Smchd1-dependent and -independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome

Abstract

X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 was recently shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation pathway that is required for long-term maintenance of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1-dependent or -independent pathways. Smchd1-dependent CGI methylation, the primary pathway, is acquired gradually over an extended period, whereas Smchd1-independent CGI methylation occurs rapidly after the onset of X inactivation. The de novo methyltransferase Dnmt3b is required for methylation of both classes of CGI, whereas Dnmt3a and Dnmt3L are dispensable. Xi CGIs methylated by these distinct pathways differ with respect to their sequence characteristics and immediate chromosomal environment. We discuss the implications of these results for understanding CGI methylation during development.

Graphical abstract

Figure 1

Fast and Slow Methylation Dynamics of Xi CGIs in Differentiating XX ES Cells (A) Southern analysis of MSRE digests using indicated restriction enzymes. Presence of methylated (m) and unmethylated (u) sites were determined in XX ES cells, in XX ES cells differentiated (diff) for 4, 7, and 10 days, and in XX somatic controls (adult kidney). Multiple sites within the same CGI are linked with a vertical line. The schematic indicates the chromosomal location of CGI-associated loci. (B) Sequenom EpiTyper analysis of CGIs associated with genes along the length of the X chromosome, as indicated on the schematic. Average CpG methylation levels were determined for XX and XY somatic tissue (adult kidney) and XX ES cells differentiated (diff) for the times shown. Error bars indicate positive SD values from three independent experiments. (C) Bisulfite DNA sequencing analysis of Mtm1 and Hprt CGIs in XX somatic cells (adult kidney), XX ES cells, and ES cells differentiated (diff) for the times shown. Schematics illustrate the regions analyzed by bisulfite sequencing (horizontal line below map), transcription start site (arrows), and CpG density (vertical lines). Bottom: Each line represents methylation on an individual DNA strand determined by sequencing subcloned PCR product from bisulfite-treated genomic DNA. Methylated and unmethylated CpGs are indicated with closed and open circles, respectively. Ambivalent sequence reads are shown as gaps. See also Table S1.

Figure 2

Allelic CpG Methylation Patterns on Xa and Xi (A) Bisulfite DNA sequencing assays of CGIs of Mtm1, Hprt, and Pdk3 CGIs in T16H/Cast XX somatic cells. Schematics illustrate the regions analyzed by bisulfite sequencing. SNPs between M. domesticus (dom) Xa and M. castaneus (cast) Xi alleles were used to assign the origin of individual strands. Average % methylation of Xa and Xi alleles is indicated. (B) Bisulfite DNA sequencing analysis of CpG sites upstream (US) and downstream (DS) of X-linked genes as indicated in T16H/Cast XX somatic cells and an XY somatic MEF cell line (E4.22.5). Schematics illustrate maps of four non-CGI regions indicating the region amplified by PCR (horizontal line below map) and CpG sites (vertical lines). M. domesticus and M. castaneus SNPs (open and closed square boxes, respectively) are indicated together with bisulfite sequencing patterns.

Figure 3

Xi CGI Methylation Dynamics In Vivo (A) Analysis of Mtm1 and Hprt CGIs in DNA from pooled XX embryos isolated at developmental stages as indicated by bisulfite DNA sequencing. The regions analyzed are as shown in Figure 1, and XX somatic cell data from Figure 1 are included for illustrative purposes. (B) Graphical representation of data in (A) illustrating methylation at Mtm1 and Hprt as a percentage of methylation levels in XX somatic cells. (C) Sequenom EpiTyper analysis of methylation at CGIs associated with Zdhhc15 and Mcts1 in E6.5 and E10.5 XX embryos and somatic cell controls (adult kidney). The % CpG methylation is averaged for sites across the region analyzed. Error bars indicate variation between individual embryos (n = 3) or adult tissue DNA samples (n = 3). See also Table S1.

Figure 4

Chromosome-wide Analysis of CGI Methylation Dynamics in Differentiating XX ES Cells (A–E) Browser screenshots of 10 kb regions illustrating examples of major classes of Xi CGI methylation patterns detected in MBD-seq analysis of XX and XY somatic cells (MEF cell lines) and XX ES cells differentiated (diff) as indicated. The y axis indicates number of reads. The CGI plots are from a previously published CAP-seq analysis of DNA from mouse cerebellum (Illingworth et al., 2010). (F) Pie chart illustrating the proportion of major CGI methylation patterns as defined in the text. (G) Illustrative examples of significant differences in gene density, % CpG, and ES cell expression level of associated genes from a comparison of class A+B genes with class C genes. See also Tables S2 and S3.

