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.     

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.     

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.     

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.     

Nonrandom X Chromosome Inactivation Is Influenced by Multiple Regions on the Murine X Chromosome

During the development of female mammals, one of the two X chromosomes is inactivated, serving as a dosage-compensation mechanism to equalize the expression of X-linked genes in females and males. While the choice of which X chromosome to inactivate is normally random, X chromosome inactivation can be skewed in F1 hybrid mice, as determined by alleles at the X chromosome controlling element (Xce), a locus defined genetically by Cattanach over 40 years ago. Four Xce alleles have been defined in inbred mice in order of the tendency of the X chromosome to remain active: Xce^a < Xce^b < Xce^c < Xce^d. While the identity of the Xce locus remains unknown, previous efforts to map sequences responsible for the Xce effect in hybrid mice have localized the Xce to candidate regions that overlap the X chromosome inactivation center (Xic), which includes the Xist and Tsix genes. Here, we have intercrossed 129S1/SvImJ, which carries the Xce^a allele, and Mus musculus castaneus EiJ, which carries the Xce^c allele, to generate recombinant lines with single or double recombinant breakpoints near or within the Xce candidate region. In female progeny of 129S1/SvImJ females mated to recombinant males, we have measured the X chromosome inactivation ratio using allele-specific expression assays of genes on the X chromosome. We have identified regions, both proximal and distal to Xist/Tsix, that contribute to the choice of which X chromosome to inactivate, indicating that multiple elements on the X chromosome contribute to the Xce.     

Understanding the X chromosome inactivation cycle in mice

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.     

Genetic Architecture of Skewed X Inactivation in the Laboratory Mouse

X chromosome inactivation (XCI) is the mammalian mechanism of dosage compensation that balances X-linked gene expression between the sexes. Early during female development, each cell of the embryo proper independently inactivates one of its two parental X-chromosomes. In mice, the choice of which X chromosome is inactivated is affected by the genotype of a cis-acting locus, the X-chromosome controlling element (Xce). Xce has been localized to a 1.9 Mb interval within the X-inactivation center (Xic), yet its molecular identity and mechanism of action remain unknown. We combined genotype and sequence data for mouse stocks with detailed phenotyping of ten inbred strains and with the development of a statistical model that incorporates phenotyping data from multiple sources to disentangle sources of XCI phenotypic variance in natural female populations on X inactivation. We have reduced the Xce candidate 10-fold to a 176 kb region located approximately 500 kb proximal to Xist. We propose that structural variation in this interval explains the presence of multiple functional Xce alleles in the genus Mus. We have identified a new allele, Xce^e present in Mus musculus and a possible sixth functional allele in Mus spicilegus. We have also confirmed a parent-of-origin effect on X inactivation choice and provide evidence that maternal inheritance magnifies the skewing associated with strong Xce alleles. Based on the phylogenetic analysis of 155 laboratory strains and wild mice we conclude that Xce^a is either a derived allele that arose concurrently with the domestication of fancy mice but prior the derivation of most classical inbred strains or a rare allele in the wild. Furthermore, we have found that despite the presence of multiple haplotypes in the wild Mus musculus domesticus has only one functional Xce allele, Xce^b. Lastly, we conclude that each mouse taxa examined has a different functional Xce allele.     

The physical phenotype of girls and women with Turner syndrome is not X-imprinted

Certain behavioral and metabolic aspects of Turner syndrome (TS) are attributed to X-chromosome genomic imprinting. To investigate the possible contribution of imprinting to the physical features of the TS phenotype in live-born individuals, we genotyped the single normal X-chromosome in subjects with TS who all underwent a comprehensive evaluation as part of the NIH genotype–phenotype protocol. All had physical examinations, auxological measurements and imaging of the renal and cardiovascular systems. Absolute height and height as a percent of predicted height was the same in X^M (n = 56) and X^P (n = 23) subjects that had reached final height and were not growth hormone treated. Interestingly, adult height was significantly correlated with maternal but not paternal heights in both X^M and X^P groups. Neck webbing was found in 35% of the X^M (n = 133) and 22% of the X^P (n = 50) groups (P = 0.11). Renal anomalies were present in 24% of X^M and 25% of X^P groups (P = 0.9). Bicuspid aortic valve was found in 26% of X^M and 24% of X^P groups (P = 0.83), and any cardiovascular anomaly (abnormal aortic valve, aortic coarctation, elongated transverse aortic arch, anomalous pulmonary venous connection, left superior vena cava) affected 55% of X^M and 52% of X^P groups. Thus, we found no evidence for X-linked genomic imprinting effects on stature or lymphatic, renal or cardiovascular development in TS. Our sample size was sufficient to exclude such effects within 95% confidence limits. We did demonstrate a selective maternal effect on final stature that was independent of X-chromosome origin, suggesting potential autosomal imprinting effects on growth revealed by X monosomy.     

The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation

The Polycomb group (PcG) proteins are thought to silence gene expression by modifying chromatin. The Polycomb repressive complex 2 (PRC2) plays an essential role in mammalian X-chromosome inactivation (XCI), a model system to investigate heritable gene silencing. In the mouse, two different forms of XCI occur. In the preimplantation embryo, all cells undergo imprinted inactivation of the paternal X-chromosome (Xp). During the peri-implantation period, cells destined to give rise to the embryo proper erase the imprint and randomly inactivate either the maternal X-chromosome or the Xp; extraembryonic cells, on the other hand, maintain imprinted XCI of the Xp. PRC2 proteins are enriched on the inactive-X during early stages of both imprinted and random XCI. It is therefore thought that PRC2 contributes to the initiation of XCI. Mouse embryos lacking the essential PRC2 component EED harbor defects in the maintenance of imprinted XCI in differentiating trophoblast cells. Assessment of PRC2 requirement in the initiation of XCI, however, has been hindered by the presence of maternally derived proteins in the early embryo. Here we show that Eed^−/− embryos initiate and maintain random XCI despite lacking any functional EED protein prior to the initiation of random XCI. Thus, despite being enriched on the inactive X-chromosome, PcGs appear to be dispensable for the initiation and maintenance of random XCI. These results highlight the lineage- and differentiation state–specific requirements for PcGs in XCI and argue against PcG function in the formation of the facultative heterochromatin of the inactive X-chromosome.