X-chromosome inactivation (XCI) is an epigenetically essential process for balancing dosage of X-linked genes between male and female eutherian. Importance of this complex and species-specific event has been highlighted recently in developmental and stem cell biology. However, the process has been confirmed only in restricted species, even though the species-specific studies are needed for comprehensive understanding of XCI in specific species. XCI is regulated by the various genes, many of which are coded on the X chromosome inactivation centre (XIC). Among the XIC-linked genes, especially non-coding RNA (ncRNA) like XIST, which is master gene for XCI, are known to regulate XIC. But the centre is not identified in various species. In this study, we identified XIC in pig and analysed the dosage differences of XIC-linked gene in porcine embryos. At first, the centre was searched in pig. The genomic length of the porcine XIC was similar to human XIC and the order and coding strand of the counterparts in pig XIC were same as the human XIC-linked genes. However, sequence comparison between human XIC-linked gene and its porcine counterpart showed that ncRNA around XIST were less conserved rather than protein-coding genes. This would be caused by rapid evolution of genomic region harboring ncRNA. The expression of XIC-linked genes was compared between male and female porcine embryonic fibroblast (PEF) to confirm that dosage compensation is completed in PEF. Most of the genes were not expressed sex-specifically, but two genes, XIST and an uncharacterized gene, LOC102165544, were expressed female preferentially in PEF. Interestingly, LOC102165544, which had low sequence homology with human JPX, was expressed about 2-fold higher in female PEF. This means that XIST and LOC102165544 are XCI-escaping genes. Among the XIC-linked genes, CHIC1, XIST, LOC102165544, and RLIM were stably expressed in embryonic stage, and XIST and LOC102165544 were up-regulated after morula formation. As XIST accumulation is a requisite for XCI initiation, expression levels of the 4 genes between male and female blastocysts were compared. Interestingly, expression levels of CHIC1 and RLIM were not different in male and female blastocysts. This means their dosage would be already compensated in porcine blastocyst. Additionally, to confirm loci of the 2 genes CHIC1 and RLIM harbor one of the inactive alleles in female blastocyst, the DNA methylation pattern was examined. One of the CHIC1 alleles was inactive but RLIM CpG site was hypo-methylated in female blastocyst. This would indicate that one of the RLIM alleles is transcriptionally inactivated by chromatin modification rather than by DNA methylation of the allele. Regulatory regions of XIST and LOC102165544 were demethylated in blastocyst and this showed XCI was not finished in porcine blastocyst. Conclusively, our results demonstrate the XCI already occurs in porcine blastocyst at least one gene but it is not completed.This work was supported by Next BioGreen21 program (PJ009493), Rural Development Administration, Republic of Korea.
DNA methylation stabilizes X chromosome inactivation in eutherians but not in marsupials: evidence for multistep maintenance of mammalian X dosage compensation.
