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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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BfER repression of BfSR by competition for EREs.A) BfER's DNA-binding domain recognizes an estrogen response element (ERE). A fusion of the BfER-DBD with a constitutive activation domain activates transcription of an ERE-driven luciferase reporter gene. Negative control (pCMV-AD vector only) and positive control (HsER, human ERα DBD) are shown for comparison. Replacement C205A in the BfER-DBD abolishes recognition of an ERE. B) Neither the BfER-DBD nor the BfSR-DBD recognize steroid response elements (SRE). Negative control (pCMV-AD vector only) and positive control (HsGR, human GR DBD) are shown for comparison. C) BfER and BfSR bind directly to EREs. An electromobility shift assay shows that each receptor binds to a biotin-labeled ERE and migrates as expected (BfER, 521 amino acids, ∼58 kD; BfSR, 616 amino acids, ∼68 kD). When the two receptors are co-transfected, ERE-binding by the BfER homodimer is so strong that it is unclear whether BfER and BfSR heterodimerize. D) Mutation C205A, which compromises DNA binding, abrogates BfER's capacity to repress BfSR-mediated transcription of an ERE-driven reporter. Full-length BfSR, wild-type BfER (BfER-WT), and the BfER C205A mutant were cotransfected. Cells were treated with no hormone (white bars) or 1 µM estradiol (black bars).
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pgen-1000191-g005: BfER repression of BfSR by competition for EREs.A) BfER's DNA-binding domain recognizes an estrogen response element (ERE). A fusion of the BfER-DBD with a constitutive activation domain activates transcription of an ERE-driven luciferase reporter gene. Negative control (pCMV-AD vector only) and positive control (HsER, human ERα DBD) are shown for comparison. Replacement C205A in the BfER-DBD abolishes recognition of an ERE. B) Neither the BfER-DBD nor the BfSR-DBD recognize steroid response elements (SRE). Negative control (pCMV-AD vector only) and positive control (HsGR, human GR DBD) are shown for comparison. C) BfER and BfSR bind directly to EREs. An electromobility shift assay shows that each receptor binds to a biotin-labeled ERE and migrates as expected (BfER, 521 amino acids, ∼58 kD; BfSR, 616 amino acids, ∼68 kD). When the two receptors are co-transfected, ERE-binding by the BfER homodimer is so strong that it is unclear whether BfER and BfSR heterodimerize. D) Mutation C205A, which compromises DNA binding, abrogates BfER's capacity to repress BfSR-mediated transcription of an ERE-driven reporter. Full-length BfSR, wild-type BfER (BfER-WT), and the BfER C205A mutant were cotransfected. Cells were treated with no hormone (white bars) or 1 µM estradiol (black bars).

Mentions: In contrast, the BfER LBD has lost the ancestral capacity to activate transcription in response to estrogens. Neither the BfER LBD nor the full-length BfER activates transcription in response to high doses of any of a large panel of vertebrate steroid hormones, including estradiol, estriol, and estrone (Figures 3A, S5). In the absence of hormone, full-length BfER (like other estrogen receptors) represses basal levels of ERE-reporter transcription, but hormone treatment does not alter this effect (Figure 4A). On an SRE-driven reporter, full-length BfER has no effect (Figure 4B). BfER does retain the ancestral DNA-recognition function: a fusion construct of the BfER-DBD with the constitutively-active NFkB activation domain yields robust transcription of an ERE-driven reporter (Figure 5A), but no activity on an SRE-driven reporter (Figure 5B). Further, BfER strongly binds EREs in an electrophoretic mobility shift assay (EMSA, Figure 5C).


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)

BfER repression of BfSR by competition for EREs.A) BfER's DNA-binding domain recognizes an estrogen response element (ERE). A fusion of the BfER-DBD with a constitutive activation domain activates transcription of an ERE-driven luciferase reporter gene. Negative control (pCMV-AD vector only) and positive control (HsER, human ERα DBD) are shown for comparison. Replacement C205A in the BfER-DBD abolishes recognition of an ERE. B) Neither the BfER-DBD nor the BfSR-DBD recognize steroid response elements (SRE). Negative control (pCMV-AD vector only) and positive control (HsGR, human GR DBD) are shown for comparison. C) BfER and BfSR bind directly to EREs. An electromobility shift assay shows that each receptor binds to a biotin-labeled ERE and migrates as expected (BfER, 521 amino acids, ∼58 kD; BfSR, 616 amino acids, ∼68 kD). When the two receptors are co-transfected, ERE-binding by the BfER homodimer is so strong that it is unclear whether BfER and BfSR heterodimerize. D) Mutation C205A, which compromises DNA binding, abrogates BfER's capacity to repress BfSR-mediated transcription of an ERE-driven reporter. Full-length BfSR, wild-type BfER (BfER-WT), and the BfER C205A mutant were cotransfected. Cells were treated with no hormone (white bars) or 1 µM estradiol (black bars).
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Related In: Results  -  Collection

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pgen-1000191-g005: BfER repression of BfSR by competition for EREs.A) BfER's DNA-binding domain recognizes an estrogen response element (ERE). A fusion of the BfER-DBD with a constitutive activation domain activates transcription of an ERE-driven luciferase reporter gene. Negative control (pCMV-AD vector only) and positive control (HsER, human ERα DBD) are shown for comparison. Replacement C205A in the BfER-DBD abolishes recognition of an ERE. B) Neither the BfER-DBD nor the BfSR-DBD recognize steroid response elements (SRE). Negative control (pCMV-AD vector only) and positive control (HsGR, human GR DBD) are shown for comparison. C) BfER and BfSR bind directly to EREs. An electromobility shift assay shows that each receptor binds to a biotin-labeled ERE and migrates as expected (BfER, 521 amino acids, ∼58 kD; BfSR, 616 amino acids, ∼68 kD). When the two receptors are co-transfected, ERE-binding by the BfER homodimer is so strong that it is unclear whether BfER and BfSR heterodimerize. D) Mutation C205A, which compromises DNA binding, abrogates BfER's capacity to repress BfSR-mediated transcription of an ERE-driven reporter. Full-length BfSR, wild-type BfER (BfER-WT), and the BfER C205A mutant were cotransfected. Cells were treated with no hormone (white bars) or 1 µM estradiol (black bars).
Mentions: In contrast, the BfER LBD has lost the ancestral capacity to activate transcription in response to estrogens. Neither the BfER LBD nor the full-length BfER activates transcription in response to high doses of any of a large panel of vertebrate steroid hormones, including estradiol, estriol, and estrone (Figures 3A, S5). In the absence of hormone, full-length BfER (like other estrogen receptors) represses basal levels of ERE-reporter transcription, but hormone treatment does not alter this effect (Figure 4A). On an SRE-driven reporter, full-length BfER has no effect (Figure 4B). BfER does retain the ancestral DNA-recognition function: a fusion construct of the BfER-DBD with the constitutively-active NFkB activation domain yields robust transcription of an ERE-driven reporter (Figure 5A), but no activity on an SRE-driven reporter (Figure 5B). Further, BfER strongly binds EREs in an electrophoretic mobility shift assay (EMSA, Figure 5C).

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