CpG methylation in the 5′-flanking region of LGP2 gene lacks association with resistance/susceptibility to GCRV but contributes to the differential expression between muscle and spleen tissues in grass carp, Ctenopharyngodon idella

Abstract

As an intracellular pattern recognition receptor (PRR), laboratory of genetics and physiology 2 (LGP2) plays a pivotal role in detecting nucleic acids of invading pathogens and simultaneously modulating signaling by retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in type I interferon (IFN–I) pathway. Nevertheless, the underlying antiviral transcription mechanism of LGP2 remains obscure. The present study attempted to reveal the methylation levels of CiLGP2 (Ctenopharyngodon idella LGP2) in muscle and spleen of grass carp and their association with the resistance against grass carp reovirus (GCRV). By prediction, the CpG island was 133 bp in length in 5′-flanking region, containing six candidate CpG loci, whose methylation statuses were investigated by virtue of the bisulfite sequencing PCR (BSP) among muscle and spleen tissues in 120 individuals that were divided into resistant/susceptible groups after a challenge experiment, and the association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was employed to ascertain the interrelation between methylation status and transcription of CiLGP2. The CpG sites at −1394, −1366, −1331 and −1314 nt were identified as hypermethylated, inversely unmethylated at −1350 CpG site. The −1411 CpG site presented six methylation patterns as well as one mentionable type of mutation triggered by spontaneous deamination. Although there was no statistically significant difference on DNA methylation with resistance against GCRV at −1411 CpG site, the methylation levels were significantly lower in spleen than those in muscle, accompanied by higher mRNA expression of CiLGP2 in spleen. Notably, DNA methylation may be conceivably serve as an essential regulatory factor for CiLGP2 antiviral transcription in spleen. This research first demonstrated the relationship between DNA methylation and LGP2 gene expression, preliminary revealed the underlying transcription mechanism of CiLGP2 against GCRV as well as provided potential references and laid a theoretical foundation for viral recognition and regulation research of LGP2 in vertebrates.