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Evolution of a new function by degenerative mutation in cephalochordate steroid receptors.

Bridgham JT, Brown JE, Rodríguez-Marí A, Catchen JM, Thornton JW - PLoS Genet. (2008)

Bottom Line: BfSR is specifically activated by estrogens and recognizes estrogen response elements (EREs) in DNA; BfER does not activate transcription in response to steroid hormones but binds EREs, where it competitively represses BfSR.These results corroborate previous findings that the ancestral steroid receptor was estrogen-sensitive and indicate that, after duplication, BfSR retained the ancestral function, while BfER evolved the capacity to negatively regulate BfSR.Our findings suggest that after duplication of genes whose functions depend on specific molecular interactions, high-probability degenerative mutations can yield novel functions, which are then exposed to positive or negative selection; in either case, the probability of neofunctionalization relative to gene loss is increased compared to existing models.

View Article: PubMed Central - PubMed

Affiliation: Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon, United States of America.

ABSTRACT
Gene duplication is the predominant mechanism for the evolution of new genes. Major existing models of this process assume that duplicate genes are redundant; degenerative mutations in one copy can therefore accumulate close to neutrally, usually leading to loss from the genome. When gene products dimerize or interact with other molecules for their functions, however, degenerative mutations in one copy may produce repressor alleles that inhibit the function of the other and are therefore exposed to selection. Here, we describe the evolution of a duplicate repressor by simple degenerative mutations in the steroid hormone receptors (SRs), a biologically crucial vertebrate gene family. We isolated and characterized the SRs of the cephalochordate Branchiostoma floridae, which diverged from other chordates just after duplication of the ancestral SR. The B. floridae genome contains two SRs: BfER, an ortholog of the vertebrate estrogen receptors, and BfSR, an ortholog of the vertebrate receptors for androgens, progestins, and corticosteroids. BfSR is specifically activated by estrogens and recognizes estrogen response elements (EREs) in DNA; BfER does not activate transcription in response to steroid hormones but binds EREs, where it competitively represses BfSR. The two genes are partially coexpressed, particularly in ovary and testis, suggesting an ancient role in germ cell development. These results corroborate previous findings that the ancestral steroid receptor was estrogen-sensitive and indicate that, after duplication, BfSR retained the ancestral function, while BfER evolved the capacity to negatively regulate BfSR. Either of two historical mutations that occurred during BfER evolution is sufficient to generate a competitive repressor. Our findings suggest that after duplication of genes whose functions depend on specific molecular interactions, high-probability degenerative mutations can yield novel functions, which are then exposed to positive or negative selection; in either case, the probability of neofunctionalization relative to gene loss is increased compared to existing models.

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Mechanism for evolution of a duplicate receptor repressor.A) Conservation and variability of ligand-contacting residues. The figure shows a generic steroid hormone surrounded by the amino acids that line the ligand-binding pocket in the crystal structures of the human ERα. For each site, residues from the human ERα (HsER), BfSR, BfER, and AncSR1 are shown, from top to bottom, numbered according to the human ERα sequence. Moieties at the 3 and 17 positions, shown as large circles, vary among steroids. B) Historical substitutions R394C and F404L, which occurred in the lineage leading to transcriptional repressor BfER, are predicted to abolish estrogen binding and activation. Left, x-ray crystal structure of the ligand pocket of the human ERα with estradiol. Arg394 and Phe404 play key roles in a network of hydrogen bonds and packing interactions that stabilize the ligand and transcriptionally active conformation. Right, mutations R394C and F404L from BfER disrupt this network. Red sphere, water molecule. C) Mutations R394C and F404L, introduced into BfSR, abolish the receptor's transcriptional capacity and generate a dose-dependent repressor of the wild-type (WT) BfSR. Numbers show the quantity of each plasmid transfected (in ng) with 1 µM estradiol (black bars) or with no hormone added (vehicle only, white bars). Mutation C205A, which disrupts DNA-binding, eliminates BfER's capacity to repress BfSR-driven transcription. D) Either mutation R394C or F404L, introduced singly into BfSR, are each sufficient to abolish transcriptional capacity and generate a repressor of BfSR-WT.
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pgen-1000191-g007: Mechanism for evolution of a duplicate receptor repressor.A) Conservation and variability of ligand-contacting residues. The figure shows a generic steroid hormone surrounded by the amino acids that line the ligand-binding pocket in the crystal structures of the human ERα. For each site, residues from the human ERα (HsER), BfSR, BfER, and AncSR1 are shown, from top to bottom, numbered according to the human ERα sequence. Moieties at the 3 and 17 positions, shown as large circles, vary among steroids. B) Historical substitutions R394C and F404L, which occurred in the lineage leading to transcriptional repressor BfER, are predicted to abolish estrogen binding and activation. Left, x-ray crystal structure of the ligand pocket of the human ERα with estradiol. Arg394 and Phe404 play key roles in a network of hydrogen bonds and packing interactions that stabilize the ligand and transcriptionally active conformation. Right, mutations R394C and F404L from BfER disrupt this network. Red sphere, water molecule. C) Mutations R394C and F404L, introduced into BfSR, abolish the receptor's transcriptional capacity and generate a dose-dependent repressor of the wild-type (WT) BfSR. Numbers show the quantity of each plasmid transfected (in ng) with 1 µM estradiol (black bars) or with no hormone added (vehicle only, white bars). Mutation C205A, which disrupts DNA-binding, eliminates BfER's capacity to repress BfSR-driven transcription. D) Either mutation R394C or F404L, introduced singly into BfSR, are each sufficient to abolish transcriptional capacity and generate a repressor of BfSR-WT.

