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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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Effects of E75 knockdown on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies at the indicated time points of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies under LD cycle. (C) PER and CLK levels in the same genotypes as above. A representative western blot is shown. HSP70 antibodies are used to control for loading. Quantification of six independent experiments shows (D) PER and (E) CLK levels in TG27 >UAS-E75 RNAi (GD) flies and 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 2: Effects of E75 knockdown on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies at the indicated time points of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies under LD cycle. (C) PER and CLK levels in the same genotypes as above. A representative western blot is shown. HSP70 antibodies are used to control for loading. Quantification of six independent experiments shows (D) PER and (E) CLK levels in TG27 >UAS-E75 RNAi (GD) flies and 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: Next we examined the effects of knocking down E75 using the same TG27 driver to express UAS-E75 RNAi (GD) constructs. As shown in Table 1B, the GD line in particular led to a strong behavioral phenotype. As predicted by the overexpression data, per transcript levels were slightly, although not significantly, increased in flies in which E75 was knocked down, although PER protein levels were significantly different at ZT08 (Figure 2A, C and D). Clk mRNA and protein levels were also significantly higher at specific times of day (Figure 2B, C and E). Notably under these conditions, per and Clk mRNA cycling did not appear to be affected (Figure 2A–E). As with E75 overexpression, knockdown had robust effects on CLK expression under freerunning conditions. CLK levels were significantly higher at CT08 and CT14 on the first day of DD in E75 knockdown flies (Supplementary Figure 2C and D).


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)

Effects of E75 knockdown on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies at the indicated time points of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies under LD cycle. (C) PER and CLK levels in the same genotypes as above. A representative western blot is shown. HSP70 antibodies are used to control for loading. Quantification of six independent experiments shows (D) PER and (E) CLK levels in TG27 >UAS-E75 RNAi (GD) flies and 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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Figure 2: Effects of E75 knockdown on the expression of per and Clk in adult heads(A)per mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies at the indicated time points of an LD cycle. (B)Clk mRNA expression in TG27 controls and TG27 >UAS-E75 RNAi (GD) flies under LD cycle. (C) PER and CLK levels in the same genotypes as above. A representative western blot is shown. HSP70 antibodies are used to control for loading. Quantification of six independent experiments shows (D) PER and (E) CLK levels in TG27 >UAS-E75 RNAi (GD) flies and 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: Next we examined the effects of knocking down E75 using the same TG27 driver to express UAS-E75 RNAi (GD) constructs. As shown in Table 1B, the GD line in particular led to a strong behavioral phenotype. As predicted by the overexpression data, per transcript levels were slightly, although not significantly, increased in flies in which E75 was knocked down, although PER protein levels were significantly different at ZT08 (Figure 2A, C and D). Clk mRNA and protein levels were also significantly higher at specific times of day (Figure 2B, C and E). Notably under these conditions, per and Clk mRNA cycling did not appear to be affected (Figure 2A–E). As with E75 overexpression, knockdown had robust effects on CLK expression under freerunning conditions. CLK levels were significantly higher at CT08 and CT14 on the first day of DD in E75 knockdown flies (Supplementary Figure 2C and D).

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