Brenig Bertram, Duan Yanyu, Xing Yuyun, Ding Nengshui, Huang Lusheng, Schütz Ekkehard
Institute of Veterinary Medicine, Georg-August-University, Burckhardtweg 2, D-37077, Göttingen, Germany.
Key Laboratory for Animal Biotechnology of Jiangxi Province and the Ministry of Agriculture of China, Jiangxi Agricultural University, 330045, Nanchang, China.
PLoS One. 2015 Oct 2;10(10):e0139583. doi: 10.1371/journal.pone.0139583. eCollection 2015.
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 18 bp 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 (Δ18) between position +247 bp and +266 bp 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 18 bp 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 Δ18 reduced expression efficiency in HEK293T, PK-15, and ATDC5 cells significantly. Transfection experiments of wild type and Δ18 SOX9 promoter luciferase constructs show a significant reduction of RNA and protein levels depending on the presence or absence of the 18 bp segment. Hence, the data presented here demonstrate that the 18 bp indel in the porcine SOX9 5'-UTR is of functional importance and may therefore indeed be a causative variation in SOX9 associated traits.
性别决定区Y盒9（SOX9）是性别和骨骼发育的重要调节因子，在多种胚胎和成年组织中表达。SOX9基因座编码区内的突变或染色体畸变导致的功能丧失或获得，会在人类和动物中引发大量有害表型。其中一种表型是所谓的雄性向雌性或雌性向雄性的性反转，在包括猪、狗、猫、山羊、马和鹿在内的几种哺乳动物中都有观察到。在38,XX性反转的法国大白猪中，一项全基因组关联研究表明SOX9是致病基因，尽管在受影响的动物中未发现功能性突变。然而，除其他因素外，通过比较受影响的动物和对照，在SOX9基因的5'非翻译区检测到一个18 bp的插入/缺失。我们在其他四个猪品种，即德国大白猪、莱芜黑猪、八眉猪和二花脸猪中，在猪SOX9基因转录起始位点下游+247 bp至+266 bp位置之间鉴定出了相同的插入/缺失（Δ18）。这些动物已经进行了基因分型，试图确定猪腹股沟疝和/或阴囊疝的候选基因。由于野生型5'-UTR中的18 bp片段含有一个高度保守的cAMP反应元件（CRE）半位点，我们在体外分析了其在SOX9表达中的作用。竞争和免疫去除电泳迁移率变动分析表明，CRE半位点被CREB特异性识别。CREB与野生型的结合以及Δ18中CRE半位点的缺失，均显著降低了HEK293T、PK-15和ATDC5细胞中的表达效率。野生型和Δ18 SOX9启动子荧光素酶构建体的转染实验表明，根据18 bp片段的存在与否，RNA和蛋白质水平会显著降低。因此，本文提供的数据表明，猪SOX9 5'-UTR中的18 bp插入/缺失具有功能重要性，因此可能确实是与SOX9相关性状的致病变异。