Mentions: Because BfSR and BfER both interact with EREs, but only the former is activated by estrogens, we hypothesized that BfER might function as a competitive inhibitor of BfSR-activated transcription. To test this hypothesis, we cotransfected full-length BfER and BfSR in varying ratios with an ERE-driven reporter in the presence of estrogens. As predicted, BfER inhibited the transcriptional activity of BfSR in a manner dependent on the ratio of BfER to BfSR (Figure 4A). BfER's inhibitory activity is mediated by competition for EREs: alanine replacement of Cys205, a residue in the DNA-binding domain crucial for response element recognition (Figure 5A, see also [64]), abolishes BfER's capacity to repress BfSR-mediated transcription, even when transfected in very high ratios (Figure 5D, see also Figure 7C). In the EMSA, BfER binds very strongly to and competes for EREs as a homodimer; whether it can also heterodimerize with BfSR is unclear (Figure 5C). Taken together, these data indicate that competition for DNA is the primary mode by which BfER represses BfSR, although we cannot rule out a minor additional role for heterodimerization.


Evolution of a new function by degenerative mutation in cephalochordate steroid receptors.

Bridgham JT, Brown JE, Rodríguez-Marí A, Catchen JM, Thornton JW - PLoS Genet. (2008)

Mechanism for evolution of a duplicate receptor repressor.A) Conservation and variability of ligand-contacting residues. The figure shows a generic steroid hormone surrounded by the amino acids that line the ligand-binding pocket in the crystal structures of the human ERα. For each site, residues from the human ERα (HsER), BfSR, BfER, and AncSR1 are shown, from top to bottom, numbered according to the human ERα sequence. Moieties at the 3 and 17 positions, shown as large circles, vary among steroids. B) Historical substitutions R394C and F404L, which occurred in the lineage leading to transcriptional repressor BfER, are predicted to abolish estrogen binding and activation. Left, x-ray crystal structure of the ligand pocket of the human ERα with estradiol. Arg394 and Phe404 play key roles in a network of hydrogen bonds and packing interactions that stabilize the ligand and transcriptionally active conformation. Right, mutations R394C and F404L from BfER disrupt this network. Red sphere, water molecule. C) Mutations R394C and F404L, introduced into BfSR, abolish the receptor's transcriptional capacity and generate a dose-dependent repressor of the wild-type (WT) BfSR. Numbers show the quantity of each plasmid transfected (in ng) with 1 µM estradiol (black bars) or with no hormone added (vehicle only, white bars). Mutation C205A, which disrupts DNA-binding, eliminates BfER's capacity to repress BfSR-driven transcription. D) Either mutation R394C or F404L, introduced singly into BfSR, are each sufficient to abolish transcriptional capacity and generate a repressor of BfSR-WT.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2527136&req=5

