A recessive mutant of Drosophila Clock reveals a role in circadian rhythm amplitude

Abstract

The transcription factor Clock (Clk) plays a critical role in animal circadian rhythms. Genetic studies defining its function have relied on two dominant negative alleles, one in Drosophila and one in mice. Here we describe a novel recessive allele of Drosophila Clock, Clk(ar). Homozygous Clk(ar) flies are viable and behaviorally arrhythmic. The Clk(ar) phenotype is caused by a splice site mutation that severely disrupts splicing and reduces Clk activity. Despite the behavioral arrhythmicity, molecular oscillations are still detectable in Clk(ar) flies. Transcription analysis indicates potent effects of Clk(ar) on levels and amplitude of transcriptional oscillations. Taken together with other data, we propose that Clk makes a major contribution to the strength and amplitude of circadian rhythms.

Fig. 1. Alterations in Clk DNA and RNA in Clk^ar flies. (A) Sequence change present at the 5′ splice site of intron 2. (B) Altered splicing of intron 2 in Clk^ar as shown by RT–PCR across intron 2. – indicates negative control (no RNA). + indicates single band in wild type corresponding to RNA without intron 2 (spliced).

Fig. 2. Analysis of splice junctions in Clk^ar transcripts. (A) Schematic of splicing of exons 2 and 3 flanking intron 2. Black box indicates the presence of intron and absence of any detectable splicing of intron 2. Altered transcripts present in Clk^ar alphabetically are indicated as well as the number of clones of that type identified out of 22 clones analyzed. Nucleotide numbers of intron (1–1281 in wild type) indicate sites of splice junctions. Asterisks indicate clones with potential in-frame methionines from the altered upstream exon 2. (B) Conceptual translation of altered transcripts. Top line indicates translation of wild-type transcript from first methionine located in exon 2. Basic DBD (basic) and helix–loop–helix DNA binding and dimerization motif is indicated (HLH) in the translated product. Potential new N-termini from altered exon 2 in Clk^ar-e and -f are indicated from Clk^ar transcripts. (C) Activation of a triplicated per circadian enhancer by CLK^ar in S2 cells. CLK, CLOCK; DBD, CLOCK without a DBD; pAC, empty pActin vector; CEx3-luc is a triplicated circadian enhancer derived from the per promoter; y-axis indicates luminescence relative to CLK where CLK = 100%. Measurements displayed are averages of two independent experiments. Error bars indicate SEM. Some error bars were not visible due to low variability. (D) Expression of CLOCK^ar in transfected cells.

Fig. 3. Abnormal diurnal and circadian locomotor behavior in Clk^ar flies. Normalized plots of locomotor activity in LD (A and B) and first two days of constant darkness (C and D; see Materials and methods) for wild type (+; A and C; n = 16) and Clk^ar flies (B and D; n = 16). Light and dark bars correspond to times of lights-on and -off. Error bars indicate SEM.

Fig. 4. Molecular oscillations in Clk^ar. Western blots of PERIOD (PER; A and B) and TIM (C–F) under 12 h light:12 h dark (A–D) and constant darkness (E and F) conditions. Zeitgeber Time (ZT; B and D) 0 is lights-on and 12 is lights-off. Circadian time (F) indicates time in constant darkness where Circadian Time 0 is 12 h after lights-off. PER quantification (B) is from four independent experiments (three for ZT 1 and 13). TIM quantification in LD (D) is from three independent experiments (two for ZT 1 and 13) and in DD (F) from two independent experiments. Error bars indicate SEM tim transcript oscillations as measured by real-time quantitative PCR (see Materials and methods). (G) Levels of tim transcript measured with respect to Actin control (tim/Actin).

Fig. 5. pdf expression in Clk^ar. Fluorescent in situ hybridization for pdf transcript in wild type (A) and Clk^ar (B) reveals labeling of large (L) and small (S) ventral lateral neurons (LNs) under low (left panel) and high (right panel) magnifications. These two groups of neurons can be detected in both wild type and Clk^ar, although levels are reduced in the small neurons in Clk^ar. High magnification image of Clk^ar is enhanced to ease visualization of the small LNs.

Fig. 6. Reduction in cycling levels and amplitude of per and tim enhancers in Clk^ar. timenh-LUC indicates transgenic flies containing a tim enhancer fused to luciferase. timenhmut-LUC is the same tim enhancer with the E-box site (CACGTG) mutated. CEx3-LUC indicates a 69 bp circadian enhancer from the per promoter triplicated and fused to luciferase. Luciferase activity is expressed in thousands of counts per minute for the tim enhancers (timenh-LUC, timenhmut-LUC) and millions of counts per minute for the per enhancer (CEx3-LUC). Error bars indicate SEM. Zeitgeber time is indicated on the x-axis with ZT0 as time of lights-on and ZT12 as time of lights-off. For timenh-LUC (A), n = 13, for timenh-LUC-Clk^ar (B), n = 16, for timenhmut-LUC (C), n = 17, for CEx3-LUC (D), n = 7 and for CEx3-LUC; Clk^ar (E), n = 5. (F) Amplitude assessment in wild type and Clk^ar. Error bars indicate the SEM across days. An asterisk indicates statistically significant result (P = 0.0001 for timenh; P = 0.002 for CEx3). (G) Phase assessment in wild type and Clk^ar. Times of average peak and trough values are indicated. SEM over several days indicated. No statistically significant differences detected between wild type and Clk^ar.