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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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E75 represses transcriptional activity of the native Clk promoter(A) Mammalian HEK-293T cells were transfected with Clk-luc (50ng) and renilla-luc (10ng) reporter (internal control) and with increasing doses of CMV-Pdp1ε (10 and 50 ng) or CMV-E75 (50, 100, and 250 ng) and in some cases with CMV-per (10, 50, 100 ng). The (+), (++) and (+++).denote 10, 50, and 100 ng of DNA respectively, except for the empty pCDNA3.1 vector. Additionally, CMV-gfp (10, 50, 100 ng) was used a control for per. The firefly luciferase activity was normalized to renilla-luc activity, and the fold induction (Y axis) was based upon comparisons with 10 ng of CMV expression vector alone transfections. Data represent an average of four experiments each performed in duplicate. Error bars depict standard error of the mean. C3m-TK luc (a mutant promoter, which cannot be activated by PDP1), was used as an additional control. PDP1 ε and E75 significantly activated and repressed Clk-luc respectively, compared to empty vector pCDNA3.1 controls. PER significantly de-repressed the E75 mediated repression of Clk promoter repression. (B) An artificial promoter, C3-TK luc, was used instead of the native Clk-luc promoter to determine if E75 acts as a co-repressor with VRI. C3-TK luc contains 3 tandem consensus binding sites for PDP1/VRI but none for E75. Other details, including the dosage used, are same to panel (A). PDP1 activates this promoter in a dose dependent manner, and this is repressed by VRI. The highest dose of E75 enhances repression by VRI. (*p < 0.05. Two-way ANOVA, Tukey’s test post-hoc comparison).
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Figure 4: E75 represses transcriptional activity of the native Clk promoter(A) Mammalian HEK-293T cells were transfected with Clk-luc (50ng) and renilla-luc (10ng) reporter (internal control) and with increasing doses of CMV-Pdp1ε (10 and 50 ng) or CMV-E75 (50, 100, and 250 ng) and in some cases with CMV-per (10, 50, 100 ng). The (+), (++) and (+++).denote 10, 50, and 100 ng of DNA respectively, except for the empty pCDNA3.1 vector. Additionally, CMV-gfp (10, 50, 100 ng) was used a control for per. The firefly luciferase activity was normalized to renilla-luc activity, and the fold induction (Y axis) was based upon comparisons with 10 ng of CMV expression vector alone transfections. Data represent an average of four experiments each performed in duplicate. Error bars depict standard error of the mean. C3m-TK luc (a mutant promoter, which cannot be activated by PDP1), was used as an additional control. PDP1 ε and E75 significantly activated and repressed Clk-luc respectively, compared to empty vector pCDNA3.1 controls. PER significantly de-repressed the E75 mediated repression of Clk promoter repression. (B) An artificial promoter, C3-TK luc, was used instead of the native Clk-luc promoter to determine if E75 acts as a co-repressor with VRI. C3-TK luc contains 3 tandem consensus binding sites for PDP1/VRI but none for E75. Other details, including the dosage used, are same to panel (A). PDP1 activates this promoter in a dose dependent manner, and this is repressed by VRI. The highest dose of E75 enhances repression by VRI. (*p < 0.05. Two-way ANOVA, Tukey’s test post-hoc comparison).

Mentions: The behavioral and molecular effects of E75 overexpression and knockdown strongly suggested an important role of this nuclear receptor in the molecular clock. To test for a possible function in the transcription of clock genes, we used cell culture assays. Given that the Drosophila E75 and mammalian REV-ERB proteins are so well conserved (~70%) in their DNA binding domains (Supplementary Figure 4A), we first tested E75 for effects on the native mouse Bmal1 promoter. We transfected Bmal1-luc (Bmal1 promoter fused to luciferase) constructs into HEK293T cells and activated expression using mammalian ROR-α. Upon co-transfection with CMV-E75, Bmal1-luc activity was significantly reduced ~ 4 fold (Supplementary Figure 4B). We then used a reporter construct for Clk in which the native promoter of Clk (~ 3.2 Kb) was fused to a luciferase reporter gene2. The Clk promoter contains multiple PDP1/VRI and E75 sites, the latter based on their homology to target sites of mammalian REV-ERB proteins, which bind AGGTCA sites in A/T rich regions20. We transfected the Clk-luc construct into HEK293T cells and assayed its expression in response to E75. As E75 is known to be a transcriptional repressor, but basal levels of Clk-luc are too low to detect further repression, we first activated expression of Clk-luc using Pdp1 (pCDNA3-CMV-Pdp1 ε) (Figure 4A). As reported previously2, we saw strong activation of the Clk promoter by PDP1ε in a dose dependent manner (Figure 4A). Next we co-transfected these cells with different doses of E75 (driven by the CMV promoter in pCDNA3.1) and found that E75 strongly repressed Clk-luc activity (Figure 4A). To verify that E75 acts on the Clk promoter, we constructed an E75-VP16 fusion protein, which is expected to turn E75 into an activator by introducing a VP16 activation domain, and tested its efficacy in regulating Clk-luc. We observed an increase of luciferase activity (Supplementary Figure 4C), suggesting that E75 binds directly to the Clk promoter.


