Cancer progenitors and epigenetic contexts: An Xisting connection

In mammals, silencing of one of the two X chromosomes is necessary to achieve dosage compensation. The non coding RNA Xist triggers X inactivation. Gene silencing by Xist is only possible in certain developmental contexts that only exist in cells of the early embryo and specific hematopoietic progenitors. Critical silencing factors may only be present in these contexts giving an explanation of why Xist is not operative outside these contexts. It has been demonstrated that Xist is functional in tumor cells, where SATB1 was identified as the first silencing factor for Xist mediated chromosome silencing.     

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.     

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.     

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.     

A chromosomal memory triggered by Xist regulates histone methylation in X inactivation

We have elucidated the kinetics of histone methylation during X inactivation using an inducible Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.     

H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non‐coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2‐dependent H3K27me3 and SETD8‐dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3‐specific intracellular antibody or H3K27me3‐mintbody. By combining live‐cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP‐seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.     

Understanding the X chromosome inactivation cycle in mice: A comprehensive view provided by nuclear transfer

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.     

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.     

Mapping the murine Xce locus with (CA)n repeats

The X Chromosome (Chr) controlling element locus (Xce) in the mouse has been shown to influence the X inactivation process. Xce maps to the central region of the X Chr, which also contains the Xist sequence, itself possibly implicated in the X inactivation process. Three microsatellite markers spanning the Xist locus have been isolated from an Xist containing YAC. All three microsatellite markers showed complete linkage with Xce in recombinants for the central span of the mouse X Chr between Ta and Mo ^blo and strong linkage disequilibrium with Xce in all but one of the inbred mouse strains tested. In the standard Xce ^b typing strain JU/Ct, the two microsatellites most closely flanking Xist fail to carry the allelic forms expected if Xist and Xce are synonymous. Alternative explanations for this finding are presented in the context of our search for understanding the relation between Xist and Xce.