Porcine SOX9 Gene Expression Is Influenced by an 18bp Indel in the 5′-Untranslated Region

Sex determining region Y-box 9 (SOX9) is an important regulator of sex and skeletal development and is expressed in a variety of embryonal and adult tissues. Loss or gain of function resulting from mutations within the coding region or chromosomal aberrations of the SOX9 locus lead to a plethora of detrimental phenotypes in humans and animals. One of these phenotypes is the so-called male-to-female or female-to-male sex-reversal which has been observed in several mammals including pig, dog, cat, goat, horse, and deer. In 38, XX sex-reversal French Large White pigs, a genome-wide association study suggested SOX9 as the causal gene, although no functional mutations were identified in affected animals. However, besides others an 18bp indel had been detected in the 5'-untranslated region of the SOX9 gene by comparing affected animals and controls. We have identified the same indel (Delta 18) between position +247bp and +266bp downstream the transcription start site of the porcine SOX9 gene in four other pig breeds; i.e., German Large White, Laiwu Black, Bamei, and Erhualian. These animals have been genotyped in an attempt to identify candidate genes for porcine inguinal and/or scrotal hernia. Because the 18bp segment in the wild type 5'-UTR harbours a highly conserved cAMP-response element (CRE) half-site, we analysed its role in SOX9 expression in vitro. Competition and immunodepletion electromobility shift assays demonstrate that the CRE half-site is specifically recognized by CREB. Both binding of CREB to the wild type as well as the absence of the CRE half-site in Delta 18 reduced expression efficiency in HEK293T, PK-15, and ATDC5 cells significantly. Transfection experiments of wild type and Delta 18 SOX9 promoter luciferase constructs show a significant reduction of RNA and protein levels depending on the presence or absence of the 18bp segment. Hence, the data presented here demonstrate that the 18bp indel in the porcine SOX9 5'-UTR is of functional importance and may therefore indeed be a causative variation in SOX9 associated traits.