A 192\xa0bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1

Xiaoyan Wang,Eduard Murani,Enrico D’Alessandro,Grazia Galeano,Klaus Wimmers,Kui Li,Chengyi Song,Cai Chen,Zixuan Chen,Yalong An

doi:10.1186/s13100-021-00248-w

Abstract

BackgroundToll-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.ResultsFive 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).ConclusionsA 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.

Highlights

Toll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses
We identified 53 large structural variations (SVs, defined as variants more than 50 bp and less than 1000 bp long) or large frameshift variants, and 32 of them were predicted as Retrotransposon insertion polymorphisms (RIPs), including 15 Short Interspersed Nuclear Elements (SINE), 11 endogenous retrovirus (ERV), and 6 LINE RIPs, which were summarized in additional file 1 (Table S1)
A single 357 bp LINE RIP in the 5’ flanking region of TLR5 was found as well as one 192 bp and one 413 bp ERV RIPs in first intron of TLR6 and TLR7, respectively. We named those insertions as TLR3SINE-RIP, TLR5-LINE-RIP, TLR6-ERV-RIP, TLR7-ERVRIP, and TLR8-SINE-RIP, respectively (Fig. 1B and 1C)

Summary

Introduction

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. 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. Toll-like receptors (TLRs) play vital roles in innate and adaptive immune responses due to their ability to recognize different types of pathogens and associated. It is commonly accepted that TLRs are important candidate genes for some human immune diseases

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