The Phospho-Code Determining Circadian Feedback Loop Closure and Output in Neurospora

Abstract

In the negative feedback loop driving fungal and animal circadian oscillators, negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) are understood to inhibit their own expression, in part by promoting the phosphorylation of their heterodimeric transcriptional activators (e.g., White Collar-1 [WC-1]-WC-2 [White Collar complex; WCC] and BMAL1/Circadian Locomotor Output Cycles Kaput [CLOCK]). However, correlations between heterodimer activity and phosphorylation are weak, contradictions exist, and mechanistic details are almost wholly lacking. We report mapping of 80 phosphosites on WC-1 and 15 on WC-2 and elucidation of the time-of-day-specific code, requiring both a group of phosphoevents on WC-1 and two distinct clusters on WC-2, that governs circadian repression, leading to feedback loop closure. Combinatorial control via phosphorylation also governs rhythmic WCC binding to the promoters of clock-controlled genes mediating the essential first step in circadian output, a group encoding both transcription factors and signaling proteins. These data provide a basic mechanistic understanding for fundamental events underlying circadian negative feedback and output, key aspects of circadian biology.

Keywords: C-box; DNA binding; FRQ; WC-1; WC-2; ccgs; clock-controlled genes; feedback loop; frq transcription; phosphorylation.

Figure 1

Identification of phosphorylation sites on WC-1 and WC-2. A A representative silver-stained gel showing WCC purified from a culture grown in the dark for 24 hr. B C. Schematic of WC-1 (B) and WC-2 (C) showing the position of phosphorylated residues identified by MS/MS. WC-1, 1,167 amino acids, contains a ZnF, zinc finger DNA binding domain; DBD, defective in DNA binding; LOV, light-, oxygen-, and voltage-sensing; and PAS, Per-Arnt-Sim domains. WC-2, 530 amino acids, contains PAS, coiled-coil, and ZnF domains. Vertical bars with numbers represent identified phosphorylation sites (above) and Ser/Thr phosphosites undetected by MS/MS (below). D Analysis of WCC activity by the frq C-box fused with firefly luciferase (frq C-box-luc transcriptional fusion). Middle two panels show circadian oscillator function with reduced amplitude and slightly shortened period (± SD) in WC-1^77A and in WC-1^109A lacking nearly all WC-1 phosphosites. Time in hours is on the X-axis. Different colored lines represent three replicates. The panel on the right shows baseline subtracted and detrended data. E Oscillator function in wc-2 mutants in which all confirmed phosphosites are converted to Ala (left, WC-2^15pA; enhanced WCC activity as noted by scale bar) or Asp (mid left, WC-2^15pD; loss of rhythmicity and severely depressed WCC activity). Right two panels show enhanced WCC activity and arrhythmicity in strains lacking phosphorylatable sites on both WC-1 and WC-2.

Figure 2

Identification of key phosphosites on WC-1 and WC-2 required for the circadian feedback loop closure. Schematic of WC-1 (A upper) and WC-2 (B upper) showing the position of phosphorylated residues as in Figure 1BC. Below these is the strategy used to identify the phosphorylation events on WC-1 (A lower) and WC-2 (B lower) essential for rhythmicity. Each horizontal bar represents a wc-1 (A) or wc-2 (B) mutant with phosphosites falling in the region of the bar mutated to Ala altogether. In the presence of wc-2^15pA (A) or wc-1^{S971A, S988A, S990A, S992A, S994A, S995A} (B) respectively, the circadian rhythms of the wc-1 (A) or wc-2 (B) phosphomutants were measured by frq C-box-luc ; phenotypes (Table S4) were as noted. C WCC activity in WT and wcc mutants (y-axis) as measured by frq C-box-luc bioluminescence. D Phosphorylation of WC-1 S971 plays a key role in repressing the circadian WCC activity. WCC activity in strains of the noted genotypes was monitored by frq C-box-luc; note scale bars. Right panel: Western blot showing FRQ, WC-1, and WC-2 levels in WT (wc-1^V5), wc-1^S971A, and wc-1^S971D in light and at 16 and 24 hrs in darkness.

Figure 3

Mutation of phosphorylation sites on both WC-1 and WC-2 together leads to constant binding to the frq promoter C-box. Quantitative ChIP PCR experiments were carried out using WC-2 antibody with C-box- specific primer sets (Table S5) in indicated wc-1 phosphomutants. Average values are plotted as a percentage of the total; error bars show SEMs (n=3).

