Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation

Abstract

During early embryogenesis of female mammals, one of the two X chromosomes is randomly chosen to be inactivated in each cell1, leading to the transcriptional silencing of thousands of genes on this chromosome. This random X-inactivation process also occurs during in vitro differentiation of female embryonic stem (ES) cells2,3,4. A locus on the X chromosome, the X inactivation centre (Xic) is initially 'counted', given that at least two copies of Xic must be present per diploid genome in order for inactivation to occur2. The counting process ensures that one X chromosome remains active in diploid cells. In the mouse, the essential functions of Xic can be assured by a 450-kb region5,6 containing the Xist gene. Xist maps within Xic (refs 7, 8, 9, 10) and is necessary in cis for inactivation11,12. The Xist transcript is a 15-kb RNA which is confined within the nucleus and coats the inactive X chromosome13. In order to characterize functional elements within Xic and the Xist gene, we created a 65-kb cre/loxP deletion extending 3′ to Xist exon 6. In undifferentiated ES cells, Xist expression from the deleted X chromosome was markedly reduced. In differentiated XX ES cells containing one deleted X chromosome, the X inactivation process still occurred but was never initiated from the unmutated X chromosome. In differentiated ES cells that were essentially XO, the mutated Xic was capable of initiating X inactivation, even in the absence of another Xic. These results demonstrate a role for the region 3′ to Xist exon 6 in the counting process and suggest that counting is mediated by a repressive mechanism which prevents inactivation of a single X chromosome in diploid cells.

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Figure 1: Strategy for deleting a 65-kb region 3′ to Xist exon 6.
Figure 2: A 65-kb deletion targeted 3′ to Xist exon 6 on the bPa X chromosome.
Figure 3: In ES cells, Xist expression from the del-bPa X chromosome is highly reduced; in differentiated ES cells, silencing occurs at the del-bPa X chromosome.
Figure 4: Initiation of inactivation of the del-bPa X chromosome is insensitive to X-chromosome counting.

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Acknowledgements

Thanks are due to D. Arnaud for generating the HP3.10 ES cell line and for technical help with the 6-TG selection and to E. Heard and F. Mongelard for establishing RNA FISH in the laboratory. F. Huetz and K. Rajewsky provided helpful advice on homologous recombination, and D. Cunningham, E. Heard, B. Robert and M. Wiles fruitful discussions. We are grateful to A. Bird, A. Bradley, S. O'Gorman, J.T. Lee and C. Vourc'h for the gifts of plasmids pBam7.6, pPGKpuro.bpA, pOG231, pXist3K, and p68. We thank F. Huetz and A. Stafford for critical reading of the manuscript. This work was supported by grants to P. A. from the Association Francaise contre la Myopathie (AFM), and the Association pour la Recherche contre le Cancer (ARC).

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Correspondence to Philippe Clerc.

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Clerc, P., Avner, P. Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation. Nat Genet 19, 249–253 (1998). https://doi.org/10.1038/924

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