…”

“…Afterward, the levels of the binding quickly decreased to low levels in LL. Significant WCC binding was also found for the frq LRE in the dark, consistent with frq being directly regulated by D-WCC in DD (Crosthwaite et al 1997;Cheng et al 2001b;Froehlich et al 2003). These data indicate that the light-induced binding of WCC to the LREs correlates with the light induction of transcription, and that there is an unknown mechanism negatively regulating the activity of WCC after light exposure.…”

“…In the dark, a heterodimeric WCC (D-WCC) binds to the C-box (the distal LRE) of the frq promoter, activating its transcription (Crosthwaite et al 1997;Cheng et al 2001b;Froehlich et al 2003;Dunlap and Loros 2004). On the other hand, FRQ and FRH (FRQinteraction RNA helicase), the negative elements in the loop, repress the transcription of frq by inhibiting WCC activity (Aronson et al 1994;Cheng et al 2001aCheng et al , 2005Denault et al 2001).…”

Transcriptional and post-transcriptional regulation of the circadian clock of cyanobacteria and Neurospora

“…The WCC is highly concentrated in the nucleus, while excess WC-2 is found in the nucleus and cytosol (Schwerdtfeger and Linden 2000;Schafmeier et al 2005). WCC binds to clock-and light-responsive elements in the frq promoter and activates frq transcription (Froehlich et al 2002(Froehlich et al , 2003. Levels of frq RNA begin to rise in the late (subjective) night (around CT 20) and peak shortly after dawn (CT 0-4) (Crosthwaite et al 1997;Garceau et al 1997;Luo et al 1998;Dunlap 1999).…”

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

“…In addition to being the circadian blue light photoreceptor (Froehlich et al 2002;He et al 2002), in the dark, WC-1 forms a heterodimeric complex with WC-2 through their PAS domains and activate frq transcription by binding its promoter (Crosthwaite et al 1997;Talora et al 1999;Cheng et al 2001bCheng et al , 2002Cheng et al , 2003Froehlich et al 2003). FRQ, the negative element of the feedback loop, inhibits its own transcription through interactions with the WC complex (Aronson et al 1994;Cheng et al 2001a;Denault et al 2001;Froehlich et al 2003). FRQ protein is immediately phosphorylated after its synthesis and becomes extensively phosphorylated prior to its degradation by the ubiquitin/proteasome pathway Liu et al 2000;He et al 2003).…”