Li Yan, Wang Lei, Zhou Jiawei, Li Fenge
Key Laboratory of Pig Genetics and Breeding of Ministry of Agriculture & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, PR China.
PeerJ. 2016 Jul 14;4:e2186. doi: 10.7717/peerj.2186. eCollection 2016.
Klotho (KL), originally discovered as an aging suppressor, is a membrane protein that shares sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might play a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311SNP (A or G) in KL intron 1 was detected in Landrace × DIV pigs using the Porcine SNP60 BeadChip. The pGL-D2-A and pGL-D2-G were constructed with KL-D2 and the intron fragment of different alleles and relative luciferase activity of pGL3-D2-G was significantly higher than that of pGL3-D2-A in the PK cells and ST cells. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immune-precipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference experiments. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1.
klotho（KL）最初被发现是一种衰老抑制因子，是一种与β-葡萄糖苷酶具有序列相似性的膜蛋白。最近的报道表明，KL可能在脂肪细胞成熟和全身葡萄糖代谢中发挥作用。然而，关于参与调节猪KL基因表达的转录因子知之甚少。缺失片段分析确定KL-D2（-418 bp至-3 bp）为猪KL核心启动子。使用猪SNP60基因芯片在长白猪×DIV猪中检测到KL内含子1中的MARC0022311SNP（A或G）。用KL-D2和不同等位基因的内含子片段构建了pGL-D2-A和pGL-D2-G，在PK细胞和ST细胞中，pGL3-D2-G的相对荧光素酶活性显著高于pGL3-D2-A。这可能是KL与转录因子有机阳离子转运体1（OCT-1）结合能力发生变化的结果，这通过电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）得到了证实。此外，通过RNA干扰实验，OCT-1调节内源性KL表达。我们的研究表明，SNP MARC0022311通过OCT-1调节猪KL的启动子活性，从而影响其表达。
