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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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Effect of E75 overexpression on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies during the indicated phases of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies under LD cycle. (C) PER and CLK levels in the genotypes indicated above. A representative western blot is shown. PER and CLK levels are significantly lower in the TG27 >UAS-E75 (II) flies than in TG27 control flies particularly at peak time points. HSP70 antibodies are used to control for loading. Quantification of four independent experiments shows significantly decreased (D) PER and (E) CLK levels in TG27 >UAS-E75 (II) flies relative to the TG27 control flies. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. A molecular marker (Precision Plus Protein™ Dual Color Standards) was run to detect the exact molecular size of different proteins.
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Figure 1: Effect of E75 overexpression on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies during the indicated phases of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies under LD cycle. (C) PER and CLK levels in the genotypes indicated above. A representative western blot is shown. PER and CLK levels are significantly lower in the TG27 >UAS-E75 (II) flies than in TG27 control flies particularly at peak time points. HSP70 antibodies are used to control for loading. Quantification of four independent experiments shows significantly decreased (D) PER and (E) CLK levels in TG27 >UAS-E75 (II) flies relative to the TG27 control flies. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. A molecular marker (Precision Plus Protein™ Dual Color Standards) was run to detect the exact molecular size of different proteins.

Mentions: To determine whether E75 affects molecular clock components, we first examined transcript levels of per and Clk in whole head extracts of flies overexpressing E75 (UAS-E75 II) via the TG27 driver. Oscillations of per and Clk were dampened by E75 over-expression, in particular through a reduction in peak levels (Figure 1A and B). We also measured PER and CLK protein levels through western blots of whole head lysates and found that these were significantly reduced in E75 overexpressing flies under LD conditions (Figure 1C,D and E). Similar effects were observed under constant dark (DD) conditions; CLK expression was significantly reduced in flies overexpressing E75 as compared to the controls (Supplementary Figure 2A 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)

Effect of E75 overexpression on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies during the indicated phases of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies under LD cycle. (C) PER and CLK levels in the genotypes indicated above. A representative western blot is shown. PER and CLK levels are significantly lower in the TG27 >UAS-E75 (II) flies than in TG27 control flies particularly at peak time points. HSP70 antibodies are used to control for loading. Quantification of four independent experiments shows significantly decreased (D) PER and (E) CLK levels in TG27 >UAS-E75 (II) flies relative to the TG27 control flies. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. A molecular marker (Precision Plus Protein™ Dual Color Standards) was run to detect the exact molecular size of different proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4269253&req=5

Figure 1: Effect of E75 overexpression on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies during the indicated phases of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 (II) flies under LD cycle. (C) PER and CLK levels in the genotypes indicated above. A representative western blot is shown. PER and CLK levels are significantly lower in the TG27 >UAS-E75 (II) flies than in TG27 control flies particularly at peak time points. HSP70 antibodies are used to control for loading. Quantification of four independent experiments shows significantly decreased (D) PER and (E) CLK levels in TG27 >UAS-E75 (II) flies relative to the TG27 control flies. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM. A molecular marker (Precision Plus Protein™ Dual Color Standards) was run to detect the exact molecular size of different proteins.
Mentions: To determine whether E75 affects molecular clock components, we first examined transcript levels of per and Clk in whole head extracts of flies overexpressing E75 (UAS-E75 II) via the TG27 driver. Oscillations of per and Clk were dampened by E75 over-expression, in particular through a reduction in peak levels (Figure 1A and B). We also measured PER and CLK protein levels through western blots of whole head lysates and found that these were significantly reduced in E75 overexpressing flies under LD conditions (Figure 1C,D and E). Similar effects were observed under constant dark (DD) conditions; CLK expression was significantly reduced in flies overexpressing E75 as compared to the controls (Supplementary Figure 2A 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