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The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence.

Huang YC, Lu YN, Wu JT, Chien CT, Pi H - PLoS Genet. (2014)

Bottom Line: We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM.Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level.We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Insitute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT
During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM.

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Association of CSN subunits and EcR in BR-Z1 repression.(A) The BR-Z1 mRNA level in wing discs 20–24 h APF, assayed by semi-quantitative RT-PCR, was upregulated in wing discs of CSN2 RNAi driven by en-GAL4 compared to en-GAL4/+ control. rp49 levels served as internal controls. (B–C′) EcRE-lacZ (red) expression was upregulated in CSN4 (B, B′) and CSN5 (C, C′) clones 20–24 h APF, but unaffected in CSN5 clones 6–8 h APF (D, D′). (E–E″) Both EcRA (red) and Myc-CSN2 (green) localized in nuclei in wing disc 20–24 h APF. (F) Western blots showing co-precipitations of Myc-EcRA (upper panel) or Myc-EcRB1 (lower panel) in Flag immunoprecipitates of Flag-USP, CSN2, CSN4 or CSN5 in S2 cell extract. Co-precipitation was not detected in Flag-GFP. * represents the non-specific bands. (G, G′) BR-Z1 levels (red) in double MARCM clones for CSN5 and DN-EcR (green) were not further elevated compared to CSN5 or DN-EcR single clones (Figure 3B, 6B).
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pgen-1004760-g007: Association of CSN subunits and EcR in BR-Z1 repression.(A) The BR-Z1 mRNA level in wing discs 20–24 h APF, assayed by semi-quantitative RT-PCR, was upregulated in wing discs of CSN2 RNAi driven by en-GAL4 compared to en-GAL4/+ control. rp49 levels served as internal controls. (B–C′) EcRE-lacZ (red) expression was upregulated in CSN4 (B, B′) and CSN5 (C, C′) clones 20–24 h APF, but unaffected in CSN5 clones 6–8 h APF (D, D′). (E–E″) Both EcRA (red) and Myc-CSN2 (green) localized in nuclei in wing disc 20–24 h APF. (F) Western blots showing co-precipitations of Myc-EcRA (upper panel) or Myc-EcRB1 (lower panel) in Flag immunoprecipitates of Flag-USP, CSN2, CSN4 or CSN5 in S2 cell extract. Co-precipitation was not detected in Flag-GFP. * represents the non-specific bands. (G, G′) BR-Z1 levels (red) in double MARCM clones for CSN5 and DN-EcR (green) were not further elevated compared to CSN5 or DN-EcR single clones (Figure 3B, 6B).

Mentions: EcR regulates BR-Z1 at the transcriptional level. To test whether the CSN also regulates BR-Z1 through transcriptional regulation, we measured the mRNA level for BR-Z1 in CSN mutants by semi-quantitative RT-PCR. While the BR-Z1 mRNA expression in en-GAL4 control pupal wing discs was undetected, expression of CSN2 RNAi by en-GAL4 induced the BR-Z1 mRNA level (Figure 7A). To test the transcriptional regulation by the CSN, the reporter EcRE-LacZ which contains seven tandem repeats of EcRE was used [51]. In CSN4 and CSN5 clones, the expression levels from EcRE-lacZ were also elevated in comparison to neighboring heterozygous cells in pupal wings (Figure 7B–C′). The regulation of EcRE-lacZ is specific to the pupal stage, as EcRE-LacZ expression was not elevated in CSN5 clones at the prepupal stage (Figure 7D, 7D′). Thus, the CSN-downregulated BR-Z1 expression is at the transcriptional level and after the prepupa-to-pupa transition.


The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence.

Huang YC, Lu YN, Wu JT, Chien CT, Pi H - PLoS Genet. (2014)

Association of CSN subunits and EcR in BR-Z1 repression.(A) The BR-Z1 mRNA level in wing discs 20–24 h APF, assayed by semi-quantitative RT-PCR, was upregulated in wing discs of CSN2 RNAi driven by en-GAL4 compared to en-GAL4/+ control. rp49 levels served as internal controls. (B–C′) EcRE-lacZ (red) expression was upregulated in CSN4 (B, B′) and CSN5 (C, C′) clones 20–24 h APF, but unaffected in CSN5 clones 6–8 h APF (D, D′). (E–E″) Both EcRA (red) and Myc-CSN2 (green) localized in nuclei in wing disc 20–24 h APF. (F) Western blots showing co-precipitations of Myc-EcRA (upper panel) or Myc-EcRB1 (lower panel) in Flag immunoprecipitates of Flag-USP, CSN2, CSN4 or CSN5 in S2 cell extract. Co-precipitation was not detected in Flag-GFP. * represents the non-specific bands. (G, G′) BR-Z1 levels (red) in double MARCM clones for CSN5 and DN-EcR (green) were not further elevated compared to CSN5 or DN-EcR single clones (Figure 3B, 6B).
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pgen-1004760-g007: Association of CSN subunits and EcR in BR-Z1 repression.(A) The BR-Z1 mRNA level in wing discs 20–24 h APF, assayed by semi-quantitative RT-PCR, was upregulated in wing discs of CSN2 RNAi driven by en-GAL4 compared to en-GAL4/+ control. rp49 levels served as internal controls. (B–C′) EcRE-lacZ (red) expression was upregulated in CSN4 (B, B′) and CSN5 (C, C′) clones 20–24 h APF, but unaffected in CSN5 clones 6–8 h APF (D, D′). (E–E″) Both EcRA (red) and Myc-CSN2 (green) localized in nuclei in wing disc 20–24 h APF. (F) Western blots showing co-precipitations of Myc-EcRA (upper panel) or Myc-EcRB1 (lower panel) in Flag immunoprecipitates of Flag-USP, CSN2, CSN4 or CSN5 in S2 cell extract. Co-precipitation was not detected in Flag-GFP. * represents the non-specific bands. (G, G′) BR-Z1 levels (red) in double MARCM clones for CSN5 and DN-EcR (green) were not further elevated compared to CSN5 or DN-EcR single clones (Figure 3B, 6B).
Mentions: EcR regulates BR-Z1 at the transcriptional level. To test whether the CSN also regulates BR-Z1 through transcriptional regulation, we measured the mRNA level for BR-Z1 in CSN mutants by semi-quantitative RT-PCR. While the BR-Z1 mRNA expression in en-GAL4 control pupal wing discs was undetected, expression of CSN2 RNAi by en-GAL4 induced the BR-Z1 mRNA level (Figure 7A). To test the transcriptional regulation by the CSN, the reporter EcRE-LacZ which contains seven tandem repeats of EcRE was used [51]. In CSN4 and CSN5 clones, the expression levels from EcRE-lacZ were also elevated in comparison to neighboring heterozygous cells in pupal wings (Figure 7B–C′). The regulation of EcRE-lacZ is specific to the pupal stage, as EcRE-LacZ expression was not elevated in CSN5 clones at the prepupal stage (Figure 7D, 7D′). Thus, the CSN-downregulated BR-Z1 expression is at the transcriptional level and after the prepupa-to-pupa transition.

Bottom Line: We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM.Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level.We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Insitute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT
During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM.

Show MeSH
Related in: MedlinePlus