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Tinkering signaling pathways by gain and loss of protein isoforms: the case of the EDA pathway regulator EDARADD.

Sadier A, Lambert E, Chevret P, Décimo D, Sémon M, Tohmé M, Ruggiero F, Ohlmann T, Pantalacci S, Laudet V - BMC Evol. Biol. (2015)

Bottom Line: Then, to gain insights into the functional relevance of this evolutionary pattern, we compared the biological function of these isoforms: i) In cellulo promoter assays showed that they are transcribed from two alternative promoters, only B exhibiting feedback regulation. ii) RT-PCR in various tissues and ENCODE data suggested that B isoform is systematically expressed whereas A isoform showed a more tissue-specific expression. iii) Both isoforms activated the NF-κB pathway in an in cellulo reporter assay, albeit at different levels and with different dynamics since A isoform exhibited feedback regulation at the protein level.These results suggest that the newly evolved A isoform enables modulating EDA signaling in specific conditions and with different dynamics.This study makes the case to pay greater attention to mosaic loss of evolutionarily speaking "young" isoforms as an important mechanism underlying phenotypic diversity and not simply as a manifestation of neutral evolution.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, UMR 5242 du CNRS, Université de Lyon, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364, Lyon, Cedex 07, France. alexa.sadier@ens-lyon.fr.

ABSTRACT

Background: Only a handful of signaling pathways are major actors of development and responsible for both the conservation and the diversification of animal morphologies. To explain this twofold nature, gene duplication and enhancer evolution were predominantly put forth as tinkering mechanisms whereas the evolution of alternative isoforms has been, so far, overlooked. We investigate here the role of gain and loss of isoforms using Edaradd, a gene of the Ecodysplasin pathway, implicated in morphological evolution. A previous study had suggested a scenario of isoform gain and loss with an alternative isoform (A) newly gained in mammals but secondarily lost in mouse lineage.

Results: For a comprehensive view of A and B Edaradd isoforms history during mammal evolution, we obtained sequences for both isoforms in representative mammals and performed in vitro translations to support functional predictions. We showed that the ancestral B isoform is well conserved, whereas the mammal-specific A isoform was lost at least 7 times independently in terminal lineages throughout mammal phylogeny. Then, to gain insights into the functional relevance of this evolutionary pattern, we compared the biological function of these isoforms: i) In cellulo promoter assays showed that they are transcribed from two alternative promoters, only B exhibiting feedback regulation. ii) RT-PCR in various tissues and ENCODE data suggested that B isoform is systematically expressed whereas A isoform showed a more tissue-specific expression. iii) Both isoforms activated the NF-κB pathway in an in cellulo reporter assay, albeit at different levels and with different dynamics since A isoform exhibited feedback regulation at the protein level. Finally, only B isoform could rescue a zebrafish edaradd knockdown.

Conclusions: These results suggest that the newly evolved A isoform enables modulating EDA signaling in specific conditions and with different dynamics. We speculate that during mammal diversification, A isoform regulation may have evolved rapidly, accompanying and possibly supporting the diversity of ectodermal appendages, while B isoform may have ensured essential roles. This study makes the case to pay greater attention to mosaic loss of evolutionarily speaking "young" isoforms as an important mechanism underlying phenotypic diversity and not simply as a manifestation of neutral evolution.

