Dissection of molecular basis on a causative mutation for ear size QTL on chromosome 7 in pigs

Abstract

The pinna，an important component of the external ear, first appeared in mammals. It plays a vital role in collecting sounds, radiating heat and signaling mood. In humans, the most common ear defect is microtia with prevalence rates ranging from 0.83 to 17.4 per 10,000 births. However, only a minority of genetic or environmental causes have been found to date. Domestic pigs have obvious diversity in pinna size, indicating a potential animal model for pinna deformity in humans. To identify causative mutations underlying pinna size, we performed a genome scan in a large scale White Duroc x Erhualian pig resource family, of which Erhualian has exceptionally large and floppy pinna and White Duroc has small and erect or semi-erect pinna. We mapped a 1% genome-wide QTL for pinna size to a 2 cM confidence interval around 58 cM on chromosome 7, which explained over 40% phenotypic variance. We further fine-mapped the QTL and finally identified that a missense mutation PPARD G32E could cause such major QTL effect on pinna size in pigs. This is the first time a novel function of PPARD on controlling pinna size has been found. After that, we continued to study the function of the causative mutation at the molecular level. We showed that the G32E substitution reduced ligand-dependent transcription activity of PPARD by transient transfection analysis. We hypothesized three underlying mechanisms, i.e. the substitution might alter (i) subcellular localization, (ii) ligand binding affinity, and/or (iii) ubiquitination according to function of the A/B domain of PPARs. To check whether the substitution altered subcellular localization, we mutated glycine 32 of wild-type PPARD into glutamic acid. Using immunofluorescence analysis, we observed that wild-type PPARD completely localized in nucleus whereas G32E mutant resided partly in cytosol in PK-15 cells and primary pinna-driven chondrocytes. To elucidate the effect of the substitution on subcellular localization, we analysed A/B domain deletion mutant and G32E mutant treated by leptomycin B, an inhibitor of CRM1 export protein. The A/B domain deletion mutant totally localized in the nucleus, whereas cytosolic localization of the G32E mutant was blocked by leptomycin B. This indicated that G32E substitution provided A/B domain a novel function to promote CRM1-mediated nuclear PPARD export. By surface plasmon resonance technology, we detected binding affinity of GW0742 towards purified His-tagged wild-type and mutated PPARD. Equilibrium dissociation constant (KD) did not show significant distinction between wild-type PPARD and G32E mutant. We also found that ubiquitination at lysine (K) 16-18 in the A/B domain of PPARD can promote ligand-induced transcription activation. The G32E substitution prevented ubiquitination of PPARD and thereby reduced transcription activity comparable with the neighbouring K16-18R mutant. In summary, the G32E substitution is a loss-of-function mutation that reduces transcription activity of PPARD by promoting nuclear export and changing non-proteolytic ubiquitination. Corresponding to this, we found that overexpression of G32E mutant led to 4.1-fold reduction of the mRNA levels of β-catenin and 40% decrease of Wnt/β-catenin signaling with TCF/LEF reporter assay compared with wild-type treatment. As we known, Wnt/β-catenin pathway inhibited chondrocyte differentiation and SOX9 expression during skeletal development. Furthermore, inactivation of SOX9 in pinna chondrocyte progenitor cells - cranial neural crest (CNC) cells resulted in a complete absence of cartilages and endochondral bones derived from the CNC. It indicated a potential mechanism underlying the G32E substitution in PPARD causing an increased pinna size in pigs. In this thesis, we also studied an 18-bp indel in the 5’-untranslated region (UTR) of SOX9, which is an essential transcription factor in all chondrocyte development. We identified an 18-bp insertion/deletion (indel) in the 5’-UTR of SOX9 in three pig breeds−Laiwu, Bamei and Large White. The 18-bp fragment harbours a CRE half-site that can interact with a transcription factor CREB by mobility and supershift analyses. We found that the variant was located in a stable stem loop within the secondary structure of 5’-UTR using RNA-folding software. The 18-bp deletion altered the secondary structure. By transient transfection analysis, we identified that the 18-bp was indispensable to the translation of SOX9. The deletion significantly reduced translation efficiency and caused mRNA destability. To determine whether the indel plays a physiological role in pinna development of pigs, we performed an association study in a White Duroc x Erhualian resource population. In this population, the indel was not associated with pinna size.