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. 2008 Oct 22;27(20):2691-701.
doi: 10.1038/emboj.2008.193. Epub 2008 Sep 25.

DNA Methylation in ES Cells Requires the Lysine Methyltransferase G9a but Not Its Catalytic Activity

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Free PMC article

DNA Methylation in ES Cells Requires the Lysine Methyltransferase G9a but Not Its Catalytic Activity

Kevin B Dong et al. EMBO J. .
Free PMC article

Abstract

Histone H3K9 methylation is required for DNA methylation and silencing of repetitive elements in plants and filamentous fungi. In mammalian cells however, deletion of the H3K9 histone methyltransferases (HMTases) Suv39h1 and Suv39h2 does not affect DNA methylation of the endogenous retrovirus murine leukaemia virus, indicating that H3K9 methylation is dispensable for DNA methylation of retrotransposons, or that a different HMTase is involved. We demonstrate that embryonic stem (ES) cells lacking the H3K9 HMTase G9a show a significant reduction in DNA methylation of retrotransposons, major satellite repeats and densely methylated CpG-rich promoters. Surprisingly, demethylated retrotransposons remain transcriptionally silent in G9a(-/-) cells, and show only a modest decrease in H3K9me2 and no decrease in H3K9me3 or HP1alpha binding, indicating that H3K9 methylation per se is not the relevant trigger for DNA methylation. Indeed, introduction of catalytically inactive G9a transgenes partially 'rescues' the DNA methylation defect observed in G9a(-/-) cells. Taken together, these observations reveal that H3K9me3 and HP1alpha recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity.