pgen-1000191-g007: Mechanism for evolution of a duplicate receptor repressor.A) Conservation and variability of ligand-contacting residues. The figure shows a generic steroid hormone surrounded by the amino acids that line the ligand-binding pocket in the crystal structures of the human ERα. For each site, residues from the human ERα (HsER), BfSR, BfER, and AncSR1 are shown, from top to bottom, numbered according to the human ERα sequence. Moieties at the 3 and 17 positions, shown as large circles, vary among steroids. B) Historical substitutions R394C and F404L, which occurred in the lineage leading to transcriptional repressor BfER, are predicted to abolish estrogen binding and activation. Left, x-ray crystal structure of the ligand pocket of the human ERα with estradiol. Arg394 and Phe404 play key roles in a network of hydrogen bonds and packing interactions that stabilize the ligand and transcriptionally active conformation. Right, mutations R394C and F404L from BfER disrupt this network. Red sphere, water molecule. C) Mutations R394C and F404L, introduced into BfSR, abolish the receptor's transcriptional capacity and generate a dose-dependent repressor of the wild-type (WT) BfSR. Numbers show the quantity of each plasmid transfected (in ng) with 1 µM estradiol (black bars) or with no hormone added (vehicle only, white bars). Mutation C205A, which disrupts DNA-binding, eliminates BfER's capacity to repress BfSR-driven transcription. D) Either mutation R394C or F404L, introduced singly into BfSR, are each sufficient to abolish transcriptional capacity and generate a repressor of BfSR-WT.
Mentions: Because BfSR and BfER both interact with EREs, but only the former is activated by estrogens, we hypothesized that BfER might function as a competitive inhibitor of BfSR-activated transcription. To test this hypothesis, we cotransfected full-length BfER and BfSR in varying ratios with an ERE-driven reporter in the presence of estrogens. As predicted, BfER inhibited the transcriptional activity of BfSR in a manner dependent on the ratio of BfER to BfSR (Figure 4A). BfER's inhibitory activity is mediated by competition for EREs: alanine replacement of Cys205, a residue in the DNA-binding domain crucial for response element recognition (Figure 5A, see also [64]), abolishes BfER's capacity to repress BfSR-mediated transcription, even when transfected in very high ratios (Figure 5D, see also Figure 7C). In the EMSA, BfER binds very strongly to and competes for EREs as a homodimer; whether it can also heterodimerize with BfSR is unclear (Figure 5C). Taken together, these data indicate that competition for DNA is the primary mode by which BfER represses BfSR, although we cannot rule out a minor additional role for heterodimerization.

Bottom Line: BfSR is specifically activated by estrogens and recognizes estrogen response elements (EREs) in DNA; BfER does not activate transcription in response to steroid hormones but binds EREs, where it competitively represses BfSR.These results corroborate previous findings that the ancestral steroid receptor was estrogen-sensitive and indicate that, after duplication, BfSR retained the ancestral function, while BfER evolved the capacity to negatively regulate BfSR.Our findings suggest that after duplication of genes whose functions depend on specific molecular interactions, high-probability degenerative mutations can yield novel functions, which are then exposed to positive or negative selection; in either case, the probability of neofunctionalization relative to gene loss is increased compared to existing models.

View Article: PubMed Central - PubMed

Affiliation: Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon, United States of America.

ABSTRACT
Gene duplication is the predominant mechanism for the evolution of new genes. Major existing models of this process assume that duplicate genes are redundant; degenerative mutations in one copy can therefore accumulate close to neutrally, usually leading to loss from the genome. When gene products dimerize or interact with other molecules for their functions, however, degenerative mutations in one copy may produce repressor alleles that inhibit the function of the other and are therefore exposed to selection. Here, we describe the evolution of a duplicate repressor by simple degenerative mutations in the steroid hormone receptors (SRs), a biologically crucial vertebrate gene family. We isolated and characterized the SRs of the cephalochordate Branchiostoma floridae, which diverged from other chordates just after duplication of the ancestral SR. The B. floridae genome contains two SRs: BfER, an ortholog of the vertebrate estrogen receptors, and BfSR, an ortholog of the vertebrate receptors for androgens, progestins, and corticosteroids. BfSR is specifically activated by estrogens and recognizes estrogen response elements (EREs) in DNA; BfER does not activate transcription in response to steroid hormones but binds EREs, where it competitively represses BfSR. The two genes are partially coexpressed, particularly in ovary and testis, suggesting an ancient role in germ cell development. These results corroborate previous findings that the ancestral steroid receptor was estrogen-sensitive and indicate that, after duplication, BfSR retained the ancestral function, while BfER evolved the capacity to negatively regulate BfSR. Either of two historical mutations that occurred during BfER evolution is sufficient to generate a competitive repressor. Our findings suggest that after duplication of genes whose functions depend on specific molecular interactions, high-probability degenerative mutations can yield novel functions, which are then exposed to positive or negative selection; in either case, the probability of neofunctionalization relative to gene loss is increased compared to existing models.

Show MeSH