Circadian clock-specific roles for the light response protein WHITE COLLAR-2

Abstract

To understand the role of white collar-2 in the Neurospora circadian clock, we examined alleles of wc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2 (ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator of frq, is critical for establishing period length and temperature compensation in this circadian system.

FIG. 1

Partially functional wc-2 allele, ER24, results in a long period, while wc-2 (ER44) and a null allele, Δwc-2, are arrhythmic. (A) Race tube analysis showing the banding rhythm at 25°C in strains possessing either a wild-type (WT) allele of wc-2, wc-2 (ER24), wc-2 (ER44), or Δwc-2. The strain possessing wc-2 (ER24) has a long period, and wc-2 (ER44) and Δwc-2 are arrhythmic. Race tubes were inoculated at the left end, incubated in constant light at room temperature for 2 days, and then transferred into darkness at 25°C, at which point the growth front (vertical white line) was marked. Growth fronts were marked at 24-h intervals thereafter. (B) Densitometric analysis of race tubes, plotting conidial density over time. The period length (τ) is indicated where relevant. (C) Distribution of period length in progeny possessing the bd mutation from a cross of bd with wc-2 (ER24). Period length cosegregated with light-induced mycelial carotenogenesis, and those progeny possessing wc-2⁺ (WT) or wc-2 (ER24) photoresponses are indicated with mean period length (τ) ± standard error of the mean (SEM) above the appropriate group.

FIG. 2

Sequence alignment of the Zn finger domain of WC-2 and other proteins from the GenBank protein and nucleic acid sequence databases. The top group of sequences are all type IVa Zn fingers, and the bottom group are all type IVb Zn fingers (using the designation of Teakle and Gilmartin [63]). A black background marks residues which are found in 90% or more of the sequences, and those which are found in 25 to 90% are shown by a gray background. An asterisk marks the mutated amino acid of allele ER24. An N or a C after the protein name indicates the N- or C-terminal Zn finger of that protein. The organism and GenBank accession number for the different sequences are mGATA4 (Mus musculus, GenBank 3183530), mGATA2 (M. musculus, 1754586), dGATAa (D. melanogaster, 709699), mGATA1 (M. musculus, 120957), ELT1 (Caenorhabditis elegans, 119299), URB1 (Ustilago maydis, 731074), NIT2 (N. crassa, 128352), aGATA2 (Arabidopsis thaliana, Y13648), aGATA4 (A. thaliana, Y13651), aGATA3 (A. thaliana, Y13650), aGATA1 (A. thaliana, Y13648), PBP (Fusarium solani, 1362526), WC-2 (N. crassa, 1835159), WC-1 (N. crassa, 2494692), NSDD (Aspergillus nidulans, 1617552), YM19 (Saccharomyces cerevisiae, 732160), and SRD1 (S. cerevisiae, 140465).

FIG. 3

Levels of frq mRNA and protein are reduced in a strain containing wc-2 (ER24) grown at 25°C. (A) Northern blots of frq mRNA in wc-2 (ER24) and a wild-type (WT) strain over one circadian cycle. Ethidium bromide staining of the rRNA bands on the agarose gel is shown below the Northern blot. (B) Western blot of FRQ protein in wc-2 (ER24) and a wild-type strain over 48 h. The amido black-stained membrane is shown below the blot of FRQ. A wild-type reference sample from DD16 was included on the ER24 blot and on the wild-type blot to allow a comparison of the levels of FRQ between the strains. The level of FRQ is greatly reduced in the wc-2 (ER24) strain compared to the wild type and cycles with an altered period. (C) Densitometric analysis plotting the amount of FRQ normalized against the wild-type DD16 reference sample versus time. Squares, wc-2 (ER24); circles, wc-2⁺. Each point corresponds to the mean of two experiments. Error bars show the SEM.

FIG. 4

Reduced temperature compensation in a strain containing wc-2 (ER24). Mean period plotted against temperature for wc-2 (ER24) and clock wild type (WT). Squares, wc-2 (ER24); circles, wc-2⁺. Error bars show the SEM. Q₁₀ from 25 to 30°C was calculated from the equation Q₁₀ = (P₂/P₁)10/T₁−T₂, where P₁ and P₂ are mean periods at temperatures T₁ and T₂, respectively (64).

FIG. 5

Elevated temperature results in increased FRQ levels in wc-2 (ER24) strains and wild type. (A) Representative Northern blots of frq mRNA in strains containing either wc-2 (ER24) or wc-2⁺ grown at 25 or 30°C. The membrane of blotted RNA was cut in half between the large and small nuclear ribosomal subunits, the top half was hybridized to a probe specific for frq, and the bottom was hybridized to a probe for rRNA. To allow comparison of relative frq mRNA levels between the two strains, reference samples corresponding to peak frq mRNA levels from wc-2⁺ (DD12) and wc-2 (ER24) (DD16) were compared in triplicate (bottom). (B) Representative Western blots of FRQ in strains containing wc-2 (ER24) or wc-2⁺ grown at 25 or 30°C. Two different exposures, 20 and 60 s, of the blots are shown for each temperature. The amido black-stained membrane is shown as an estimate of loading. Wild-type samples were included on each blot for reference to allow a comparison of the levels of FRQ between the strains. (C) Densitometric analysis plotting the relative amounts of frq mRNA and FRQ protein versus time. Each point corresponds to the mean of two experiments for FRQ and three experiments for frq mRNA ±, SEM. For each sample, the density of the frq or FRQ signal was divided by the corresponding density of the rRNA or amido black-stained protein. These values were then normalized to the reference sample for each blot. Squares, wc-2⁺; circles, wc-2 (ER24); solid symbols, 25°C; open symbols, 30°C.