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 intra- and 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.

Figure 1.

Comparative methylation profiling of the X chromosome in 45,X and 46,XX individuals shows that X inactivation results in increased methylation of CpG islands at gene promoters. Regions containing high densities of CpG dinucleotides, corresponding with the transcription start sites of genes subject to X inactivation, show raised methylation levels on the X_i. The image shows the screenshot of a 250-kb region of Xp11.3 (chrX:46,475,000–46,725,000, hg18), with methylation data uploaded as custom tracks in the UCSC Genome Browser.

Figure 2.

Methylation patterns of genes subject to and escaping X inactivation on active and inactive X chromosomes. (A) For genes subject to XCI, methylation differences between 46,XX and 45,X individuals occur specifically at gene promoters, with 46,XX individuals showing increased methylation compared to 45,X cases. As 83% of RefSeq transcripts listed on the X chromosome have a CGI within 1 kb of their TSS and CGIs are heavily methylated on the X_i versus the X_a, this enrichment of methylation at gene promoters on the X_i is a correlate of CGI methylation. (B) In contrast, for genes escaping XCI, there is a generalized increase in methylation in the bodies of genes in both 45,X and 46,XX individuals compared to genes subject to XCI, with the biggest difference between 46,XX and 45,X occurring at the promoter region. Additionally, a slight but consistent increase in methylation in the bodies of genes escaping XCI is evident in 46,XX compared to 45,X individuals. Two hundred sixteen RefSeq genes listed on chrX scored as subject to XCI (score = 0) and 52 RefSeq genes scored as escaping XCI (score = 8 or 9) by Carrel and Willard (2005) were analyzed. Where multiple splice forms were listed for a single gene, we chose the maximal boundary of all isoforms to define the transcribed region. Probes were then assigned into one of 40 bins depending on their relative position along the gene body, defined by the maximal transcription start and end coordinates. Similar data were observed when considering only those genes of intermediate length (10–100 kb) (Supplemental Figs. 11, 12).

Figure 3.

Differential methylation between active and inactive X chromosomes occurs specifically at CpG islands. Each panel shows a composite plot of mean methylation levels within and flanking 3060 CGIs on the X chromosome in (A) 46,XX females; (B) 45,X Turner syndrome cases, corresponding to the X_a; and (C) the difference between these two groups, corresponding to the X_i. Three thousand sixty CGIs defined using epigenetic criteria (Bock et al. 2007) were analyzed. Those separated by <500 bp (n = 726) were merged into single regions, and the mean methylation level both within CGIs, and +5 kb and −5 kb was calculated. The apparent increase in methylation in flanking regions in 46,XX individuals is likely due to the clustering of CGIs on the X chromosome, which results in neighboring CGIs being sampled in the 5-kb flanking regions (10% of CGIs are separated by <1 kb, 36% by <5 kb). (Gray dashes) Borders of the CGIs. Mean methylation within CGIs was calculated by assigning probes into 10 windows proportional to the length of each CGI. Mean methylation in flanking regions was plotted in 250-bp windows. Underlying mean log₂ values with variance and associated P-values are shown in Supplemental Table 2.

Figure 4.

X inactivation results in highly variable changes in methylation of CpG islands that correlate with the location of genes escaping X inactivation. While the majority of CGIs (61%) show increased methylation on the X_i (yellow circles), 7% of CGIs have significantly lower levels of methylation in 46,XX compared to 45,X individuals (blue squares; p < 0.01), contradicting the notion that XCI is always associated with increased methylation. The location of these sites of reduced methylation highly correlates with the physical position of genes known to escape XCI (r = −0.81). (Gray diamonds) The remaining 32% of CGIs showed no significant difference in methylation (p > 0.01). Each point represents the change in mean methylation between 45,X and 46,XX individuals at a CGI (Bock et al. 2007), with the black line showing the moving average of methylation at 10 CGIs. The heat map shows genes expressed from the X_i (blue), silent genes (yellow), pseudoautosomal genes (purple), and untested genes (white) in each of nine cell lines (adapted from Carrel and Willard 2005 and reprinted with permission from Nature Publishing Group © 2005).

Figure 5.

Methylation status of CpG islands at gene promoters varies depending on gene inactivation status. (A) CGIs at promoters of genes subject to XCI show much higher methylation levels in 46,XX compared to 45,X individuals (mean 46,XX log₂ methylation = 0.99, mean log₂ methylation difference = 1.04, p = 1.3 × 10⁻²³). (B) In contrast, the majority of CGIs at promoters of genes escaping XCI show much lower methylation levels in 46,XX individuals, similar to those seen in 45,X individuals (mean 46,XX log₂ methylation = 0.54, mean log₂ methylation difference = 0.34, p = 0.00013). Each point represents the mean log₂ methylation of a CGI located within 1 kb of the TSS of a RefSeq gene scored by Carrel and Willard (2005) as being (A) silenced on the X_i (expressed in 0 of 9 hybrids containing an inactive X, n = 135), or (B) expressed from the X_i (expressed in ≥8 of 9 hybrids containing an inactive X, n = 29). Colored lines show the moving average of methylation at 10 and 3 CGIs for genes subject to and escaping XCI, respectively. Data for CGIs that lie outside of RefSeq gene promoters are shown in Supplemental Figure 14.

Figure 6.

Difference in methylation levels between 46,XX and 45,X individuals at transcription start sites is inversely correlated with X-inactivation status. Boxplots show the mean methylation of CGIs located within 1 kb of the TSS of 363 RefSeq genes scored by Carrel and Willard (2005) for their expression status on the X_i (Supplemental Table 3). Genes are divided based on their expression status on the X_i, with a score from 0 to 9 corresponding to the number of somatic cell hybrids containing an inactive X chromosome that express that gene. Genes with score 0 are always subject to XCI, genes with score 9 always escape XCI, and genes with intermediate scores show polymorphic or unstable inactivation. The XIST gene is unique in being transcribed only from the X_i, and shows lower methylation in 46,XX versus 45,X individuals. Linear regression analysis of XCI score with methylation difference between 46,XX and 45,X yields R² = 0.18, p < 0.001. ANOVA followed by Tukey analysis showed that genes with scores 4–9 showed statistically significant methylation differences compared to genes with score 0 (Bonferroni-adjusted p < 0.001, indicated by an asterisk within each box). Boxplots define lower and upper quartiles of each distribution, the internal band shows the median, while whiskers correspond to top and bottom deciles. Categories containing less than 15 genes were combined to yield sufficient sample sizes for meaningful comparison.