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.
Highlights
X-chromosome inactivation (XCI) occurs early in embryo development, and involves silencing one of the X chromosomes in mammalian female cells to compensate gene dosage [1]
The DNA sequence ranging from nucleotides 289,233 to 257,103 in the pig scaffold was well-aligned with the three gene models
reverse transcriptase (RT)-PCR using these primer sets confirmed that the candidate XIST gene was transcribed only in female porcine embryonic fibroblast (PEF), and the target regions were not detected in male PEFs (Figure S2)
Summary
X-chromosome inactivation (XCI) occurs early in embryo development, and involves silencing one of the X chromosomes in mammalian female cells to compensate gene dosage [1]. The importance of XIST RNA for early embryo development and embryonic stem cell research has been highlighted. The identification of abnormal expression of Xist and X-chromosome-linked genes in a cloned mouse was reported [12], and RNA interference (RNAi)-mediated downregulation of Xist during embryo development in cloned mice was shown to dramatically elevate birth rates [13]. These reports suggest an important role for Xist in early embryo development. While the identified porcine XIST sequences could be used for research related to early embryo development and stem cells, identification of a full XIST sequence using gene prediction methods may be difficult [18] because of the low sequence conservation of XIST genes among species [19,20]
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