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An amphioxus orthologue of the estrogen receptor that does not bind estradiol: insights into estrogen receptor evolution.

Paris M, Pettersson K, Schubert M, Bertrand S, Pongratz I, Escriva H, Laudet V - BMC Evol. Biol. (2008)

Bottom Line: In contrast, we found that although amphiER binds EREs, it is unable to bind E2 and to activate transcription in response to E2.This result does not support a previous analysis based on ancestral sequence reconstruction that proposed the ancestral steroid receptor to bind estradiol.Taken together, our results highlight the importance of comparative experimental approaches vs ancestral reconstructions for the evolutionary study of endocrine systems: comparative analysis of extant ERs suggests that the ancestral ER did not bind estradiol and that it gained the ability to be regulated by estradiol specifically in the vertebrate lineage, before lamprey split.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, Molecular Zoology team, Université de Lyon, Ecole Normale Supérieure de Lyon, Université Lyon 1, CNRS, INRA, Institut Fédératif 128 Biosciences Gerland Lyon Sud, France. mathilde.paris@ens-lyon.fr

ABSTRACT

Background: The origin of nuclear receptors (NRs) and the question whether the ancestral NR was a liganded or an unliganded transcription factor has been recently debated. To obtain insight into the evolution of the ligand binding ability of estrogen receptors (ER), we comparatively characterized the ER from the protochordate amphioxus (Branchiostoma floridae), and the ER from lamprey (Petromyzon marinus), a basal vertebrate.

Results: Extensive phylogenetic studies as well as signature analysis allowed us to confirm that the amphioxus ER (amphiER) and the lamprey ER (lampER) belong to the ER group. LampER behaves as a "classical" vertebrate ER, as it binds to specific DNA Estrogen Responsive Elements (EREs), and is activated by estradiol (E2), the classical ER natural ligand. In contrast, we found that although amphiER binds EREs, it is unable to bind E2 and to activate transcription in response to E2. Among the 7 natural and synthetic ER ligands tested as well as a large repertoire of 14 cholesterol derivatives, only Bisphenol A (an endocrine disruptor with estrogenic activity) bound to amphiER, suggesting that a ligand binding pocket exists within the receptor. Parsimony analysis considering all available ER sequences suggest that the ancestral ER was not able to bind E2 and that this ability evolved specifically in the vertebrate lineage. This result does not support a previous analysis based on ancestral sequence reconstruction that proposed the ancestral steroid receptor to bind estradiol. We show that biased taxonomic sampling can alter the calculation of ancestral sequence and that the previous result might stem from a high proportion of vertebrate ERs in the dataset used to compute the ancestral sequence.

Conclusion: Taken together, our results highlight the importance of comparative experimental approaches vs ancestral reconstructions for the evolutionary study of endocrine systems: comparative analysis of extant ERs suggests that the ancestral ER did not bind estradiol and that it gained the ability to be regulated by estradiol specifically in the vertebrate lineage, before lamprey split.

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Sequence alignment of several ERs including the amphioxus ER and the lamprey ER, as well as ancestral steroid receptors. The DBD is highlighted with light grey. The 12 helices from the LBD are indicated, based on the known 3D structure of human ERα [49]. Amino acids from human ERα making direct hydrogen bonds with E2 are indicated in green. Amino acids making hydrophobic bonds with E2 are highlighted in purple. Amino acids known to be involved in co-activator interaction have been indicated with a star on top of each site [55]. The more divergent A/B domain as well as the F domain have been omitted from the alignment. However, the numbering of the sites along the alignment starts at the beginning of each protein. The exon-intron limits of amphiER and humanERα have been indicated with small red strokes. The sequences of AncSRa and AncSRb have been inferred in this study. The sequence of AncSR1 was retrieved from a previous analysis [11].
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Figure 1: Sequence alignment of several ERs including the amphioxus ER and the lamprey ER, as well as ancestral steroid receptors. The DBD is highlighted with light grey. The 12 helices from the LBD are indicated, based on the known 3D structure of human ERα [49]. Amino acids from human ERα making direct hydrogen bonds with E2 are indicated in green. Amino acids making hydrophobic bonds with E2 are highlighted in purple. Amino acids known to be involved in co-activator interaction have been indicated with a star on top of each site [55]. The more divergent A/B domain as well as the F domain have been omitted from the alignment. However, the numbering of the sites along the alignment starts at the beginning of each protein. The exon-intron limits of amphiER and humanERα have been indicated with small red strokes. The sequences of AncSRa and AncSRb have been inferred in this study. The sequence of AncSR1 was retrieved from a previous analysis [11].