Figure 5

Dnmt3b Is Required for Xi CGI Methylation (A) Western blot analysis illustrating levels of Dnmt3a isoforms a1 and a2, and the major Dnmt3b isoform during differentiation of XX ES cells in vitro. Detection of Lamin B was used as a loading control. (B) Examples of IF for Dnmt3a and Dnmt3b, as indicated, in XX ES cells and following in vitro differentiation for the times shown. DNA was counterstained with DAPI. Staining patterns reveal overall nuclear localization of Dnmt3a/b and, at specific times, focal staining that colocalizes with DAPI dense pericentric heterochromatin. (C) Immuno-RNA FISH for Dnmt3b (green) and Xist RNA (red) in XX ES cells and following differentiation for the indicated times. Dnmt3b foci adjacent to Xist RNA domains, likely corresponding to the X chromosome centromere, were seen at day 3 (short arrows). At later stages, Xist RNA domains stain negatively for Dnmt3b as shown in expanded boxes (long arrows). (D) Sequenom EpiTyper analysis of CGIs along the length of the X chromosome (indicated in schematic) in WT, Dnmt3a, Dnmt3b, and Dnmt3L mutant XX E9.5 embryos as indicated. Average values shown were determined from a minimum of two embryos of each genotype. Error bars indicate the SD between values for individual embryos. See also Table S1.

Figure 6

Smchd1-Dependent and -Independent DNA Methylation of Xi CGIs (A) Examples of IF analysis illustrating staining for GFP-tagged Smchd1 (green) and H3K27me3 (red) in XX ES cells lines differentiated (diff) for the times indicated. Xi domains, detected as strong H3K27me3 foci, that are also enriched for GFP-Smchd1 are indicated with arrows. DNA is counterstained with DAPI. (B) Scoring of IF data illustrates the percentage of cells with Xi domains detected using antibody to H3K27me3 and Smchd1-GFP at 5 days (n = 230), 7 days (n = 195), and 9 days (n = 166) of differentiation. (C) Histogram plots showing average CpG methylation levels at selected CGIs that are either unmethylated (Hprt and Ndufa1) or methylated (Mtm1, Maob, Nup62cl, Nlgn3, and Pnck) in differentiated XX ES cells. Sequenom EpiTyper analysis of bisulfite-treated DNA was carried out on XX (Fwt), Smchd1^−/− XX (Fmut), XY WT (Mwt), and XY Smchd1^−/− (Mmut) E10.5 embryos. Error bars indicate the SD of average CpG methylation in individual embryos (n = 3). (D) Histogram plots showing average CpG methylation levels at Hprt, Maob, Nup62cl, Nlgn3, and Pnck CGIs determined by Sequenom EpiTyper analysis of bisulfite-treated DNA from two independent XX MEF cell lines derived from E10.5 WT (Fwt1 and Fwt2) and Smchd1^−/− (Fmut1 and Fmut2) embryos. Error bars indicate the SD of average CpG methylation levels from independent determinations (n = 3). See also Figure S1 and Table S1.

Figure 7

Chromosome-wide Analysis of Smchd1-Independent Xi CGI Methylation (A) Browser screenshots showing MBD-seq data over 10 kb regions, illustrating examples of patterns of methylation in CGIs associated with X-linked genes as indicated. MBD-seq tracks show data for WT and Smchd1^−/− (mutant) XX and XY MEF cell lines. The y axis indicates number of reads. (B) Pie chart illustrating the proportion of Xi CGIs with high, intermediate, and low levels of CGI methylation (as defined in the text) in Smchd1^−/− XX MEFs. (C) Illustrative examples of significant differences in gene density, % CpG, and ES cell expression level of associated genes from a comparison of unmethylated (unmeth) and methylated (meth) CGIs in Smchd1^−/− XX MEFs. (D) Schematic illustrating the relation of CGI methylation kinetics, Smchd1 recruitment, and Dnmt3b expression in differentiating XX ES cells. XX ES cell differentiation to form embryoid bodies is triggered by withdrawal of LIF from the culture medium. Methylation kinetics of fast and slow CGIs are represented by the dashed blue line. Upregulation of Xist RNA (green dashed line) occurs in a large proportion of cells between days 1 and 4 of differentiation, and this is closely followed by early markers of X inactivation, such as recruitment of Polycomb repressors PRC1/2 (red dashed line). Fast and intermediate methylating CGIs gain methylation progressively during the period in which Dnmt3b levels ([Dnmt3b]) remain high, and this occurs independently of Smchd1 enrichment on Xi at days 7–9 (yellow dashed line). Smchd1 enrichment occurs concurrently with recruitment of the variant histone macroH2A (purple dashed line). See also Figure S2 and Tables S2, and S5.