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The two isoforms of EDARADD exhibit different activities and stabilities in cellulo. a Constructs. HA constructs: HA tag was N-terminally fused to EDARADD A or B protein. Myc constructs: Myc tag was C-terminally fused to EDARADD A or B protein. These constructs were generated using recombination so that 1A and 1B constructs only differ for A and B peptide coding region. NF-κB reporter gene: NF-κB are located upstream a minimal promoter and a luciferase reporter gene. b EDARADD A and B activate the NF-κB pathway in a dose-dependent manner. Increasing amounts of constructs encoding A or B isoforms were transfected in HEK cells with NF-κB reporter plasmid that harbors NF-κB response elements upstream luciferase reporter gene. The luciferase activity was normalized against β-gal and the empty vector. The luciferase activity was assayed 24 hours after transfection. In yellow: isoform A, in brown: isoform B. c A isoform is twice more active than B isoform when luciferase activity is corrected for Edaradd quantity. HEK cells were transfected with constructs encoding EDARADD A or B C-terminally tagged with myc (EDD-myc A or B) and the luciferase reporter gene as in A. 24 h after transfection, three wells were pooled in order to have enough material for Western Blotting and three others were used for luciferase assays. Proteins were detected by Western Blot with an anti-Myc antibody. Histone H3 was used as a control. Luciferase activity corrected for edaradd quantity is presented on the right. d EDARADD A and B reach different steady state levels. HEK cells were transfected with constructs encoding EDARADD A or B N-terminally tagged with HA (HA-EDD A or B) or C-terminally tagged with myc (EDD-myc A or B). Proteins were detected by Western Blot 12 h, 24 h, 36 h and 48 h after transfection. Histone H3 was used as a control. e EDARADD A but not B is destabilized by NF-κB activators. Western Blot for Myc C-terminally-tagged A and B isoforms following co-transfection of NF-κB activators such as EDAR or TRAF6, at 24 or 48 h. Histone H3 is used as a control
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Fig4: The two isoforms of EDARADD exhibit different activities and stabilities in cellulo. a Constructs. HA constructs: HA tag was N-terminally fused to EDARADD A or B protein. Myc constructs: Myc tag was C-terminally fused to EDARADD A or B protein. These constructs were generated using recombination so that 1A and 1B constructs only differ for A and B peptide coding region. NF-κB reporter gene: NF-κB are located upstream a minimal promoter and a luciferase reporter gene. b EDARADD A and B activate the NF-κB pathway in a dose-dependent manner. Increasing amounts of constructs encoding A or B isoforms were transfected in HEK cells with NF-κB reporter plasmid that harbors NF-κB response elements upstream luciferase reporter gene. The luciferase activity was normalized against β-gal and the empty vector. The luciferase activity was assayed 24 hours after transfection. In yellow: isoform A, in brown: isoform B. c A isoform is twice more active than B isoform when luciferase activity is corrected for Edaradd quantity. HEK cells were transfected with constructs encoding EDARADD A or B C-terminally tagged with myc (EDD-myc A or B) and the luciferase reporter gene as in A. 24 h after transfection, three wells were pooled in order to have enough material for Western Blotting and three others were used for luciferase assays. Proteins were detected by Western Blot with an anti-Myc antibody. Histone H3 was used as a control. Luciferase activity corrected for edaradd quantity is presented on the right. d EDARADD A and B reach different steady state levels. HEK cells were transfected with constructs encoding EDARADD A or B N-terminally tagged with HA (HA-EDD A or B) or C-terminally tagged with myc (EDD-myc A or B). Proteins were detected by Western Blot 12 h, 24 h, 36 h and 48 h after transfection. Histone H3 was used as a control. e EDARADD A but not B is destabilized by NF-κB activators. Western Blot for Myc C-terminally-tagged A and B isoforms following co-transfection of NF-κB activators such as EDAR or TRAF6, at 24 or 48 h. Histone H3 is used as a control

Mentions: To decipher the biochemical role of the two EDARADD isoforms, we studied the function of the two human proteins in cellulo (Fig. 4) using the same HEK cells. We used recombination methods to generate expression plasmids for the two proteins that are rigorously identical (Fig. 4A), except for the small N-terminal peptide characterizing the A or B isoform (notably, translation initiation sites were identical). Since the first known function of the EDA pathway is to activate the NF-κB pathway we tested the ability of myc tagged-versions of both isoforms to activate the NF-κB pathway in a NF-κB reporter luciferase assay. As previously described, the B isoform activated the NF-κB pathway reporter in a dose-dependent manner [19] (Fig. 4B). The A isoform also activated the NF-κB pathway reporter in a dose-dependent manner. Examination of the protein level by western blot showed that the amount of B isoform was 2.5 higher than the amount of A isoform (Fig. 4C and D). Similar results were obtained whenever an N-terminal or a C-terminal tagged version of the proteins was used. Thus, to compare the activity of both isoforms, the luciferase activity needed to be corrected by the amount of EDARADD protein detected in the lysates. In so doing, the A isoform appeared to be twice more active than the B isoform (Fig. 4C). The co-transfection of the two constructs together at different concentrations did not exhibit any synergistic nor antagonistic effect [see Additional file 3: Figure S3]. We then followed the accumulation of the two proteins for 48 hours and found that the B isoform accumulated more rapidly than the A isoform (Fig. 4D). Considering the steady-state level of the two proteins, at 48 hours, the A isoform disappeared when co-transfected with EDAR or other NF-κB activators (Fig. 4E) whereas the B isoform remained stable suggesting a difference of regulation at the protein level. Similar results were obtained in presence of a proteasome inhibitor (MG132), presumably ruling out proteasome-dependent degradation (data not shown). In summary, the two human isoforms of EDARADD exhibit different functionalities in cellulo: they both activate the NF-κB pathway but exhibit differences in their dynamics: the B isoform accumulates more rapidly whereas the A isoform is more active but downregulated at the protein level following pathway activation. Such differences could impact their in vivo functions in transducing the NF-κB pathway both quantitatively and qualitatively.Fig. 4


Tinkering signaling pathways by gain and loss of protein isoforms: the case of the EDA pathway regulator EDARADD.

