The circadian output gene takeout is regulated by Pdp1epsilon

Abstract

The circadian clock controls many circadian outputs. Although a large number of transcripts are affected by the circadian oscillator, very little is known about their regulation and function. We show here that the Drosophila takeout gene, one of the output genes of the circadian oscillator, is regulated similarly to the circadian clock genes Clock (Clk) and cry. takeout RNA levels are at constant high levels in Clk(JRK) mutants. The circadian transcription factor PAR domain protein 1 (Pdp1epsilon) is a transcription factor that had previously been postulated to control clock output genes, particularly genes regulated similarly to Clk. In agreement with this, we show here that Pdp1epsilon is a regulator of takeout. Takeout levels are low in flies with reduced Pdp1epsilon and high in flies with increased amounts of Pdp1epsilon. Furthermore, flies with reduced or elevated Pdp1epsilon levels in the fat body display courtship defects, identifying Pdp1epsilon as an important transcriptional regulator in that tissue.

Fig. 1.

(A and B) Circadian takeout RNA expression in Clk^JRK(CS) males is constant at high levels. takeout qPCR analysis of male head RNA from flies collected at the indicated times under (A) LD (ZT) and (B) DD conditions (CT). Relative to mRNA levels were quantified as described in Materials and Methods. RNA levels were normalized to the amount in control CS flies at ZT1. Data are from three independent repeats. Original Clk^JRK mutants, mutants carrying a high-expressing takeout allele Clk^JRK(CS), and CS control flies were examined. (C and D) Takeout (TO) protein levels are under posttranscriptional control. Quantification of TO levels from Western blots. TO levels were quantified as described in Materials and Methods and normalized to wild-type levels. Data are from three independent repeats. Proteins from heads of wild-type CS and Clk^JRK(CS) males were examined. (C) Flies were collected at the indicated times under LD conditions (ZT). (D) Flies were entrained for 3 days and collected at the indicated times on the first day of DD.

Fig. 2.

takeout RNA and protein levels are low in flies with reduced Pdp1ε levels. takeout RNA (A) and protein (B) levels are strongly reduced under constant low levels of PDP1ε. PDP1 levels were reduced by expression of UAS-Pdp1i using the timGal4 driver. RNA and protein from heads of timGal4/UAS-Pdp1i males was compared to that of the corresponding control genotypes (+/UAS-Pdp1i and +/timGal4). Flies were entrained for 3 days and collected at the indicated times on the first day of DD. Data are from three independent repeats.

Fig. 3.

takeout RNA and protein levels are elevated in Pdp1ε over-expressing flies. takeout RNA (A) and protein (B) levels are increased in flies that overexpress PDP1ε. PDP1 was over-expressed by expression of UAS-Pdp1 by the timGal4 driver. RNA and protein from heads of timGal4/UAS-Pdp1 males was compared to that of the corresponding control genotypes (+/UAS-Pdp1 and +/timGal4). Flies were entrained for 3 days and collected at the indicated times on the first day of DD. Data are from three independent repeats.

Fig. 4.

Males with decreased or increased Pdp1ε levels or disrupted fat body clock show reduced courtship. Courtship indices (± SEM) of males toward wild-type virgin females. (A) timGal4 driven Pdp1 overexpression (timGal4/UAS-Pdp1) or reduction (timGal4/UAS-Pdp1i) results in reduced male courtship indices. Mutant males are compared to the corresponding control males. (B) Males with decreased or increased levels of PDP in the fat body show reduced courtship. The fat body specific Lsp2-Gal4 driver was used to express UAS-Pdp1i or UAS-Pdp1. n = 10 for all genotypes. (C and D) The courtship indices of males expressing a dominant negative form of Clk (dnClk) in the fat body is shown. (C) The fat body specific Lsp2-Gal4 driver was used to express dnClk. The presence of Gal80^ts allows induction of dnClk only in adult males. Expression was induced by exposing mature males to 32 °C overnight. Experimental and control males were treated equally. Testing occurred 2–4 h later. (D) The to-Gal4 driver was used to express dnClk predominantly in fat body. (E) Activity assay of the genotypes in D. The number of line crossings was counted.