Abstract
Peroxisome proliferator-activated receptor beta/delta (PPARD) is a crucial and multifaceted determinant of diverse biological functions including lipid metabolism, embryonic development, inflammatory response, wound healing and cancer. Recently, we proposed a novel function of porcine PPARD (sPPARD) in external ear development. A missense mutation (G32E) in an evolutionary conservative domain of sPPARD remarkably increases external ear size in pigs. Here, we investigated the underlying molecular mechanism of the causal mutation at the cellular level. Using a luciferase reporter system, we showed that the G32E substitution reduced transcription activity of sPPARD in a ligand-dependent manner. By comparison of the subcellular localization of wild-type and mutated sPPARD in both PK-15 cells and pinna cartilage-derived primary chondrocytes, we found that the G32E substitution promoted CRM-1 mediated nuclear exportation of sPPARD. With the surface plasmon resonance technology, we further revealed that the G32E substitution had negligible effect on its ligand binding affinity. Finally, we used co-immunoprecipitation and luciferase reporter assays to show that the G32E substitution greatly reduced ubiquitination level by blocking ubiquitination of the crucial A/B domain and consequently decreased transcription activity of sPPARD. Taken together, our findings strongly support that G32E is a functional variant that plays a key role in biological activity of sPPARD, which advances our understanding of the underlying mechanism of sPPARD G32E for ear size in pigs.
Highlights
Deciphering the genetic architecture of complex traits is a big challenge for geneticists
The result showed that activation of peroxisome proliferatoractivated receptor beta/delta (PPARD) by GW0742 inhibited the growth of primary ear chondrocytes of wild-type pigs in vitro, especially after 5-days treatment (Figure S1)
No potential phosphorylation site was found in the A/B domain of sPPARD according to the prediction method described in [24]
Summary
Deciphering the genetic architecture of complex traits is a big challenge for geneticists. Only a handful of causal variants underlying quantitative traits have been unequivocally identified in domestic animals [1], such as the DGAT1 K232A [2,3,4] and ABCG2 Y581S [5] mutations affecting milk in cattle, the porcine IGF2 intron g.3072. We have successfully identified a missense mutation in the peroxisome proliferatoractivated receptor beta/delta (PPARD) that contributes to external ear size in pigs [9]. The protein-altering mutation explains a proportion of ear size across diverse Chinese pigs. Our findings further suggest a novel biological role of PPARD in external ear development. Our results clearly show that G32E is a functional variant affecting activity of sPPARD, providing novel insights into the biology of the important nuclear hormone receptor
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