X-inactivation in female mammals is triggered by the association of non-coding Xist RNA in cis with the X chromosome. Although it has been suggested that the A-repeat located in the proximal part of the Xist RNA is required for chromosomal silencing in ES cells, its role in mouse has not yet been addressed. Here, we deleted the A-repeat in mouse and studied its effects on X-inactivation during embryogenesis. The deletion, when paternally transmitted, caused a failure of imprinted X-inactivation in the extraembryonic tissues, demonstrating the essential role of the A-repeat in X-inactivation in the mouse embryo. Unexpectedly, the failure of X-inactivation was caused by a lack of Xist RNA rather than by a defect in the silencing function of the mutated RNA, which we expected to be expressed from the mutated X. Interestingly, the normally silent paternal copy of Tsix, which is an antisense negative regulator of Xist, was ectopically activated in the preimplantation embryo. Furthermore, CpG sites in the promoter region of paternal Xist, which are essentially unmethylated in the extraembryonic tissues of the wild-type female embryo, acquire a significant level of methylation on the mutated paternal X. These findings demonstrate that the DNA sequence deleted on the mutated X, most probably the A-repeat, is essential as a genomic element for the appropriate transcriptional regulation of the Xist/Tsix loci and subsequent X-inactivation in the mouse embryo.

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.

The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist CAG ) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist CAG resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist CAG in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4-to 8-cell stages when paternally inherited, and Xist CAG was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.

Some species of the family Ascidiidae accumulate vanadium in concentrations in excess of 350 mM, which is about 10 (7)-fold higher than the concentration of vanadium in seawater. In these species, signet ring cells with a single large vacuole in which vanadium ions are contained function as vanadium-accumulating cells. These have been termed vanadocytes. We recently isolated five vanadium-binding proteins, which we named Vanabin1, Vanabin2, Vanabin3, Vanabin4, and VanabinP, from vanadocytes of the vanadium-rich ascidian Ascidia sydneiensis samea. In this study, we analyzed localization of the Vanabins in the blood cells of A. sydneiensis samea using monoclonal antibodies and confocal microscopy. The Vanabin1 and Vanabin2 proteins were found in the cytoplasm and/or in some organelles of vanadocytes. Vanabin3 was also detected in the cytoplasm, while Vanabin4 was found exclusively in the cytoplasmic membrane.

Mouse blastocyst stage embryo stained for histone H3 lysine-27 trimethylation (red) and DNA (blue). H3K27me3 marks the inactive X chromosome. The study by Chiba et al. in this issue suggests that de novo DNA methyltransferases are dispensable for setting the imprint on the maternally-derived X chromsome in growing oocytes. See Chiba et al. in this issue.