Figure 4

FRQ promotes phosphorylation of certain residues in WC-1 and WC-2. A Western blot showing complete loss of phosphorylation in wc-1^113A. Western blots were performed using the 149:1 gel containing 20 μM Phos-tag (See Methods). B FRQ-induced phosphorylation. In WC-1^{113A A603-966p back}, all Ser/Thr between aa 603 and aa 966 were returned to normal (phosphorylatable) sequence, whereas all other WC-1 Ser/Thr remained converted to Ala. These strains were either backcrossed to introduce Δfrq or transformed with qa-2 promoter-driven frq at the csr locus. To cultures in constant light, inducer (10⁻² M QA) was added and the cultures immediately moved to dark for 16 hr. Phos-tag Western blots as in A are shown. C In the context of unphosphorylatable WC-1^113A, Ser was restored at indicated sites; e.g. in wc-1^{113A, A971S} the only Ser is at 971. Mutants were placed in a Δfrq or QA-inducible frq background and assayed for WC-1 phosphorylation as in B. Note differences in amount loaded in the top panel. D In the context of the unphosphorylatable wc-2^15pA, Ser was introduced to aa 433 (wc-2^{15pA, A433S}). This allele was placed in an inducible frq background in the presence or absence of inducer (10⁻² M QA) and assayed for WC-2 phosphorylation as in B.

Figure 5

Loss of overt rhythmicity and reduction of oscillator amplitude in strains lacking phosphorylation around the WC-1 ZnF. A Race tube assays of WT (328-4) and wc-1 phosphomutants as indicated; WC-1^1-602A retained S363, S373, S390, and Y525 to maintain WC-1 at a level sufficient for core oscillator rhythmicity. B Effects of loss of WCC phosphorylation on levels of WC-1, WC-2, and FRQ. Left panel: Western blot showing reduced levels of WC-1 and WC-2 with increased FRQ in constant light (L), DD16 (~CT4, subjective morning), and DD24 (~CT14, subjective evening). Right panel: Same as left, but examining more restricted regions of WC-1. C Mutation of WCC residues essential for feedback repression has dramatic effects on WCC and FRQ levels; the level of FRQ is inversely proportional to the amount of WCC. Experimental conditions as in B.

Figure 6

Mutations of phosphosites on the WCC that impact repression within the core oscillator also have direct effects on rhythmic binding to DNA targets in the first step of circadian output. A Identification of novel direct targets of the WCC. G-browse data for ncu06593 (encoding MAP kinase activator prk-5) from a series of ChIP-seq experiments using anti-WC-2. Top; gene map with the transcription unit circled. Below this are Δwc-1 (negative control for WCC binding) and WC-2 binding assessed at 4 hr intervals from DD4 to DD32. The peak of binding is marked by the box; arrows indicate the primer set (Table S5) used to detect the region. Right; ChIPqPCR data assessing WC-2 binding to this site and to the promoter of ncu07024, in WT and four mutants as shown, revealing that mutant strains that have lost circadian repression within the feedback loop are also altered for binding related to circadian output. Binding was assessed at DD16 (~CT4, subjective morning) and DD24 (~CT14, subjective evening). Error bars are SEMs (n=3). B Binding of WC-2 to two other direct targets of the WCC within the first step of circadian output in WT and mutants as shown. See Figure S9 for other examples of output genes.

Figure 7

Re-assessment of kinases and a phosphatase previously implicated in regulation of WCC activity, and a summary model for the role of phosphorylation in regulating WCC activity. A PKA and PP2A are not required for WCC circadian activity. WCC activity was monitored by frq C-box-luc in the Δpkac-1, Δpkac-2, and in the rgb-1^RIP strain that is defective in PP2A activity on the WCC. B Working model for the role of phosphorylation in regulating WCC activity. In a circadian cycle, FRQ/FRH/CK1 promotes phosphorylation of key residues (red circles) on both WC-1 and WC-2 to repress WCC activity in the feedback loop of the core clock, and also induces phosphorylation near the ZnF region of WC-1 (purple circles) that acts to depress the circadian amplitude as an aid to enhance robustness of circadian outputs.