Relaxed constraint and functional divergence of the progesterone receptor (PGR) in the human stem-lineage

Abstract

The steroid hormone progesterone, acting through the progesterone receptor (PR), a ligand-activated DNA-binding transcription factor, plays an essential role in regulating nearly every aspect of female reproductive biology. While many reproductive traits regulated by PR are conserved in mammals, Catarrhine primates evolved several derived traits including spontaneous decidualization, menstruation, and a divergent (and unknown) parturition signal, suggesting that PR may also have evolved divergent functions in Catarrhines. There is conflicting evidence, however, whether the progesterone receptor gene (PGR) was positively selected in the human lineage. Here we show that PGR evolved rapidly in the human stem-lineage (as well as other Catarrhine primates), which likely reflects an episode of relaxed selection intensity rather than positive selection. Coincident with the episode of relaxed selection intensity, ancestral sequence resurrection and functional tests indicate that the major human PR isoforms (PR-A and PR-B) evolved divergent functions in the human stem-lineage. These results suggest that the regulation of progesterone signaling by PR-A and PR-B may also have diverged in the human lineage and that non-human animal models of progesterone signaling may not faithfully recapitulate human biology.

Fig 1. Phylogeny of species used to characterize the strength and direction of selection on PGR genes.

Branch lengths are drawn proportional to the number of substitutions per site [Subs per site, see inset scale bar] under the GTR model. Substitutions per site are a measure of sequence divergence–long branches are more diverged than shorter branches. The human stem-lineage, modern human, Denisovan, and Neanderthal lineages are shown in magenta; the Hominini clade is shown in cyan. Asterisk (*) indicates the human stem-lineage, which has an ω = 1.06 and k = 0.00 (LRT = 5.01, P = 0.01) under the RELAX a priori model. Branches are colored black if k is not significantly different than 1, red if k is significantly greater than 1 and blue if k is significantly less than 1 in the ad hoc RELAX analyses.

Fig 2. Distribution of human-specific amino acid substitutions in PGR.

(A) Functional domains of the progesterone receptor isoforms A (PR-A) and B (PR-B). AF1-3, activation function domains 1–3. IF, inhibitory function domain. DBD, DNA-binding domain. H, hinge region. LBD, ligand-binding domain. (B) Conservation of PGR across 119 Eutherian mammals shown as a logo [colors are arbitrary]. Bits are a measure of randomness; random sequences have a low bit score while non-random [conserved] sequences have a high bit score [max 4.2 for amino acid sequences]. (C) Site-specific d_N/d_S ratios (ω) inferred by MEME (red), FUBAR (pink), SLAC (light blue), and FEL (dark blue). (D) Logo showing conservation of human-specific substitutions within modern and archaic humans. (E) CADD scores for human-specific substitutions. (F) Logo showing conservation at sites with human-specific substitutions in non-human mammals.

Fig 3. Functional divergence of the human progesterone receptor.

(A) Hominini (African ape) phylogeny, nodes with ancestral sequence reconstructions are indicated. (B) The dPRL-332 luciferase reporter vector with the location of experimentally verified transcription factor binding sites. (See main text for references). (C) Strip chart showing relative luminescence values, standardized to negative control (log scale). Mean is depicted as a white dot; 95% confidence intervals are indicated by the ends of the black vertical error bars. (D) The mean difference in luminescence of AncHuman against AncHominini shown as a Cumming estimation plot (log scale). Mean differences are plotted as bootstrap sampling distributions (n = 5,000). Each mean difference is depicted as a dot, 95% confidence intervals are indicated by the ends of the black vertical error bars. The AncHuman PR-A was a weaker trans-activator than the AncHominini PR-A (Mann-Whitney P = 0.035), while the AncHuman PR-B was a stronger trans-activator than the AncHominini PR-B (Mann-Whitney P = 9.46×10⁻⁴). The ability of AncHuman PR-A to repress AncHuman PR-B was weaker than AncHominini PR-A on AncHominini PR-B (Mann-Whitney P = 3.06×10⁻⁶).

Fig 4. Model of progesterone receptor action under the isoform switching hypothesis.

Progesterone signaling through PR-B promotes the maintenance of pregnancy and inhibits parturition, while PR-A induces the cessation of pregnancy and parturition by repressing the actions of PR-B. PR-B evolved stronger trans-activation ability in the human lineage, while the ability of PR-A to trans-repress PR-B is weaker in the human lineage, suggesting the pregnancy maintenance functions of PR-B are stronger and the parturition inhibiting functions of PR-A are weaker in the human lineage than other species.