Sadier A, Lambert E, Chevret P, Décimo D, Sémon M, Tohmé M, Ruggiero F, Ohlmann T, Pantalacci S, Laudet V - BMC Evol. Biol. (2015)

The two isoforms of EDARADD exhibit different activities and stabilities in cellulo. a Constructs. HA constructs: HA tag was N-terminally fused to EDARADD A or B protein. Myc constructs: Myc tag was C-terminally fused to EDARADD A or B protein. These constructs were generated using recombination so that 1A and 1B constructs only differ for A and B peptide coding region. NF-κB reporter gene: NF-κB are located upstream a minimal promoter and a luciferase reporter gene. b EDARADD A and B activate the NF-κB pathway in a dose-dependent manner. Increasing amounts of constructs encoding A or B isoforms were transfected in HEK cells with NF-κB reporter plasmid that harbors NF-κB response elements upstream luciferase reporter gene. The luciferase activity was normalized against β-gal and the empty vector. The luciferase activity was assayed 24 hours after transfection. In yellow: isoform A, in brown: isoform B. c A isoform is twice more active than B isoform when luciferase activity is corrected for Edaradd quantity. HEK cells were transfected with constructs encoding EDARADD A or B C-terminally tagged with myc (EDD-myc A or B) and the luciferase reporter gene as in A. 24 h after transfection, three wells were pooled in order to have enough material for Western Blotting and three others were used for luciferase assays. Proteins were detected by Western Blot with an anti-Myc antibody. Histone H3 was used as a control. Luciferase activity corrected for edaradd quantity is presented on the right. d EDARADD A and B reach different steady state levels. HEK cells were transfected with constructs encoding EDARADD A or B N-terminally tagged with HA (HA-EDD A or B) or C-terminally tagged with myc (EDD-myc A or B). Proteins were detected by Western Blot 12 h, 24 h, 36 h and 48 h after transfection. Histone H3 was used as a control. e EDARADD A but not B is destabilized by NF-κB activators. Western Blot for Myc C-terminally-tagged A and B isoforms following co-transfection of NF-κB activators such as EDAR or TRAF6, at 24 or 48 h. Histone H3 is used as a control
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Related In: Results  -  Collection

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Fig4: The two isoforms of EDARADD exhibit different activities and stabilities in cellulo. a Constructs. HA constructs: HA tag was N-terminally fused to EDARADD A or B protein. Myc constructs: Myc tag was C-terminally fused to EDARADD A or B protein. These constructs were generated using recombination so that 1A and 1B constructs only differ for A and B peptide coding region. NF-κB reporter gene: NF-κB are located upstream a minimal promoter and a luciferase reporter gene. b EDARADD A and B activate the NF-κB pathway in a dose-dependent manner. Increasing amounts of constructs encoding A or B isoforms were transfected in HEK cells with NF-κB reporter plasmid that harbors NF-κB response elements upstream luciferase reporter gene. The luciferase activity was normalized against β-gal and the empty vector. The luciferase activity was assayed 24 hours after transfection. In yellow: isoform A, in brown: isoform B. c A isoform is twice more active than B isoform when luciferase activity is corrected for Edaradd quantity. HEK cells were transfected with constructs encoding EDARADD A or B C-terminally tagged with myc (EDD-myc A or B) and the luciferase reporter gene as in A. 24 h after transfection, three wells were pooled in order to have enough material for Western Blotting and three others were used for luciferase assays. Proteins were detected by Western Blot with an anti-Myc antibody. Histone H3 was used as a control. Luciferase activity corrected for edaradd quantity is presented on the right. d EDARADD A and B reach different steady state levels. HEK cells were transfected with constructs encoding EDARADD A or B N-terminally tagged with HA (HA-EDD A or B) or C-terminally tagged with myc (EDD-myc A or B). Proteins were detected by Western Blot 12 h, 24 h, 36 h and 48 h after transfection. Histone H3 was used as a control. e EDARADD A but not B is destabilized by NF-κB activators. Western Blot for Myc C-terminally-tagged A and B isoforms following co-transfection of NF-κB activators such as EDAR or TRAF6, at 24 or 48 h. Histone H3 is used as a control
Mentions: To decipher the biochemical role of the two EDARADD isoforms, we studied the function of the two human proteins in cellulo (Fig. 4) using the same HEK cells. We used recombination methods to generate expression plasmids for the two proteins that are rigorously identical (Fig. 4A), except for the small N-terminal peptide characterizing the A or B isoform (notably, translation initiation sites were identical). Since the first known function of the EDA pathway is to activate the NF-κB pathway we tested the ability of myc tagged-versions of both isoforms to activate the NF-κB pathway in a NF-κB reporter luciferase assay. As previously described, the B isoform activated the NF-κB pathway reporter in a dose-dependent manner [19] (Fig. 4B). The A isoform also activated the NF-κB pathway reporter in a dose-dependent manner. Examination of the protein level by western blot showed that the amount of B isoform was 2.5 higher than the amount of A isoform (Fig. 4C and D). Similar results were obtained whenever an N-terminal or a C-terminal tagged version of the proteins was used. Thus, to compare the activity of both isoforms, the luciferase activity needed to be corrected by the amount of EDARADD protein detected in the lysates. In so doing, the A isoform appeared to be twice more active than the B isoform (Fig. 4C). The co-transfection of the two constructs together at different concentrations did not exhibit any synergistic nor antagonistic effect [see Additional file 3: Figure S3]. We then followed the accumulation of the two proteins for 48 hours and found that the B isoform accumulated more rapidly than the A isoform (Fig. 4D). Considering the steady-state level of the two proteins, at 48 hours, the A isoform disappeared when co-transfected with EDAR or other NF-κB activators (Fig. 4E) whereas the B isoform remained stable suggesting a difference of regulation at the protein level. Similar results were obtained in presence of a proteasome inhibitor (MG132), presumably ruling out proteasome-dependent degradation (data not shown). In summary, the two human isoforms of EDARADD exhibit different functionalities in cellulo: they both activate the NF-κB pathway but exhibit differences in their dynamics: the B isoform accumulates more rapidly whereas the A isoform is more active but downregulated at the protein level following pathway activation. Such differences could impact their in vivo functions in transducing the NF-κB pathway both quantitatively and qualitatively.Fig. 4

