DNA methylation profiles of human active and inactive X chromosomes

Abstract

X-chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell. To characterize epigenetic changes that accompany this process, we measured DNA methylation levels in 45,X patients carrying a single active X chromosome (X a ), and in normal females, who carry one X a and one inactive X (X i ). Methylated DNA was immunoprecipitated and hybridized to high-density oligonucleotide arrays covering the X chromosome, generating epigenetic profiles of active and inactive X chromosomes. We observed that XCI is accompanied by changes in DNA methylation specifically at CpG islands (CGIs). While the majority of CGIs show increased methylation levels on the X i , XCI actually results in significant reductions in methylation at 7% of CGIs. Both intraand inter-genic CGIs undergo epigenetic modification, with the biggest increase in methylation occurring at the promoters of genes silenced by XCI. In contrast, genes escaping XCI generally have low levels of promoter methylation, while genes that show inter-individual variation in silencing show intermediate increases in methylation. Thus, promoter methylation and susceptibility to XCI are correlated. We also observed a global correlation between CGI methylation and the evolutionary age of X-chromosome strata, and that genes escaping XCI show increased methylation within gene bodies. We used our epigenetic map to predict 26 novel genes escaping XCI, and searched for parent-of-origin-specific methylation differences, but found no evidence to support imprinting on the human X chromosome. Our study provides a detailed analysis of the epigenetic profile of active and inactive X chromosomes.[Supplemental material is available for this article.] X-chromosome inactivation (XCI) is a mechanism of dosage compensation that equalizes the expression of sex-linked genes between 46,XY males and 46,XX females (Lyon 1961). This results in the silencing of the majority of genes on one of the two X chromosomes in each somatic cell of females (Carrel and Willard 2005) and a transition to a heterochromatic state. XCI is accompanied by several epigenetic modifications to the inactive X (X i ), such as an accumulation of variant histones and transcripts from the XIST gene (Clemson et al. 1996;Costanzi and Pehrson 1998), a delay in replication timing (Willard and Latt 1976), and relocalization to the nuclear periphery (Barr and Bertram 1949).Several studies have shown that DNA methylation of the X i plays an important role in the maintenance of its inactive state. While the active X (X a ) and X i have very similar global levels of methylation (Bernadino et al. 1996), studies have shown that CpG islands (CGIs) have a tendency to be methylated on the X i and unmethylated on the X a (Tribioli et al. 1992;Hellman and Chess 2007). In contrast, the CGIs of genes escaping XCI often remain unmethylated on both the X i and X a (Weber et al. 2007). Mouse knockouts for the DNA methyltransferase enzyme Dnmt1 show defects in X inactivat...

Human active X-specific DNA methylation events showing stability across time and tissues

“…We generated longitudinal DNA methylation data from 38 wholeblood spots (including 26 male and 12 female samples) sampled at birth (14) and 18 years (24 samples) from singletons and from 56 buccal cell samples (25 males and 31 females) sampled at birth (31 samples) and 18 months (25 samples) of age from monozygotic and dizygotic twin pairs (Supplementary Table 1). Twenty-eight of the blood samples were collected from the same individuals at birth and 18 years old (ie, 14 pairs).…”

“…The study reported methylation profiles predictive of XCI escape, identifying a total 313 candidate genes 14 with either low methylation (o0.65) in females or low difference (o0.39) between Turner syndrome cases and control females.…”

“…Xa-specific methylation is partially concordant with Turner syndrome Xa-sites Sharp and co-workers 14 have previously examined XCI in peripheral blood comparing methylation levels of seven unrelated female patients carrying a single X chromosome (defined by the 45,XO karyotype characteristic of Turner syndrome) with three unrelated control female samples. The study reported methylation profiles predictive of XCI escape, identifying a total 313 candidate genes 14 with either low methylation (o0.65) in females or low difference (o0.39) between Turner syndrome cases and control females.…”

“…Nevertheless, recent DNA methylation profiling of the human active and Xi chromosome using an affinity-based approach revealed multiple regions of apparently lower levels of methylation on the X chromosome in XX females relative to XO females, suggesting an association with active X-specific methylation. 14 In the current study, we used high-resolution DNA methylation BeadChip arrays to investigate the extent of Xa-specific methylation in tissues of different origin collected longitudinally from birth.…”

A review of Rett syndrome (RTT) with induced pluripotent stem cells

“…Upon Xist upregulation, the Xi is heavily epigenetically remodeled, in many ways similar to other silenced genes throughout the genome. Epigenetic marks associated with the Xi include CpG island promoter DNA methylation (96,97), incorporation of histone variant MacroH2A, and modification of core histones (98-100). An early event that follows XIST accumulation is the recruitment of the polycomb complex PRC2 that induces histone H3 trimethylation at lysine 27 (101-103).…”