Mentions: Using degenerate primers designed to match motifs in the most conserved part of vertebrate ERs in the DNA binding domain, a single gene fragment from total RNA of an adult Branchiostoma floridae was amplified, cloned and sequenced. Rapid amplification of cDNA ends (RACE) was utilized to obtain the full-length cDNA. From this sequence, a new set of specific primers were designed and used to amplify the full length open reading frame of this gene. The obtained cDNA [GenBank: ACF16007] is 2118 bp long and encodes a 705 aa long putative protein (Figure 1) that harbors the classical features of an ER with the 5 main functional domains (Figure 2A), among which a highly conserved DNA binding domain (DBD) and a less conserved ligand binding domain (LBD). The DBD shares an 82% sequence identity with the human ERα one (83% with human ERβ) and much less with the other NR3 receptors (<62%). The same pattern is observed for the LBD, although this domain is less conserved since it exhibits only 34% amino acid identity with human ERα (35% for human ERβ) and about 20% with other steroid receptors (Figure 2A). The three other domains, namely the A/B region in the N-terminal part, the hinge between DBD and LBD, and the short C-terminal end of the protein, are more divergent, which is a general pattern for NRs [2] (Figure 2A). The recent release of the amphioxus genome confirmed the presence of a single ER gene [35]. In contrast the previously described lamprey ER is more similar to the human ERα with its DBD sharing a 93% sequence identity (93% for human ERβ) and its LBD sharing 55% sequence identity (56% for human ERβ) [8].


An amphioxus orthologue of the estrogen receptor that does not bind estradiol: insights into estrogen receptor evolution.

Paris M, Pettersson K, Schubert M, Bertrand S, Pongratz I, Escriva H, Laudet V - BMC Evol. Biol. (2008)

Sequence alignment of several ERs including the amphioxus ER and the lamprey ER, as well as ancestral steroid receptors. The DBD is highlighted with light grey. The 12 helices from the LBD are indicated, based on the known 3D structure of human ERα [49]. Amino acids from human ERα making direct hydrogen bonds with E2 are indicated in green. Amino acids making hydrophobic bonds with E2 are highlighted in purple. Amino acids known to be involved in co-activator interaction have been indicated with a star on top of each site [55]. The more divergent A/B domain as well as the F domain have been omitted from the alignment. However, the numbering of the sites along the alignment starts at the beginning of each protein. The exon-intron limits of amphiER and humanERα have been indicated with small red strokes. The sequences of AncSRa and AncSRb have been inferred in this study. The sequence of AncSR1 was retrieved from a previous analysis [11].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529310&req=5

