Identification of the Porcine XIST Gene and Its Differential CpG Methylation Status in Male and Female Pig Cells

Abstract

XIST, a long non-coding RNA, plays an important role in triggering X chromosome inactivation in eutherians, and is used extensively for qualifying stem cells and cloned embryos. However, a porcine XIST has not yet been thoroughly identified despite its biological importance in a wide variety of research fields. Here, we present a full-length porcine XIST sequence assembled using known sequences (GenBank), RNA-Seq data (NCBI SRA), and PCR/sequencing. The proposed porcine XIST gene model encodes a 25,215-bp transcript consisting of 7 exons, including two conserved and two porcine-specific repeat regions. Transcription covering the entire XIST region was observed specifically in female cells, but not in male cells. We also identified eight transcription starting sites (TSSs) and evaluated CpG methylation patterns in the upstream (+2.0 kb) and downstream (-2.0 kb) regions. Sixty-seven CG di-nucleotides identified in the target region were considered to be candidate CpG sites, and were enriched in the following two regions: -284 to +53 bp (13 sites) and +285 to +1,727 bp (54 sites) from the selected TSS. Male 5' region of XIST (64.5 sites, 96.26%) had a higher level of CpG methylation than female DNA (33.4 sites, 49.85%). Taken together, our results revealed that the porcine XIST gene is expressed exclusively in female cells, which is influenced by the lower level of CpG methylation in the putative promoter region compared with male cells. The porcine XIST presented in this study represents a useful tool for related research areas such as porcine embryology and stem cell biology. © 2013 Hwang et al.