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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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Incorporating EcR and E75 in the molecular clockEcR is expressed in clock cells of larval (A) and adult brains (B). EcR is detected with two different mouse anti-EcR antibodies (EcR-A and EcR-C) and PDF is stained with a rabbit anti-PDF antibody. EcR-C antibody detects all isoforms of EcR (A, B1 and B2), whereas EcR-A detects the RA specific isoform of EcR. Scale bar = 10μm. (C) Model for the role of E75 in the Drosophila molecular clock. E75 represses Clk transcription, and this repression is inhibited by PER, which thus acts as a de-repressor of Clk. PER can also modulate Clk expression through VRI (as VRI is a transcriptional target of CLK, which is regulated by PER), but this is not shown here for the sake of simplicity. In addition, E75 also regulates VRI expression in such a way that overexpression or knockdown of E75 increases or reduces the VRI levels respectively, thus indirectly affecting the CLK expression. Under stress (nutritional and temperature) conditions, E75 is required to maintain robust rhythms.
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Figure 6: Incorporating EcR and E75 in the molecular clockEcR is expressed in clock cells of larval (A) and adult brains (B). EcR is detected with two different mouse anti-EcR antibodies (EcR-A and EcR-C) and PDF is stained with a rabbit anti-PDF antibody. EcR-C antibody detects all isoforms of EcR (A, B1 and B2), whereas EcR-A detects the RA specific isoform of EcR. Scale bar = 10μm. (C) Model for the role of E75 in the Drosophila molecular clock. E75 represses Clk transcription, and this repression is inhibited by PER, which thus acts as a de-repressor of Clk. PER can also modulate Clk expression through VRI (as VRI is a transcriptional target of CLK, which is regulated by PER), but this is not shown here for the sake of simplicity. In addition, E75 also regulates VRI expression in such a way that overexpression or knockdown of E75 increases or reduces the VRI levels respectively, thus indirectly affecting the CLK expression. Under stress (nutritional and temperature) conditions, E75 is required to maintain robust rhythms.

Mentions: We first established that the ecdysone receptor (EcR) is expressed in circadian neurons. EcR has three isoforms A, B1 and B2, which differ in their N termini26. We obtained antibodies to EcR isoforms and verified that they recognize these specific proteins, based on their reduced levels in EcR RNAi lines and increased levels in flies that over-express EcR (data not shown). The EcR-A antibody is specific for EcR-A isoform, and the EcR-C antibody is known to detect all 3 isoforms of EcR27. Through immunohistochemistry experiments, we identified distinct expression of the EcR-A specific isoform and perhaps other isoforms (as detected by EcR-C) in adult LNvs as well as in the 3rd instar larval stage (Figure 6A, B). To alter EcR activity in clock cells, we utilized RNAi, dominant negative and overexpression approaches, as mutations of EcR are lethal. The dominant negative form of EcR (EcRΔ) cannot be activated by ecdysone and interferes with the activity of endogenous EcR, leading to deficiencies in EcR function26. Expression of EcR-B1Δ by Pdf-Gal4 resulted in a significant increase in period and decreased rhythm strength (Table 2A). Using TUG, UAS-EcR-B1Δ was expressed in broader sets of clock cells, and resulted in a much longer period (~26 hrs) and ~ 30% arrhythmicity, but surprisingly less of an effect on the strength of rhythms in rhythmic flies (Table 2A). We also expressed EcR-B1Δ using the even stronger clock cell Gal4 (TG27), which resulted in 100% lethality. As reported by Itoh et al15, knockdown of EcR using RNAi also yielded circadian phenotypes. EcR-A RNAi in PDF positive cells did not alter period but significantly reduced rhythm strength (Table 2A). On the other hand, EcR-A RNAi using the TUG driver led to a significantly longer period (Table 2A), although again, with less of an effect on rhythm strength.


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)

Incorporating EcR and E75 in the molecular clockEcR is expressed in clock cells of larval (A) and adult brains (B). EcR is detected with two different mouse anti-EcR antibodies (EcR-A and EcR-C) and PDF is stained with a rabbit anti-PDF antibody. EcR-C antibody detects all isoforms of EcR (A, B1 and B2), whereas EcR-A detects the RA specific isoform of EcR. Scale bar = 10μm. (C) Model for the role of E75 in the Drosophila molecular clock. E75 represses Clk transcription, and this repression is inhibited by PER, which thus acts as a de-repressor of Clk. PER can also modulate Clk expression through VRI (as VRI is a transcriptional target of CLK, which is regulated by PER), but this is not shown here for the sake of simplicity. In addition, E75 also regulates VRI expression in such a way that overexpression or knockdown of E75 increases or reduces the VRI levels respectively, thus indirectly affecting the CLK expression. Under stress (nutritional and temperature) conditions, E75 is required to maintain robust rhythms.
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Related In: Results  -  Collection

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Figure 6: Incorporating EcR and E75 in the molecular clockEcR is expressed in clock cells of larval (A) and adult brains (B). EcR is detected with two different mouse anti-EcR antibodies (EcR-A and EcR-C) and PDF is stained with a rabbit anti-PDF antibody. EcR-C antibody detects all isoforms of EcR (A, B1 and B2), whereas EcR-A detects the RA specific isoform of EcR. Scale bar = 10μm. (C) Model for the role of E75 in the Drosophila molecular clock. E75 represses Clk transcription, and this repression is inhibited by PER, which thus acts as a de-repressor of Clk. PER can also modulate Clk expression through VRI (as VRI is a transcriptional target of CLK, which is regulated by PER), but this is not shown here for the sake of simplicity. In addition, E75 also regulates VRI expression in such a way that overexpression or knockdown of E75 increases or reduces the VRI levels respectively, thus indirectly affecting the CLK expression. Under stress (nutritional and temperature) conditions, E75 is required to maintain robust rhythms.
Mentions: We first established that the ecdysone receptor (EcR) is expressed in circadian neurons. EcR has three isoforms A, B1 and B2, which differ in their N termini26. We obtained antibodies to EcR isoforms and verified that they recognize these specific proteins, based on their reduced levels in EcR RNAi lines and increased levels in flies that over-express EcR (data not shown). The EcR-A antibody is specific for EcR-A isoform, and the EcR-C antibody is known to detect all 3 isoforms of EcR27. Through immunohistochemistry experiments, we identified distinct expression of the EcR-A specific isoform and perhaps other isoforms (as detected by EcR-C) in adult LNvs as well as in the 3rd instar larval stage (Figure 6A, B). To alter EcR activity in clock cells, we utilized RNAi, dominant negative and overexpression approaches, as mutations of EcR are lethal. The dominant negative form of EcR (EcRΔ) cannot be activated by ecdysone and interferes with the activity of endogenous EcR, leading to deficiencies in EcR function26. Expression of EcR-B1Δ by Pdf-Gal4 resulted in a significant increase in period and decreased rhythm strength (Table 2A). Using TUG, UAS-EcR-B1Δ was expressed in broader sets of clock cells, and resulted in a much longer period (~26 hrs) and ~ 30% arrhythmicity, but surprisingly less of an effect on the strength of rhythms in rhythmic flies (Table 2A). We also expressed EcR-B1Δ using the even stronger clock cell Gal4 (TG27), which resulted in 100% lethality. As reported by Itoh et al15, knockdown of EcR using RNAi also yielded circadian phenotypes. EcR-A RNAi in PDF positive cells did not alter period but significantly reduced rhythm strength (Table 2A). On the other hand, EcR-A RNAi using the TUG driver led to a significantly longer period (Table 2A), although again, with less of an effect on rhythm strength.

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