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DNA methylation demonstrates spread of X-chromosome inactivation to human transgenes Yang, Christine

Abstract

X-chromosome inactivation is the process by which mammalian females achieve dosage compensation with males by silencing one of the two X chromosomes in female cells. Despite the chromosome-wide inactivation, a significant proportion of genes on the X chromosome in humans remain expressed on the inactive X chromosome. It has been long hypothesized that the genomic context plays an important role in influencing whether a gene is subject to or escapes from X-chromosome inactivation; however, cis-regulatory elements involved in X-chromosome inactivation have not yet been identified. The objective of this thesis was to identify DNA elements that promote the escape of genes from X-chromosome inactivation in the human genome, through analyzing the X-chromosome inactivation statuses of human transgenes integrated at the Hprt locus on the mouse X chromosome and identifying the transgenes that escape from X-chromosome inactivation. DNA methylation was used to assess the inactivation status of 74 human reporter constructs comprising over 1.5 Mb of DNA. Transgenes that show low promoter DNA methylation in males and females would be potential escape genes. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X-chromosome inactivation was verified by the demonstration of expression from the inactive X chromosome in females with non-random X-chromosome inactivation. Analysis on the repeat element content of five BAC-derived transgenes subject to X-chromosome inactivation suggested that local LINE-1 and Alu densities were insufficient to determine whether a gene would be subject to X-inactivation. Interestingly, CpG islands not associated with promoters also showed female-specific DNA hypermethylation, suggesting a dominant effect of X-chromosome inactivation on the regulation of DNA methylation. Different human transgenes show a differential capacity to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, indicating the presence of additional cis-acting epigenetic modulators. As only one of the human transgenes analyzed escaped from X-chromosome inactivation, we conclude that elements involved in ongoing expression from the inactive X are rare in the human genome and that mouse X-chromosome inactivation is very effective in silencing human transgenes.

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