Goto, Tetsuya; (1998) Imprinting of X-chromosome inactivation and functional studies of the regulation of Xist promoter activity in preimplantation embryos and transgenic mice. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Xist (X-inactive specific transcript) gene, which maps to the X-inactivation centre (Xic), is expressed from the inactive X chromosome and is required in cis for X-chromosome inactivation. The expression of Xist is imprinted in mouse preimplantation embryos in that only the paternally-inherited allele is expressed. This early imprinted expression correlates with preferential paternal X-chromosome inactivation in extraembryonic tissues in mice. Differential DNA methylation of the Xist promoter in sperm and eggs has been suggested as the mechanism for imprinted expression. In this study, Xist promoter activity is analysed in mouse preimplantation embryos microinjected with Xist promoter-containing plasmid constructs and in transgenic mice. Following microinjection, it is shown that a 233 bp (nt-220 to +13) Xist promoter fragment is sufficient to drive transcription of the firefly luciferase reporter gene in 2-cell embryos. Promoter activity is repressed by in vitro methylation of the construct with site-specific DNA methylases before microinjection. In transgenic mice, transgene Xist promoter activity is only observed in the testes of males and correlates with undermethylation of the transgene. Following paternal transmission, the transgene Xist promoter is active in morula-stage embryos provided that it is extensively undermethylated in sperm. These results support the role played by DNA methylation for the regulation of imprinted expression of the endogenous Xist in preimplantation embryos. Imprinted preferential paternal X-chromosome inactivation is also investigated in human development. The inactive X chromosome is identified by methylation-sensitive restriction enzyme digestion followed by PCR. Using DNA isolated from fresh chorionic villus samples at 10-12 weeks of gestation, it is shown that, as in mice, the human paternal X chromosome is preferentially inactivated in human trophoblastic cells but not in mesodermal cells.

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