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DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity

2008; Springer Nature; Volume: 27; Issue: 20 Linguagem: Inglês

10.1038/emboj.2008.193

1460-2075

Kevin Dong, Irina A. Maksakova, Fabio Mohn, Danny Leung, Ruth Appanah, Sandra Lee, Hao Yang, Lucia L.C. Lam, Dixie L. Mager, Dirk Schübeler, Makoto Tachibana, Yoichi Shinkai, Matthew C. Lorincz,

Genetics and Neurodevelopmental Disorders

Article25 September 2008free access DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity Kevin B Dong Kevin B Dong Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Irina A Maksakova Irina A Maksakova Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada Search for more papers by this author Fabio Mohn Fabio Mohn Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Danny Leung Danny Leung Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Ruth Appanah Ruth Appanah Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Sandra Lee Sandra Lee Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Hao W Yang Hao W Yang Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Lucia L Lam Lucia L Lam Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Dixie L Mager Dixie L Mager Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada Search for more papers by this author Dirk Schübeler Dirk Schübeler Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Makoto Tachibana Makoto Tachibana Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto, Japan Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Yoichi Shinkai Yoichi Shinkai Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto, Japan Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Matthew C Lorincz Corresponding Author Matthew C Lorincz Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Kevin B Dong Kevin B Dong Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Irina A Maksakova Irina A Maksakova Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada Search for more papers by this author Fabio Mohn Fabio Mohn Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Danny Leung Danny Leung Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Ruth Appanah Ruth Appanah Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Sandra Lee Sandra Lee Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Hao W Yang Hao W Yang Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Lucia L Lam Lucia L Lam Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Dixie L Mager Dixie L Mager Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada Search for more papers by this author Dirk Schübeler Dirk Schübeler Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Makoto Tachibana Makoto Tachibana Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto, Japan Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Yoichi Shinkai Yoichi Shinkai Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto, Japan Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Matthew C Lorincz Corresponding Author Matthew C Lorincz Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Author Information Kevin B Dong1, Irina A Maksakova1,2, Fabio Mohn3, Danny Leung1, Ruth Appanah1, Sandra Lee1, Hao W Yang1, Lucia L Lam1, Dixie L Mager1,2, Dirk Schübeler3, Makoto Tachibana4,5, Yoichi Shinkai4,5 and Matthew C Lorincz 1 1Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada 2Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada 3Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 4Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto, Japan 5Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan *Corresponding author. Department of Medical Genetics, Life Sciences Institute Room 5-507, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. Tel.: +604 827 3965; Fax: +604 822 5348; E-mail: [email protected] The EMBO Journal (2008)27:2691-2701https://doi.org/10.1038/emboj.2008.193 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 HP1α 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 HP1α recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity. Introduction Retrotransposons, including long terminal repeat (LTR) and non-LTR elements, are widely dispersed in the euchromatic compartment in higher mammals (Kuff and Lueders, 1988; