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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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PER interacts with E75 to de-repress expression of the Clk promoter(A) Co-immunoprecipitation assay showed that PER physically interacts with E75. 100 ng of CMV-per HA was transfected with 200 ng of CMV-E75 V5 (with two different isoforms RC and RA), CMV-cry V5, CMV-GABA-Transaminase (GABAT) V5 and empty CMV vector. Anti-V5 antibody was used to pull down protein complexes. PER specifically binds with CRY but not with GABA-T, PER also binds strongly with E75-RC. The RA isoform was poorly expressed, hence weaker interaction with PER (lane 5 in IP: αV5). (B) Knockdown of E75 increases Clk mRNA expression in wild type and per0 flies. As in Figure 2, E75 knock down by TG27 significantly increases Clk mRNA levels in wild type flies relative to TG27 controls at ZT14. This effect is more striking in per0 flies where baseline Clk mRNA levels are quite low. (C) Overexpression of E75 reduces Clk mRNA expression in wild type and per0 flies. As in Figure 1, E75 overexpression by TG27 significantly decreases Clk mRNA levels in wild type and per0 flies relative to TG27 controls at ZT02. (D) CLK levels in TG27 control, TG27 >UAS-E75 RNAi (GD) and TG27 > UAS-E75 (II) in wild type and per0 backgrounds are shown for two time points. HSP70 antibodies are used to control for loading. Molecular marker (Precision Plus Protein™ Dual Color Standards) were run to facilitate detection of different proteins. (D) Quantification of four independent western blots is shown. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM and indicate variability across flies of a specific genotype.
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Figure 5: PER interacts with E75 to de-repress expression of the Clk promoter(A) Co-immunoprecipitation assay showed that PER physically interacts with E75. 100 ng of CMV-per HA was transfected with 200 ng of CMV-E75 V5 (with two different isoforms RC and RA), CMV-cry V5, CMV-GABA-Transaminase (GABAT) V5 and empty CMV vector. Anti-V5 antibody was used to pull down protein complexes. PER specifically binds with CRY but not with GABA-T, PER also binds strongly with E75-RC. The RA isoform was poorly expressed, hence weaker interaction with PER (lane 5 in IP: αV5). (B) Knockdown of E75 increases Clk mRNA expression in wild type and per0 flies. As in Figure 2, E75 knock down by TG27 significantly increases Clk mRNA levels in wild type flies relative to TG27 controls at ZT14. This effect is more striking in per0 flies where baseline Clk mRNA levels are quite low. (C) Overexpression of E75 reduces Clk mRNA expression in wild type and per0 flies. As in Figure 1, E75 overexpression by TG27 significantly decreases Clk mRNA levels in wild type and per0 flies relative to TG27 controls at ZT02. (D) CLK levels in TG27 control, TG27 >UAS-E75 RNAi (GD) and TG27 > UAS-E75 (II) in wild type and per0 backgrounds are shown for two time points. HSP70 antibodies are used to control for loading. Molecular marker (Precision Plus Protein™ Dual Color Standards) were run to facilitate detection of different proteins. (D) Quantification of four independent western blots is shown. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM and indicate variability across flies of a specific genotype.

Mentions: To determine whether E75 and PER physically interact, we co-transfected them into mammalian HEK293T cells as well as into Drosophila S2 cells and conducted co-immunoprecipitation assays. E75 tagged with V5 pulled down PER in HEK 293T cells (Figure 5A and Supplementary Figure 6A). CRY-V5 also pulled down PER under these conditions, whereas a non-specific GABA-T-V5 (GABA-transaminase fused to a V5 tag) protein did not, indicating specificity of the interaction between E75 and PER and also between CRY and PER (Figure 5A and Supplementary Figure 6A). Similar results were obtained in Drosophila S2 cells, where PER pulled down V5-tagged E75 (Supplementary Figure 6B).


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)

PER interacts with E75 to de-repress expression of the Clk promoter(A) Co-immunoprecipitation assay showed that PER physically interacts with E75. 100 ng of CMV-per HA was transfected with 200 ng of CMV-E75 V5 (with two different isoforms RC and RA), CMV-cry V5, CMV-GABA-Transaminase (GABAT) V5 and empty CMV vector. Anti-V5 antibody was used to pull down protein complexes. PER specifically binds with CRY but not with GABA-T, PER also binds strongly with E75-RC. The RA isoform was poorly expressed, hence weaker interaction with PER (lane 5 in IP: αV5). (B) Knockdown of E75 increases Clk mRNA expression in wild type and per0 flies. As in Figure 2, E75 knock down by TG27 significantly increases Clk mRNA levels in wild type flies relative to TG27 controls at ZT14. This effect is more striking in per0 flies where baseline Clk mRNA levels are quite low. (C) Overexpression of E75 reduces Clk mRNA expression in wild type and per0 flies. As in Figure 1, E75 overexpression by TG27 significantly decreases Clk mRNA levels in wild type and per0 flies relative to TG27 controls at ZT02. (D) CLK levels in TG27 control, TG27 >UAS-E75 RNAi (GD) and TG27 > UAS-E75 (II) in wild type and per0 backgrounds are shown for two time points. HSP70 antibodies are used to control for loading. Molecular marker (Precision Plus Protein™ Dual Color Standards) were run to facilitate detection of different proteins. (D) Quantification of four independent western blots is shown. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM and indicate variability across flies of a specific genotype.
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Figure 5: PER interacts with E75 to de-repress expression of the Clk promoter(A) Co-immunoprecipitation assay showed that PER physically interacts with E75. 100 ng of CMV-per HA was transfected with 200 ng of CMV-E75 V5 (with two different isoforms RC and RA), CMV-cry V5, CMV-GABA-Transaminase (GABAT) V5 and empty CMV vector. Anti-V5 antibody was used to pull down protein complexes. PER specifically binds with CRY but not with GABA-T, PER also binds strongly with E75-RC. The RA isoform was poorly expressed, hence weaker interaction with PER (lane 5 in IP: αV5). (B) Knockdown of E75 increases Clk mRNA expression in wild type and per0 flies. As in Figure 2, E75 knock down by TG27 significantly increases Clk mRNA levels in wild type flies relative to TG27 controls at ZT14. This effect is more striking in per0 flies where baseline Clk mRNA levels are quite low. (C) Overexpression of E75 reduces Clk mRNA expression in wild type and per0 flies. As in Figure 1, E75 overexpression by TG27 significantly decreases Clk mRNA levels in wild type and per0 flies relative to TG27 controls at ZT02. (D) CLK levels in TG27 control, TG27 >UAS-E75 RNAi (GD) and TG27 > UAS-E75 (II) in wild type and per0 backgrounds are shown for two time points. HSP70 antibodies are used to control for loading. Molecular marker (Precision Plus Protein™ Dual Color Standards) were run to facilitate detection of different proteins. (D) Quantification of four independent western blots is shown. Asterisks above the bars denote significant differences between genotypes. (*) P < 0.05 using unpaired Student’s t-test. Error bars depict SEM and indicate variability across flies of a specific genotype.
Mentions: To determine whether E75 and PER physically interact, we co-transfected them into mammalian HEK293T cells as well as into Drosophila S2 cells and conducted co-immunoprecipitation assays. E75 tagged with V5 pulled down PER in HEK 293T cells (Figure 5A and Supplementary Figure 6A). CRY-V5 also pulled down PER under these conditions, whereas a non-specific GABA-T-V5 (GABA-transaminase fused to a V5 tag) protein did not, indicating specificity of the interaction between E75 and PER and also between CRY and PER (Figure 5A and Supplementary Figure 6A). Similar results were obtained in Drosophila S2 cells, where PER pulled down V5-tagged E75 (Supplementary Figure 6B).

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