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An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila.

Kumar S, Chen D, Jang C, Nall A, Zheng X, Sehgal A - Nat Commun (2014)

Bottom Line: PER inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of PER on Clk transcription.The ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms.We find that E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Little is known about molecular links between circadian clocks and steroid hormone signalling, although both are important for normal physiology. Here we report a circadian function for a nuclear receptor, ecdysone-induced protein 75 (Eip75/E75), which we identified through a gain-of-function screen for circadian genes in Drosophila melanogaster. Overexpression or knockdown of E75 in clock neurons disrupts rest:activity rhythms and dampens molecular oscillations. E75 represses expression of the gene encoding the transcriptional activator, CLOCK (CLK), and may also affect circadian output. PER inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of PER on Clk transcription. The ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms. We find that E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila.

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Knockdown of E75 increases PER expression in brain clock cellsFlies from TG27 and TG27> E75 RNAi genotypes were tested for their circadian behavior under DD conditions on 6th day and 8–10 rhythmic TG27 controls and 8–10 arrhythmic E75 knockdown flies were used for IHC at each of the indicated time points. PER expression at different times of day in TG27>UAS-E75 RNAi (GD) flies and TG27 controls in the (A) small and large LNvs and (B) Dorsal LNs (LNds). Quantification of PER staining from (C) s-LNvs and (D) l-LNvs subset of neurons in the TG27 >UAS-E75 RNAi (GD) flies (n=10) relative to TG27 control flies (n=9). Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. Time is indicated as CT (circadian time) where CT0 is 12h after lights-off of the last LD cycle. Scale bar = 10 μm.
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Figure 3: Knockdown of E75 increases PER expression in brain clock cellsFlies from TG27 and TG27> E75 RNAi genotypes were tested for their circadian behavior under DD conditions on 6th day and 8–10 rhythmic TG27 controls and 8–10 arrhythmic E75 knockdown flies were used for IHC at each of the indicated time points. PER expression at different times of day in TG27>UAS-E75 RNAi (GD) flies and TG27 controls in the (A) small and large LNvs and (B) Dorsal LNs (LNds). Quantification of PER staining from (C) s-LNvs and (D) l-LNvs subset of neurons in the TG27 >UAS-E75 RNAi (GD) flies (n=10) relative to TG27 control flies (n=9). Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. Time is indicated as CT (circadian time) where CT0 is 12h after lights-off of the last LD cycle. Scale bar = 10 μm.

Mentions: Clock proteins in adult head extracts are derived largely from the eyes, which do not contribute to the behavioral rhythm. Therefore we also assayed PER levels through immunohistochemistry in circadian behavior-relevant brain clock neurons of flies with reduced levels of E75. As the behavioral phenotype produced by E75 knockdown was somewhat variable (perhaps due to inefficient knockdown), we first selected arrhythmic flies by assaying their behavior and then collected 8–10 flies from each experimental and control group at four different times of day. TG27 mediated knockdown of E75 resulted in dampened cycling of PER in constant darkness in different subsets of clock neurons (Figure 3A and, B). The dampening appeared to arise from significantly higher expression at trough time points (CT8 and CT14) (Figure 3A and B). As the PDF cells are the ones most relevant for free-running behavior, we quantified PER expression in these cells, and found significant differences at CT8 and 14 in the small LNvs and at CT8 in the large LNvs (Figure 3C, D). PER cycling was also dampened under light:dark (LD) cycles; in fact, under these conditions, PER levels appeared to be relatively higher at all times in LNvs and LNds of the E75 knockdown flies (Supplementary Figure 3A and B).


An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila.

Kumar S, Chen D, Jang C, Nall A, Zheng X, Sehgal A - Nat Commun (2014)

Knockdown of E75 increases PER expression in brain clock cellsFlies from TG27 and TG27> E75 RNAi genotypes were tested for their circadian behavior under DD conditions on 6th day and 8–10 rhythmic TG27 controls and 8–10 arrhythmic E75 knockdown flies were used for IHC at each of the indicated time points. PER expression at different times of day in TG27>UAS-E75 RNAi (GD) flies and TG27 controls in the (A) small and large LNvs and (B) Dorsal LNs (LNds). Quantification of PER staining from (C) s-LNvs and (D) l-LNvs subset of neurons in the TG27 >UAS-E75 RNAi (GD) flies (n=10) relative to TG27 control flies (n=9). Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. Time is indicated as CT (circadian time) where CT0 is 12h after lights-off of the last LD cycle. Scale bar = 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4269253&req=5

Figure 3: Knockdown of E75 increases PER expression in brain clock cellsFlies from TG27 and TG27> E75 RNAi genotypes were tested for their circadian behavior under DD conditions on 6th day and 8–10 rhythmic TG27 controls and 8–10 arrhythmic E75 knockdown flies were used for IHC at each of the indicated time points. PER expression at different times of day in TG27>UAS-E75 RNAi (GD) flies and TG27 controls in the (A) small and large LNvs and (B) Dorsal LNs (LNds). Quantification of PER staining from (C) s-LNvs and (D) l-LNvs subset of neurons in the TG27 >UAS-E75 RNAi (GD) flies (n=10) relative to TG27 control flies (n=9). Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. Time is indicated as CT (circadian time) where CT0 is 12h after lights-off of the last LD cycle. Scale bar = 10 μm.
Mentions: Clock proteins in adult head extracts are derived largely from the eyes, which do not contribute to the behavioral rhythm. Therefore we also assayed PER levels through immunohistochemistry in circadian behavior-relevant brain clock neurons of flies with reduced levels of E75. As the behavioral phenotype produced by E75 knockdown was somewhat variable (perhaps due to inefficient knockdown), we first selected arrhythmic flies by assaying their behavior and then collected 8–10 flies from each experimental and control group at four different times of day. TG27 mediated knockdown of E75 resulted in dampened cycling of PER in constant darkness in different subsets of clock neurons (Figure 3A and, B). The dampening appeared to arise from significantly higher expression at trough time points (CT8 and CT14) (Figure 3A and B). As the PDF cells are the ones most relevant for free-running behavior, we quantified PER expression in these cells, and found significant differences at CT8 and 14 in the small LNvs and at CT8 in the large LNvs (Figure 3C, D). PER cycling was also dampened under light:dark (LD) cycles; in fact, under these conditions, PER levels appeared to be relatively higher at all times in LNvs and LNds of the E75 knockdown flies (Supplementary Figure 3A and B).

Bottom Line: PER inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of PER on Clk transcription.The ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms.We find that E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Little is known about molecular links between circadian clocks and steroid hormone signalling, although both are important for normal physiology. Here we report a circadian function for a nuclear receptor, ecdysone-induced protein 75 (Eip75/E75), which we identified through a gain-of-function screen for circadian genes in Drosophila melanogaster. Overexpression or knockdown of E75 in clock neurons disrupts rest:activity rhythms and dampens molecular oscillations. E75 represses expression of the gene encoding the transcriptional activator, CLOCK (CLK), and may also affect circadian output. PER inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of PER on Clk transcription. The ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms. We find that E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila.

Show MeSH