Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior

Abstract

In Drosophila, a number of key processes such as emergence from the pupal case, locomotor activity, feeding, olfaction, and aspects of mating behavior are under circadian regulation. Although we have a basic understanding of how the molecular oscillations take place, a clear link between gene regulation and downstream biological processes is still missing. To identify clock-controlled output genes, we have used an oligonucleotide-based high-density array that interrogates gene expression changes on a whole genome level. We found genes regulating various physiological processes to be under circadian transcriptional regulation, ranging from protein stability and degradation, signal transduction, heme metabolism, detoxification, and immunity. By comparing rhythmically expressed genes in the fly head and body, we found that the clock has adapted its output functions to the needs of each particular tissue, implying that tissue-specific regulation is superimposed on clock control of gene expression. Finally, taking full advantage of the fly as a model system, we have identified and characterized a cycling potassium channel protein as a key step in linking the transcriptional feedback loop to rhythmic locomotor behavior.

Fig. 1.

A, Output genes peak throughout the day in the fly head and body. Genes were grouped according to the phase calculated by COSOPT; each cluster represents genes peaking at the specified time ± 2 hr. ZT0 refers to the time when lights were switched on. Cluster size is represented as percentage of the total number of cycling genes. B, The clock controls different subsets of genes in head and body fractions. Distribution of genes cycling in both tissues, cycling and expressed in only one, and expressed to mid-high levels in both but cycling in one is displayed.

Fig. 2.

Northern blot analysis of several clock-controlled genes. Northern blot analysis of independent head and body time courses confirms both the cyclic nature of the candidates tested and their respective phases of expression. Gene ID refers to the cDNA used as probes. The right columns indicate the expected peak time and the corresponding p value predicted by the array experiment, respectively. Theasterisk indicates a gene that is outside of thep < 0.01 cutoff and still appears rhythmic. According to our experiments, the CT18196 transcript has smaller size than predicted.

Fig. 3.

Cyclic patterns of clock-controlled genes in the fly head and body under entrained conditions. Complementary RNA samples were prepared as described and hybridized to duplicate DNA GeneChips. Data were normalized such that the mean expression level for each particular gene over the course of all time points equals 1. The average signal strength at each time point was then expressed as a ratio over the median signal strength for that particular gene. Representative traces of cycling transcripts implicated in various physiologies and metabolic pathways are shown inA–F. A, Clock control of protein stability. pros26 (light blue),pros26.4 (green),rpn9 (purple), and ubiquitin thiolesterase (red). B, Heme metabolism. The gene encoding alas is in red; heme-oxygenase is in light blue. C, Genes implicated in detoxification are under circadian regulation. Phase I cytochrome P450s are colored as follows: cyp4e3 (green), cyp6a2 (light blue), cyp6a17 (brown),cyp6a21 (red), cyp6d5 (dark blue), and cyp18 (pink). D, Phase II genes:ugt35b in red, GST3 inpurple, and two uncharacterized GSTs (CT38753 and CT38747) in light blue and green, respectively. (E) Neurotransmission.ple expression under entrained and free-running conditions is depicted in red and blue, respectively. F, Immunity. A number of genes involved in different aspects of innate immunity are under circadian control and are indicated as follows: in recognition and phagocytosis: Agr5 (light brown), a peptidoglycan protein (light green), lectin galC (aqua), Idgf4 (brown), CT29102 (deep blue); antimicrobial peptides: lysX (red) and CT 30310 (pink); Chitinase-like molecules: Chit (light blue) and CT5624 (dark green). Relative intensities ± SEM at each time point are shown.White and black boxes on theabscissa represent the duration of the light and dark periods. Hatched boxes (E) indicate subjective day.

Fig. 4.

slo and slob cycle in LD and DD in wild-type flies. Shown is circadian pattern of expression of slob (gray) andslo (black) under entrained (A) and free-running (B) conditions. Relative intensities ± SEM at each time point are shown. C, Representative example of a Western blot to detect the SLO protein under LD conditions. Flies were entrained and collected as described. The first and last lanes correspond to protein extracts collected at ZT16 fromslo4 and tim⁰ flies, respectively. The graph indicates the quantification of SLO levels throughout the day (normalized to HSP-70).

Fig. 5.

slowpoke is required for sustained rhythmic behavior. Representative actograms of wild-type CS andy w flies (left) and the mutantsper⁰, Clk^jrk, and arrhythmicslo I and slo 4 flies are displayed. Flies were entrained for 5 d before the onset of the experiment. During the experiments, flies were kept in LD for 3–4 d and then switched to DD and monitored for at least another week. Rhythmicity and total activity in LD and DD conditions were determined using the Clocklab software package.

Fig. 6.

Mutations in slowpoke disrupt the consolidation of activity around the transitions. Average activity plots for CS, slo 4, and slo I mutant flies are shown. Activity records of the LD portion of the experiment for 53 wild-type, 28 slo 4, and 56 slo I flies were used for the analysis. To superimpose the separate animal records, the levels of activity were normalized per fly per day. Each vertical bar represents the mean ± SD.