Figures

Figure 1
Figure 1
DNA methylation of MLV, IAP and LINE1 retrotransposons is reduced in G9a−/− cells. Genomic DNA isolated from G9a−/−, G9a−/−Tg, Dnmt1−/−, Suv39h1/2−/− (Suv−/−) and the wt parent lines TT2, J1 and R1, respectively, was digested with MspI (M) or the methylation-sensitive restriction enzyme HpaII (H) and subject to Southern blotting using probes specific for (A) IAP, (B) MLV or (C) LINE1 retrotransposons. The G9a−/− line shows a dramatic decrease in DNA methylation at each of these repetitive elements that is reversed in the G9a−/−Tg line. In contrast, the Suv39h1/2−/− line shows no DNA methylation defect at IAP or MLV repeats. (D, E) Bisulphite analysis of the 5′LTR regions of IAP and MLV elements was conducted on TT2, G9a−/−, G9a−/−Tg (15-3), Dnmt1−/− and Dnmt3a/b−/− cells. For each molecule sequenced (horizontal bar), filled ovals represent the presence of an mCpG. The mean number of mCpGs per molecule sequenced is shown to the right of each set of sequenced samples. The mean % of mCpGs relative to the wild-type line is also shown (in parentheses).
Figure 2
Figure 2
DNA methylation of promoter regions is reduced in G9a−/− cells. (A) MeDIP followed by quantitative PCR of nine CpG-rich promoter regions and two imprinting control loci (ICR) shown previously to be methylated in ES cells (Mohn et al, 2008) was conducted on wt, G9a−/− and G9a−/−Tg lines. IAP and MusD amplicons were included as positive controls. An active housekeeping gene (Gapdh) and a CpG-poor intergenic region (Interg) were included as negative controls. A bar graph illustrating DNA methylation changes in G9a−/− and G9a−/−Tg ES cells relative to wt ES cells (set to 1) is shown. The fold change is normalized to an unmethylated control gene (Hprt). Numbers in parentheses indicate the enrichment in MeDIP relative to Hprt. Error bars indicate the s.e.m. of at least three independent experiments. A lower level of methylation was detected in the G9a−/− line than the wt or rescued lines for all of the genes that show a high level of methylation in the TT2 parent line. (B) DNA methylation status of the germline-specific Dazl and Tuba3 genes in wt, G9a−/−, Dnmt1−/− and Dnmt3a/b−/− cells was confirmed by bisulphite sequencing. The mean number of mCpGs per molecule sequenced is shown, along with the mean % of mCpGs relative to the wild-type line (in parentheses). Both promoters show an ∼40% reduction in DNA methylation density in the G9a−/− line.
Figure 3
Figure 3
Expression of DNMTs in G9a−/− cells. (A) RNA was isolated from TT2 wt and G9a−/− cells and expression levels of Dnmt1, Dnmt3a, Dnmt3b and Dnmt3L were determined by real-time quantitative RT–PCR, normalized to β-actin (RT–reverse transcriptase). Values represent the mean (±s.d.) expression level relative to the wild-type line, from three independent experiments. (B) Western blot analyses using quantitative two-colour fluorescence imaging was performed on nuclear extracts isolated from TT2 and G9a−/− cells, using antibodies specific for Dnmt1, Dnmt3a and DNMT3b and TFII-I as an internal control. Extract isolated from DNMT-deficient cells was used as a control for antibody specificity. Relative protein expression levels, normalized to TFII-I, are shown beneath each blot.
Figure 4
Figure 4
G9a−/− ES cells show defects in recruitment of Dnmt3a to ERVs and de novo methylation of introduced retroviruses. (A) Formaldehyde-fixed chromatin was isolated from TT2 and G9a−/− lines and ChIP was conducted using nonspecific IgG or antisera raised against Dnmt3a or unmodified histone H3. Real-time PCR using primers specific for the LTR regions of MLV, IAP and MusD ERVs was carried out and values are presented as percentage of input precipitated (±s.d.) relative to the input in the representative experiment shown. A significant reduction in Dnmt3a enrichment in the G9a−/− line relative to the wt control is clearly apparent. (B) TT2 wt, G9a−/−, J1 wt and Dnmt3a/b−/− (3a/b−/−) lines were infected with the retroviral vector MFG–GFP and passaged in the absence of selection. Genomic DNA was isolated on day 18 post-infection and analysed by bisulphite genomic sequencing.
Figure 5
Figure 5
ERVs are not aberrantly expressed in G9a−/− cells. RNA was isolated from TT2 wt, G9a−/−, G9a−/−Tg, J1 wt, Dnmt3a/b−/− and Dnmt1−/− cells and analysed by northern blotting. RNA isolated from a cell line harbouring an active MLV-based retroviral vector was used as a positive control. 18 and 28s RNA loading controls are shown for each blot. (A) No expression of MLV was detected in any of the lines tested, using a probe specific for the MLV LTR region. (B) A high level of aberrant expression of the three subtypes (I, IΔ1 and II) of IAP elements was detected in the Dnmt1−/− line, but not the G9a−/− line, using a probe specific for the IAP LTR region. (C) Quantitative RT–PCR (+/−RT) using primers specific for the Pol region of full-length IAP elements revealed no increase in expression in the parent or G9a−/− lines, but a significant increase in expression in the Dnmt1−/− and Dnmt3a/b−/− lines.
Figure 6
Figure 6
ERVs show a reduction in H3K9 dimethylation in G9a−/− cells, but no reduction in H3K9 trimethylation or HP1α binding. TT2 wt and G9a−/− ES cells were analysed using ChIP with specific for H3K9me2, H3K9me3, HP1α, unmodified H3 and nonspecific IgG (IgG) as a control. Quantitative real-time PCR was conducted using primers specific for IAP or MusD retrotransposons or major satellite repeats. (A) Mean enrichment values are presented as percentage of input precipitated (±s.d.), relative to the input in the representative experiment shown. (B) Plotting the mean relative enrichment (±s.d.) of H3K9me2 and H3 from three independent experiments reveals an ∼2-fold decrease in H3K9me2 in the G9a−/− line relative to the parent line (*P<0.05, by Student's t-test) but no difference in H3 occupancy at these elements. (C) Relative to the R1 wt parent line, Suv39h1/2−/− (Suv−/−) ES cells show a dramatic decrease in H3K9me3 only at major satellite repeats.
Figure 7
Figure 7
H3K9 methylation and HP1α binding at MusD, IAP and major satellite repeats in Dnmt1−/− cells. ChIP was conducted on J1 wt and Dnmt1−/− ES cells using antibodies specific for H3K9me2, H3K9me3, HP1α and unmodified H3. Nonspecific IgG was used as a control. Real-time PCR of reverse-crosslinked material using primers specific for IAP, MusD or major satellite repeats was conducted in triplicate and enrichment (±s.d.) is presented as the mean percentage of input material immunoprecipitated, normalized to unmodified H3. IAP elements show a modest reduction in H3K9me3 and HP1α binding in the Dnmt1−/− line, but no change in H3K9me2 enrichment. No significant difference in any of these features was detected at MusD or major satellite repeats.
Figure 8
Figure 8
Stable expression of catalytically inactive G9a transgenes is sufficient to rescue the DNA methylation defect observed in G9a−/− ES cells. The G9a−/− line 2-3 was stably transfected with constructs encoding a wt G9a transgene G9a−/−Tg(wt), or the mutant G9a transgenes G9a−/−Tg(C1168A) and G9a−/−Tg(Y1120V;Y1207F). Each of the latter transgenes harbour amino-acid substitutions in the SET domain that reduce the catalytic activity of the encoded protein to <1% of that of the wt protein. (A) Western blot analysis of cell lines expressing each of these transgenes reveals that the mutant proteins are expressed at the expected molecular weight. An antibody specific for TFII-I was used as a loading control. (B) Quantitative western blot analysis of GLP expression in these lines was determined by normalizing to the signal obtained for endogenous TFII-I on the same blot. (C) Genomic DNA isolated from wt (TT2), G9a−/− (2-3), G9a−/−Tg(wt), G9a−/−Tg(C1168A), G9a−/−Tg(Y1120V;Y1207F) and Dnmt1−/− ES cells was digested with HpaII (H) and subject to Southern blotting using an IAP-specific probe. MspI (M) was used as a control. (D) Bisulphite analysis using primers specific for the IAP 5′LTR confirms that expression of catalytically inactive G9a partially rescues the DNA methylation defect. (E) A bar graph showing the mean no. of mCpGs per molecule sequenced is shown for the bisulphite data presented in (D) and Figure 1D. (F) ChIP was conducted on the wt (TT2), G9a−/− (2-3), G9a−/−Tg(wt) and G9a−/−Tg(C1168A) lines using antisera raised against H3K9me2, Dnmt3a or unmodified H3. Nonspecific IgG was included as a control. Real-time PCR of reverse-crosslinked material using IAP-specific primers was carried out in triplicate and enrichment (±s.d.) is presented as the mean percentage of input material immunoprecipitated, normalized to unmodified H3.

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