Bottom Line: Then, to gain insights into the functional relevance of this evolutionary pattern, we compared the biological function of these isoforms: i) In cellulo promoter assays showed that they are transcribed from two alternative promoters, only B exhibiting feedback regulation. ii) RT-PCR in various tissues and ENCODE data suggested that B isoform is systematically expressed whereas A isoform showed a more tissue-specific expression. iii) Both isoforms activated the NF-κB pathway in an in cellulo reporter assay, albeit at different levels and with different dynamics since A isoform exhibited feedback regulation at the protein level.These results suggest that the newly evolved A isoform enables modulating EDA signaling in specific conditions and with different dynamics.This study makes the case to pay greater attention to mosaic loss of evolutionarily speaking "young" isoforms as an important mechanism underlying phenotypic diversity and not simply as a manifestation of neutral evolution.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, UMR 5242 du CNRS, Université de Lyon, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364, Lyon, Cedex 07, France. alexa.sadier@ens-lyon.fr.

ABSTRACT

Background: Only a handful of signaling pathways are major actors of development and responsible for both the conservation and the diversification of animal morphologies. To explain this twofold nature, gene duplication and enhancer evolution were predominantly put forth as tinkering mechanisms whereas the evolution of alternative isoforms has been, so far, overlooked. We investigate here the role of gain and loss of isoforms using Edaradd, a gene of the Ecodysplasin pathway, implicated in morphological evolution. A previous study had suggested a scenario of isoform gain and loss with an alternative isoform (A) newly gained in mammals but secondarily lost in mouse lineage.

Results: For a comprehensive view of A and B Edaradd isoforms history during mammal evolution, we obtained sequences for both isoforms in representative mammals and performed in vitro translations to support functional predictions. We showed that the ancestral B isoform is well conserved, whereas the mammal-specific A isoform was lost at least 7 times independently in terminal lineages throughout mammal phylogeny. Then, to gain insights into the functional relevance of this evolutionary pattern, we compared the biological function of these isoforms: i) In cellulo promoter assays showed that they are transcribed from two alternative promoters, only B exhibiting feedback regulation. ii) RT-PCR in various tissues and ENCODE data suggested that B isoform is systematically expressed whereas A isoform showed a more tissue-specific expression. iii) Both isoforms activated the NF-κB pathway in an in cellulo reporter assay, albeit at different levels and with different dynamics since A isoform exhibited feedback regulation at the protein level. Finally, only B isoform could rescue a zebrafish edaradd knockdown.

Conclusions: These results suggest that the newly evolved A isoform enables modulating EDA signaling in specific conditions and with different dynamics. We speculate that during mammal diversification, A isoform regulation may have evolved rapidly, accompanying and possibly supporting the diversity of ectodermal appendages, while B isoform may have ensured essential roles. This study makes the case to pay greater attention to mosaic loss of evolutionarily speaking "young" isoforms as an important mechanism underlying phenotypic diversity and not simply as a manifestation of neutral evolution.

Show MeSH