Figure 1: Sequence alignment of several ERs including the amphioxus ER and the lamprey ER, as well as ancestral steroid receptors. The DBD is highlighted with light grey. The 12 helices from the LBD are indicated, based on the known 3D structure of human ERα [49]. Amino acids from human ERα making direct hydrogen bonds with E2 are indicated in green. Amino acids making hydrophobic bonds with E2 are highlighted in purple. Amino acids known to be involved in co-activator interaction have been indicated with a star on top of each site [55]. The more divergent A/B domain as well as the F domain have been omitted from the alignment. However, the numbering of the sites along the alignment starts at the beginning of each protein. The exon-intron limits of amphiER and humanERα have been indicated with small red strokes. The sequences of AncSRa and AncSRb have been inferred in this study. The sequence of AncSR1 was retrieved from a previous analysis [11].
Mentions: Using degenerate primers designed to match motifs in the most conserved part of vertebrate ERs in the DNA binding domain, a single gene fragment from total RNA of an adult Branchiostoma floridae was amplified, cloned and sequenced. Rapid amplification of cDNA ends (RACE) was utilized to obtain the full-length cDNA. From this sequence, a new set of specific primers were designed and used to amplify the full length open reading frame of this gene. The obtained cDNA [GenBank: ACF16007] is 2118 bp long and encodes a 705 aa long putative protein (Figure 1) that harbors the classical features of an ER with the 5 main functional domains (Figure 2A), among which a highly conserved DNA binding domain (DBD) and a less conserved ligand binding domain (LBD). The DBD shares an 82% sequence identity with the human ERα one (83% with human ERβ) and much less with the other NR3 receptors (<62%). The same pattern is observed for the LBD, although this domain is less conserved since it exhibits only 34% amino acid identity with human ERα (35% for human ERβ) and about 20% with other steroid receptors (Figure 2A). The three other domains, namely the A/B region in the N-terminal part, the hinge between DBD and LBD, and the short C-terminal end of the protein, are more divergent, which is a general pattern for NRs [2] (Figure 2A). The recent release of the amphioxus genome confirmed the presence of a single ER gene [35]. In contrast the previously described lamprey ER is more similar to the human ERα with its DBD sharing a 93% sequence identity (93% for human ERβ) and its LBD sharing 55% sequence identity (56% for human ERβ) [8].

Bottom Line: In contrast, we found that although amphiER binds EREs, it is unable to bind E2 and to activate transcription in response to E2.This result does not support a previous analysis based on ancestral sequence reconstruction that proposed the ancestral steroid receptor to bind estradiol.Taken together, our results highlight the importance of comparative experimental approaches vs ancestral reconstructions for the evolutionary study of endocrine systems: comparative analysis of extant ERs suggests that the ancestral ER did not bind estradiol and that it gained the ability to be regulated by estradiol specifically in the vertebrate lineage, before lamprey split.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, Molecular Zoology team, Université de Lyon, Ecole Normale Supérieure de Lyon, Université Lyon 1, CNRS, INRA, Institut Fédératif 128 Biosciences Gerland Lyon Sud, France. mathilde.paris@ens-lyon.fr

ABSTRACT

Background: The origin of nuclear receptors (NRs) and the question whether the ancestral NR was a liganded or an unliganded transcription factor has been recently debated. To obtain insight into the evolution of the ligand binding ability of estrogen receptors (ER), we comparatively characterized the ER from the protochordate amphioxus (Branchiostoma floridae), and the ER from lamprey (Petromyzon marinus), a basal vertebrate.

Results: Extensive phylogenetic studies as well as signature analysis allowed us to confirm that the amphioxus ER (amphiER) and the lamprey ER (lampER) belong to the ER group. LampER behaves as a "classical" vertebrate ER, as it binds to specific DNA Estrogen Responsive Elements (EREs), and is activated by estradiol (E2), the classical ER natural ligand. In contrast, we found that although amphiER binds EREs, it is unable to bind E2 and to activate transcription in response to E2. Among the 7 natural and synthetic ER ligands tested as well as a large repertoire of 14 cholesterol derivatives, only Bisphenol A (an endocrine disruptor with estrogenic activity) bound to amphiER, suggesting that a ligand binding pocket exists within the receptor. Parsimony analysis considering all available ER sequences suggest that the ancestral ER was not able to bind E2 and that this ability evolved specifically in the vertebrate lineage. This result does not support a previous analysis based on ancestral sequence reconstruction that proposed the ancestral steroid receptor to bind estradiol. We show that biased taxonomic sampling can alter the calculation of ancestral sequence and that the previous result might stem from a high proportion of vertebrate ERs in the dataset used to compute the ancestral sequence.

Conclusion: Taken together, our results highlight the importance of comparative experimental approaches vs ancestral reconstructions for the evolutionary study of endocrine systems: comparative analysis of extant ERs suggests that the ancestral ER did not bind estradiol and that it gained the ability to be regulated by estradiol specifically in the vertebrate lineage, before lamprey split.

Show MeSH
Related in: MedlinePlus