An embryonic story: Analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cells

In mice, dosage compensation of X‐linked gene expression is achieved through the inactivation of one of the two X‐chromosomes in XX female cells. The complex epigenetic process leading to X‐inactivation is largely controlled by Xist and Tsix, two non‐coding genes of opposing function. Xist RNA triggers X‐inactivation by coating the inactive X, while Tsix is critical for the designation of the active X‐chromosome through cis‐repression of Xist RNA accumulation. Recently, a plethora of trans‐acting factors and cis‐regulating elements have been suggested to act as key regulators of either Xist, Tsix or both; these include ubiquitous factors such as Yy1 and Ctcf, developmental proteins such as Nanog, Oct4 and Sox2, and X‐linked regulators such as Rnf12. In this paper we summarise recent advances in our knowledge of the regulation of Xist and Tsix in embryonic stem (ES) and differentiating ES cells.     

Higher order chromatin structure at the X-inactivation center via looping DNA

In mammals, the silencing step of the X-chromosome inactivation (XCI) process is initiated by the non-coding Xist RNA. Xist is known to be controlled by the non-coding Xite and Tsix loci, but the mechanisms by which Tsix and Xite regulate Xist are yet to be fully elucidated. Here, we examine the role of higher order chromatin structure across the 100-kb region of the mouse X-inactivation center (Xic) and map domains of specialized chromatin in vivo. By hypersensitive site mapping and chromosome conformation capture (3C), we identify two domains of higher order chromatin structure. Xite makes looping interactions with Tsix, while Xist makes contacts with Jpx/Enox, another non-coding gene not previously implicated in XCI. These regions interact in a developmentally-specific and sex-specific manner that is consistent with a regulatory role in XCI. We propose that dynamic changes in three-dimensional architecture leads to formation of separate chromatin hubs in Tsix and Xist that together regulate the initiation of X-chromosome inactivation.     

Identification of an Imprinted Gene Cluster in the X-Inactivation Center

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.     

X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells

X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells.     

Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome

To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.     

Molecular and DNA methylation analysis of <Emphasis Type="Italic">Peg10</Emphasis> and <Emphasis Type="Italic">Xist</Emphasis> gene in sheep

Peg10 is a maternally imprinted gene located in the imprinted domain of human chromosome 7q21 and mouse proximal chromosome 6. It is predominantly expressed in, and participates in the formation of, the placenta. Moreover, Peg10 is overexpressed in hepatocellular carcinoma, and is involved in hepatocarcinogenesis. The large noncoding RNA Xist has been shown to direct the female mammalian X chromatosome dosage compensation pathway. In the present study, we obtained partial cDNA sequences of sheep Peg10 and Xist. mRNA expression analysis in nine organs showed that they were universally expressed in two-day old lambs. The mRNA expression profile of Peg10 showed similar tissue specificity to pig, but was different compared with human and mouse. We concluded that the Peg10 mRNA expression profile was species specific. However, there was little difference in Xist expression between nine tissues of female lambs. Using bisulfite sequencing, we revealed that the first exon of Xist was either completely methylated or completely unmethylated, indicating that the newly obtained fragment of Xist was also differentially methylated in sheep as the DMR of Peg10. We did not find tissue specific DNA methylation of Xist, consistent with the Xist mRNA expression profile.     

When X-inactivation meets pluripotency: An intimate rendezvous

The integration of X-inactivation with development is a crucial aspect of this classical paradigm of epigenetic regulation. During early female mouse development, X-inactivation reprogramming occurs in pluripotent cells of the inner cell mass of the blastocyst and in pluripotent primordial germ cells. Here we discuss the developmental strategies which ensure the coupling of the regulation of X-inactivation to the acquisition of pluripotency through the regulation of the master of X-inactivation, the non-coding Xist gene, by the key factors which support pluripotency Nanog, Oct4 and Sox2.     

Dosage compensation in the mouse balances up-regulation and silencing of X-linked genes

Dosage compensation in mammals involves silencing of one X chromosome in XX females and requires expression, in cis, of Xist RNA. The X to be inactivated is randomly chosen in cells of the inner cell mass (ICM) at the blastocyst stage of development. Embryonic stem (ES) cells derived from the ICM of female mice have two active X chromosomes, one of which is inactivated as the cells differentiate in culture, providing a powerful model system to study the dynamics of X inactivation. Using microarrays to assay expression of X-linked genes in undifferentiated female and male mouse ES cells, we detect global up-regulation of expression (1.4- to 1.6-fold) from the active X chromosomes, relative to autosomes. We show a similar up-regulation in ICM from male blastocysts grown in culture. In male ES cells, up-regulation reaches 2-fold after 2–3 weeks of differentiation, thereby balancing expression between the single X and the diploid autosomes. We show that silencing of X-linked genes in female ES cells occurs on a gene-by-gene basis throughout differentiation, with some genes inactivating early, others late, and some escaping altogether. Surprisingly, by allele-specific analysis in hybrid ES cells, we also identified a subgroup of genes that are silenced in undifferentiated cells. We propose that X-linked genes are silenced in female ES cells by spreading of Xist RNA through the X chromosome territory as the cells differentiate, with silencing times for individual genes dependent on their proximity to the Xist locus.     

Paternal RLIM/Rnf12 is a survival factor for milk-producing alveolar cells

In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), whereas extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12, which encodes the ubiquitin ligase Rnf12/RLIM. We find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy, with alveolar cells that lack RLIM undergoing apoptosis as they begin to differentiate. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom maternal XCI in mammary epithelial cells. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and, together with population models, reveal implications of transgenerational epigenetic inheritance.