A 192 bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1

Abstract

Background Toll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR genes polymorphisms with disease susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs), as a new type of molecular markers developed recently, have great potential in population genetics and quantitative trait locus mapping. In this study, bioinformatic prediction combined with PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution was examined, and for one RIP the impact on gene activity and phenotype was further evaluated. Results Five RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was shown to act as an enhancer increasing the activities of TLR6 putative promoter and two mini-promoters. Furthermore, real-time quantitative polymerase chain reaction (qPCR) analysis revealed significant association (p < 0.05) of the ERV insertion with increased mRNA expression of TLR6, the neighboring gene TLR1, and genes downstream in the TLR signaling pathway such as MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) as well as the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in tissues of 30 day-old piglet. In addition, serum IL6 and TNFα concentrations were also significantly upregulated by the ERV insertion (p < 0.05). Conclusions A total of five RIPs were identified in five different TLR loci. The 192 bp ERV insertion in the first intron of TLR6 was associated with higher expression of TLR6, TLR1, and several genes downstream in the signaling cascade. Thus, the ERV insertion may act as an enhancer affecting regulation of the TLR signaling pathways, and can be potentially applied in breeding of disease resistant animals.

SINE Insertion May Act as a Repressor to Affect the Expression of Pig LEPROT and Growth Traits

“…As the bait, 3 genes in 15 assembled non-reference genomes (Landrace, Yorkshire, Pietrain, Berkshire, Hampshire, cross-breed of Yorkshire/Landrace/Duroc, Wuzhishan, Tibetan, Rongchang, Meishan, Bamei, Bama, Jinhua, Goettingen, and Ellegaard Gottingen minipigs) were downloaded from the NCBI database (version Annotation release 106, , accessed on 1 July 2020) to screen for large structural variations (more than 50 bp) by sequence alignment using the ClustalX program. Retrotransposon insertion including SINE, LINE, and ERV among these predicted structural variations were annotated by RepeatMasker (version RepeatMasker 4.1.0 Released, , accessed on 3 July 2020) with a customer-constructed library [ 28 , 32 ] and they were designated as RIPs. These RIPs were further validated by PCR amplification (Vazyme, Nanjing, China) in seven Chinese native pig breeds (Erhualian, Fengjing, Diannan small-ear, Wuzhishan, Bama, Tibetan, Meishan), three commercial pig breeds (Duroc, Landrace, Large White), one cross-breed (Sujiang), and wild boars.…”

“…The primers are listed in Table S2 . Then, the 852 bp and 566 bp sequences were inserted into the pGL3-basic vectors (Promega, Madison, WI, USA) to construct LEPROT SINE+-Luc+(En) vector and LEPROT SINE—Luc+ (En) vector, which contain β-globin and Oct4 mini promoters, respectively, for enhancer/repressor activity evaluation [ 32 , 35 ]. In 24-well plates, PK15 and 3T3-L1 cells were cultured to approximately 70% confluence and transfected with the luciferase reporter vector using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA).…”

“…Recently, research works from our laboratory have indicated RIPs distribution in the pig genome [ 27 , 28 ]; their role in evaluating population structure [ 29 , 30 ]; and their insertion function on protein-coding genes and pig phenotypic variations such as GH , TLR1 , and TLR6 [ 31 , 32 , 33 ]. Therefore, considering the importance of LEPTIN , LEPR , and LEPROT on mammalian metabolism and growth, the RIPs in these three genes were identified, and the correlation between the RIPs and some pig economic performance was investigated in this study.…”

Two Retrotransposon Elements in Intron of Porcine BMPR1B Is Associated with Phenotypic Variation

“…All the primers in this study were synthesized by Beijing Liuhe Huada Gene Technology Co. Ltd. (Beijing, China). PCR was carried out using ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA), and the reaction system was prepared as described previously by using the 12 DNA pools as template [ 38 ].…”

“…Based on the retrotransposon insertion polymorphism, several types of molecular markers have been developed in plant breeding [ 30 , 31 ]. Recently, RIPs were also identified as a causative mutation of the phenotype, or closely related to the performance of domestic animals [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. Considering the importance of the BMPR1B protein in several biological process, the RIPs in BMPR1B were excavated and verified.…”