High-resolution analysis of epigenetic changes associated with X inactivation - PubMed (original) (raw)
High-resolution analysis of epigenetic changes associated with X inactivation
Hendrik Marks et al. Genome Res. 2009 Aug.
Abstract
Differentiation of female murine ES cells triggers silencing of one X chromosome through X-chromosome inactivation (XCI). Immunofluorescence studies showed that soon after Xist RNA coating the inactive X (Xi) undergoes many heterochromatic changes, including the acquisition of H3K27me3. However, the mechanisms that lead to the establishment of heterochromatin remain unclear. We first analyze chromatin changes by ChIP-chip, as well as RNA expression, around the X-inactivation center (Xic) in female and male ES cells, and their day 4 and 10 differentiated derivatives. A dynamic epigenetic landscape is observed within the Xic locus. Tsix repression is accompanied by deposition of H3K27me3 at its promoter during differentiation of both female and male cells. However, only in female cells does an active epigenetic landscape emerge at the Xist locus, concomitant with high Xist expression. Several regions within and around the Xic show unsuspected chromatin changes, and we define a series of unusual loci containing highly enriched H3K27me3. Genome-wide ChIP-seq analyses show a female-specific quantitative increase of H3K27me3 across the X chromosome as XCI proceeds in differentiating female ES cells. Using female ES cells with nonrandom XCI and polymorphic X chromosomes, we demonstrate that this increase is specific to the Xi by allele-specific SNP mapping of the ChIP-seq tags. H3K27me3 becomes evenly associated with the Xi in a chromosome-wide fashion. A selective and robust increase of H3K27me3 and concomitant decrease in H3K4me3 is observed over active genes. This indicates that deposition of H3K27me3 during XCI is tightly associated with the act of silencing of individual genes across the Xi.
Figures
Figure 1.
Overview of the epigenetic landscape, as well as gene expression within and centered around the Xic. A screen shot from the UCSC Genome Browser showing the distribution of the ChIP-chip profiles across a 100 kb region of mouse chromosome X in mouse embryonic cells (UCSC Mouse [mm9], July 2007; chromosome X genomic coordinates 100.615–100.715 Mb). Expression was determined by hybridizing polyA RNA to the tiling array. _Y_-axis, log2 ChIP/input ratio or log2 cDNA/input ratio; _x_-axis, 50 bp oligonucleotides from sequence included on the NimbleGen mouse chromosome X tiling array. The quantification, as shown below the profiles, was obtained from the XT67E1 ChIP-seq experiments as described later. For quantifications, ChIP-seq tags were counted within the indicated regions. For the E14 cells, the tags are obtained from one X chromosome, while in XT67E1 cells they are obtained from both X chromosomes. (A) Overview of the Tsix_–_Xist interplay. Tsix and Xist noncoding RNAs, and direction of transcription, are indicated below all profiles. (B) Enlargement of the expression profiles of the LF2 cells, showing a clear correlation between the expression signal and the Xist exons after 4-d atRA treatment or 10-d EB formation. The arrows shown for the undifferentiated LF2 cells indicate PCR amplicons used for RT-qPCR validation (Supplemental Fig. S1b).
Figure 2.
Kinetics of the two H3K27me3 hotspots (which are boxed) surrounding the Xic in LF2 and E14 ES cells as obtained by H3K27me3 ChIP-chip. Screen shots from the UCSC Genome Browser showing the distribution of the H3K27me3 ChIP-chip profiles across a ∼0.5 Mb region of mouse chromosome X in mouse embryonic stem cells (UCSC Mouse [mm9], July 2007; chromosome X genomic coordinates 100.68–101.25 and 98.88–99.31 Mb for the known hotspot and the novel hotspot, respectively). The MM9 coordinates of the Xic are ∼100.3–101 Mb. Annotation is provided below the profiles. For further details, see Figure 1. For quantification using the ChIP-Seq tags of the XT67E1 cells, the “known” hotspot was split into two parts based on their differential kinetics. For comparison, tags were also counted in equally sized neighboring regions.
Figure 3.
Specific enrichment of H3K27me3 on the X chromosome after 10-d EB formation in mouse female XT67E1 ES cells, but not in male ES cells. (A) H3K27me3 tag distribution (representing amounts of H3K27me3) over autosomes or the X chromosome in XT67E1 or E14 cells (ratio of 4-d atRA or 10-d EB versus undifferentiated cells in percent). See Supplemental material and Supplemental Figure S7 for more details. (B) H3K4me3 tag distribution (representing amounts of H3K4me3) over autosomes or the X chromosome in XT67E1 cells (ratio of 4-d atRA or 10-d EB versus undifferentiated cells in percent). See Supplemental material and Supplemental Figure S7 for more details.
Figure 4.
Allele-specific mapping of tags obtained during the ChIP-seq experiments. (A) Genetic make-up of XT67E1 mouse ES cells, showing the contribution of the parental PGK, C3H, or 129 mouse strains to the individual chromosomes of the XT67E1 cells. For individual chromosomes, the contribution of each strain was calculated by dividing the number of uniquely identified SNPs in the ChIP-seq experiments by the total number of SNPs, as determined from the SNP database (
mouse.perlegen.com/mouse/index.html
). (B) Distribution of the uniquely identified PGK SNPs within the total pool of XT67E1 ChIP-seq tags in 1 Mb bins over the X chromosome (represented by the _x_-axis). Density plots of the total number of PGK-specific SNPs (114,781 for the complete X chromosome), as well as of the total number of mapped tags, are plotted below the graph. The genomic locations of the Xic and the Pgk locus are indicated. (C) Schematic representation of the X chromosomes in the undifferentiated and the 10-d EB XT67E1 cells, with the Xist deletion in the 129 derived X chromosome (which will become the Xa during XCI). The number of total H3K27me3 tags mapped over both chromosomes, as well as the number of tags that could be mapped allele-specific, is indicated for both stages.
Figure 5.
Kinetics of H3K27me3 over chromosome X during XCI in XT67E1 cells, showing a chromosome-wide increase of H3K27me3 during XCI. (A) H3K27me3 profiles over chromosome X in XT67E1 cells. Chromosome X is subdivided in bins of 1 Mb, followed by counting of the number of tags per bin. undiff, undifferentiated. (B) Subtraction tracks of the H3K27me3 profiles shown in A at a higher resolution (bins of 100 kb). Increase of H3K27me3 as compared to undifferentiated cells is indicated in blue, decrease in red.
Figure 6.
Boxplots of the occupancies of H3K27me3 and H3K4me3 over various repeats of chromosome X in female XT67E1 and male E14 cells. (A) All tags on chromosome X; a boxplot representation of the graphs shown in Fig. 5A. (B–D) Distributions over various repeat classes (LINE, LTR, and SINE repeats).
Figure 7.
Boxplots of the occupancies of H3K27me3 and H3K4me3 over promoters and gene bodies of chromosome X. Genes were binned in three equally sized groups of 212 genes according to expression level, representing not/lowly-, moderately-, and highly-expressed genes, respectively. The red boxplots represent H3K27me3 occupancies for the female XT67E1 cells, while the yellow boxplots represent H3K4me3 occupancies. The blue boxplots represent H3K27me3 occupancies for the male E14 cells. (A–D) Distribution of H3K27me3 and H3K4me3 over gene bodies (from +500 to the end of Ensemble genes). (E–H) Distributions of H3K27me3 and H3K4me3 over gene promoters (from −700 to +300 of Ensemble genes).
References
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