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Ancestral Resurrection of Anthropoid Estrogen Receptor β Demonstrates Functional Consequences of Positive Selection

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Ancestral Resurrection of Anthropoid Estrogen Receptor β Demonstrates Functional Consequences of Positive Selection

Amy Weckle et al. Mol Phylogenet Evol.

Abstract

Anthropoid primates arose during the Eocene approximately 55 million years ago (mya), and extant anthropoids share a most recent common ancestor ∼40mya. Paleontology has been very successful at describing the morphological phenotypes of extinct anthropoids. Less well understood is the molecular biology of these extinct species as well as the phenotypic consequences of evolutionary variation in their genomes. Here we resurrect the most recent common ancestral anthropoid estrogen receptor β gene (ESR2) and demonstrate that the function of this ancestral estrogen receptor has been maintained during human descent but was altered during early New World monkey (NWM) evolution by becoming a more potent transcriptional activator. We tested hypotheses of adaptive evolution in the protein coding sequences of ESR2, and determined that ESR2 evolved via episodic positive selection on the NWM stem lineage. We separately co-transfected ESR2 constructs for human, NWM, and the anthropoid ancestor along with reporter gene vectors and performed hormone binding dose response experiments that measure transactivation activity. We found the transactivation potentials of the ancestral and human sequences to be significantly lower (p<0.0001 in each comparison) than that of the NWM when treated with estradiol, the most prevalent estrogen. We conclude the difference in fold activation is due to positive selection in the NWM ERβ ligand binding domain. Our study validates inferential methods for detecting adaptive evolution that predict functional consequences of nucleotide substitutions and points a way toward examining the functional consequences of positive Darwinian selection.

Keywords: Adaptive evolution; Ateles fusciceps; Endogenous; Exogenous; New World monkey; Old World monkey.

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Phylogenetic relationships of mammals included in this analysis. The black circles indicate the ancestral crown anthropoid (ACA). The tree represented on the left depicts branching in a two-ratio model, red branches denote stem and crown NWMs. The tree on the right depicts branches in the three-ratio model, red branches denote stem NWMs and blue branches denote crown NWMs.
Figure 2
Figure 2
Structure of ERβ. Numbering indicates the C-terminal end of each domain. Shaded domains denote the region of the receptor that was transfected into CHO-K1 cells for functional comparison.
Figure 3
Figure 3
a: Depiction of luciferase reporter assay. Upon estrogen (purple hexagon) binding, human ERβ (pCMV-huERβ construct depicted in pink containing a GAL4 DNA binding domain denoted by the turquoise downward arrow) enters the cell nucleus and regulates gene expression by binding to a DNA response element (dark orange band within the circular depiction of the luciferase reporter vector) triggering luciferase production quantifiable as luminescence (yellow stars). b: Depiction of luciferase reporter assay. Upon estrogen (purple hexagon) binding, spider monkey ERβ (pCMV-ateERβ construct depicted in brown containing a GAL4 DNA binding domain denoted by the turquoise downward arrow) enters the cell nucleus and regulates gene expression by binding to a DNA response element (dark orange band within the circular depiction of the luciferase reporter vector) triggering luciferase production quantifiable as luminescence (yellow stars). c: Depiction of luciferase reporter assay. Upon estrogen (purple hexagon) binding, ancestral ERβ (pCMV-ancERβ construct depicted in green containing a GAL4 DNA binding domain denoted by the turquoise downward arrow) enters the cell nucleus and regulates gene expression by binding to a DNA response element (dark orange band within the circular depiction of the luciferase reporter vector) triggering luciferase production quantifiable as luminescence (yellow stars).
Figure 4
Figure 4
a: Luciferase production in response to estradiol. The spider monkey ERβ transactivation potential (brown curve) was significantly different from the human ERβ (pink curve) (p<0.0001). b: Luciferase production in response to estradiol. The spider monkey ERβ transactivation potential (brown curve) was significantly different from the ACA ERβ (green curve) (p<0.0001). c: Luciferase production in response to estradiol. The transactivation potential has been maintained between human and the ACA (p=0.5299).

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