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)

E75 represses transcriptional activity of the native Clk promoter(A) Mammalian HEK-293T cells were transfected with Clk-luc (50ng) and renilla-luc (10ng) reporter (internal control) and with increasing doses of CMV-Pdp1ε (10 and 50 ng) or CMV-E75 (50, 100, and 250 ng) and in some cases with CMV-per (10, 50, 100 ng). The (+), (++) and (+++).denote 10, 50, and 100 ng of DNA respectively, except for the empty pCDNA3.1 vector. Additionally, CMV-gfp (10, 50, 100 ng) was used a control for per. The firefly luciferase activity was normalized to renilla-luc activity, and the fold induction (Y axis) was based upon comparisons with 10 ng of CMV expression vector alone transfections. Data represent an average of four experiments each performed in duplicate. Error bars depict standard error of the mean. C3m-TK luc (a mutant promoter, which cannot be activated by PDP1), was used as an additional control. PDP1 ε and E75 significantly activated and repressed Clk-luc respectively, compared to empty vector pCDNA3.1 controls. PER significantly de-repressed the E75 mediated repression of Clk promoter repression. (B) An artificial promoter, C3-TK luc, was used instead of the native Clk-luc promoter to determine if E75 acts as a co-repressor with VRI. C3-TK luc contains 3 tandem consensus binding sites for PDP1/VRI but none for E75. Other details, including the dosage used, are same to panel (A). PDP1 activates this promoter in a dose dependent manner, and this is repressed by VRI. The highest dose of E75 enhances repression by VRI. (*p < 0.05. Two-way ANOVA, Tukey’s test post-hoc comparison).
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Related In: Results  -  Collection

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Figure 4: E75 represses transcriptional activity of the native Clk promoter(A) Mammalian HEK-293T cells were transfected with Clk-luc (50ng) and renilla-luc (10ng) reporter (internal control) and with increasing doses of CMV-Pdp1ε (10 and 50 ng) or CMV-E75 (50, 100, and 250 ng) and in some cases with CMV-per (10, 50, 100 ng). The (+), (++) and (+++).denote 10, 50, and 100 ng of DNA respectively, except for the empty pCDNA3.1 vector. Additionally, CMV-gfp (10, 50, 100 ng) was used a control for per. The firefly luciferase activity was normalized to renilla-luc activity, and the fold induction (Y axis) was based upon comparisons with 10 ng of CMV expression vector alone transfections. Data represent an average of four experiments each performed in duplicate. Error bars depict standard error of the mean. C3m-TK luc (a mutant promoter, which cannot be activated by PDP1), was used as an additional control. PDP1 ε and E75 significantly activated and repressed Clk-luc respectively, compared to empty vector pCDNA3.1 controls. PER significantly de-repressed the E75 mediated repression of Clk promoter repression. (B) An artificial promoter, C3-TK luc, was used instead of the native Clk-luc promoter to determine if E75 acts as a co-repressor with VRI. C3-TK luc contains 3 tandem consensus binding sites for PDP1/VRI but none for E75. Other details, including the dosage used, are same to panel (A). PDP1 activates this promoter in a dose dependent manner, and this is repressed by VRI. The highest dose of E75 enhances repression by VRI. (*p < 0.05. Two-way ANOVA, Tukey’s test post-hoc comparison).
Mentions: The behavioral and molecular effects of E75 overexpression and knockdown strongly suggested an important role of this nuclear receptor in the molecular clock. To test for a possible function in the transcription of clock genes, we used cell culture assays. Given that the Drosophila E75 and mammalian REV-ERB proteins are so well conserved (~70%) in their DNA binding domains (Supplementary Figure 4A), we first tested E75 for effects on the native mouse Bmal1 promoter. We transfected Bmal1-luc (Bmal1 promoter fused to luciferase) constructs into HEK293T cells and activated expression using mammalian ROR-α. Upon co-transfection with CMV-E75, Bmal1-luc activity was significantly reduced ~ 4 fold (Supplementary Figure 4B). We then used a reporter construct for Clk in which the native promoter of Clk (~ 3.2 Kb) was fused to a luciferase reporter gene2. The Clk promoter contains multiple PDP1/VRI and E75 sites, the latter based on their homology to target sites of mammalian REV-ERB proteins, which bind AGGTCA sites in A/T rich regions20. We transfected the Clk-luc construct into HEK293T cells and assayed its expression in response to E75. As E75 is known to be a transcriptional repressor, but basal levels of Clk-luc are too low to detect further repression, we first activated expression of Clk-luc using Pdp1 (pCDNA3-CMV-Pdp1 ε) (Figure 4A). As reported previously2, we saw strong activation of the Clk promoter by PDP1ε in a dose dependent manner (Figure 4A). Next we co-transfected these cells with different doses of E75 (driven by the CMV promoter in pCDNA3.1) and found that E75 strongly repressed Clk-luc activity (Figure 4A). To verify that E75 acts on the Clk promoter, we constructed an E75-VP16 fusion protein, which is expected to turn E75 into an activator by introducing a VP16 activation domain, and tested its efficacy in regulating Clk-luc. We observed an increase of luciferase activity (Supplementary Figure 4C), suggesting that E75 binds directly to the Clk promoter.

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