Epigenetics of X-chromosome inactivation and its relation to pluripotent genes in porcine early embryogenesis

Abstract

X-chromosome inactivation (XCI) is an essential epigenetic process observed in female eutherian embryos to equalize expression levels of X-linked genes between male and female adults. A number of studies have been confirmed the dynamic processes and molecular mechanisms of XCI in mice. Its significance on epigenetic regulation during embryo development and as an indicator for evaluating pluripotent status in stem cells originated from pre- or post-implantation embryos has been highlighted recently. Despite its importance and broad applications in stem cells and developmental studies, the insight of epigenetic phenomenon accumulated for about fifty years has been restricted in mice. However, several studies have shown evolutionary diversities on XCI and its regulators, and suggested that analyzing XCI should be carried out with species-specific manner. Therefore, in this study, XCI in pigs were studied by 1) identifying main regulators for XCI, XIST and X-chromosome inactivation center (XIC), 2) confirming epigenetic changes of XCI in preimplantation embryos, and 3) examining the relation between XCI and pluripotent genes.

The aim of the first study was the identification of XIST orthologs, which are the main inducer for XCI by coating inactive X-chromsome, in pigs. Although this non-coding RNA (ncRNA) and its functions for inactivating X-chromosome have been confirmed in mice and humans about twenty years ago, the ortholog in pigs has yet to be identified in pigs recently. For this, sequence comparison and expression analysis were performed in porcine embryonic fibroblasts (PEFs). The identified ncRNA was 25 Kb lengths, and its exon composition was similar to XIST of human and mouse. However, general sequence homologies between the transcript and XIST in other species were very low. Newly identified gene in the first study was expressed only in female PEFs. The gene contains four repeat regions and their monomers in the first and second repeat region were same to repeat A and repeat B in mouse Xist, respectively but monomers in other two repeat regions were porcine specific. Furthermore, CpG sites on its promoter were hypermethylated in male PEFs, which only have active X-chromsome. These results demonstrated that identified gene is porcine XIST.

X-linked genes located on close distance with Xist are reported to regulate XCI in mouse. The genomic region has been called XIC. However, even though importance of the region has been highlighted because of various regulators in the region for XCI by activating (Jpx, Rlim, and Ftx) or repressing (Tsix) Xist expressions, the genomic region was not confirmed in pigs. Therefore, in the second study, porcine XIC was searched and expression patterns of XIC-linked genes were analyzed in porcine preimplantation embryos to examine the XCI process in pigs. XICs are generally composed to protein coding genes and ncRNAs within about 0.5 Mb and 1.1 Mb of genomic range in mice and human, respectively. The ncRNAs have been evolved by disruption of ancestral protein coding genes and insertion of mobile element during divergence of eutherian and marsupials. Proportion and transcribing strand of XIC-linked genes are conserved but the sequences of ncRNAs in XIC are less conserved among the eutherians. 1.1 Mb lengths of porcine genomic region which was a synteny with human XIC was determined to be a porcine XIC. Porcine XIC contained porcine XIST, and sequences of the ncRNAs in the region were less conserved compared to those of protein coding genes. These results suggest that porcine XIC shares the evolutionary properties observed in eutherian XIC. Among the XIC-linked genes, CHIC1, XIST, uncharacterized ncRNAs, LOC102165544, and RLIM were stably expressed in embryonic stages. Expression levels of protein coding genes, CHIC1 and RLIM were decreased during morula to blastocyst development in females and expression levels of these genes were not different between male and female blastocysts. This would mean decrease of expression levels of the genes in female blastocysts resulted in compensating dosages of the genes between males and females. However, other two ncRNAs, XIST and LOC102165544, showed increase of expression levels after morula stage in females and sexual dimorphic expressions were observed in blastocysts. One of the CHIC1 promoter alleles was methylated in female blastocyst. These results mean dosage compensation of several genes would be achieved in porcine blastocysts but chromosome wide inactivation of X-chromosome would not be accomplished at the embryonic stage because both allele of XIST promoter were demethylated in female blastocysts.

After examining XCI in embryonic stages in pigs, relations between OCT4, which is a gate-keeper for maintaining pluripotencty and one of the XCI regulator, and XIC-linked genes, were examined. Before examining the function of OCT4 in XIC-linked genes, it was required to confirm the presence of OCT4 variants in pigs because they have been reported to induce false-positive detection. Therefore, the presence of OCT4 variants and differential expressions patterns were examined in the third study. Porcine OCT4 transcribed additional two polyadenylated RNAs, OCT4B and OCT4B1 like human OCT4, and they share the majority of exons consisting of OCT4A, which was confirmed to be a pluripotent factor, except its first exon. And OCT4B expressed all somatic tissues examined in the study and mainly located in cytoplasm of embryos contrary to OCT4A which was detected in nucleus specifically.

Next, the relation between OCT4A and XIC-linked genes was examined by overexpressing human OCT4A in developing embryos in fourth study. Contrary to the previous studies in mice, overexpression of OCT4A ortholog induced elevation of XIST expression in blastocyst when the transgene was passed to cleaved embryos. And correlative expression analysis in last study suggested that other pluripotent factors, typically NANOG and REX1, might have relation with XCI inducer, XIST, and its trans activator RLIM, respectively. Although more studies are needed, it was considered that molecular networking of pluripotent factors and XCI would be related during embryonic stages in pigs.

In conclusion, the present study demonstrated XCI in porcine preimplantation embryos with identifying key regulators of the process and their genomic cluster. Results in here suggested that the factors would be associated with XCI in pigs. Collectively, this study suggests new insights of porcine XCI. And also, the results obtained in the present study will advance the studies in the area of stem cells and developmental biology in pigs.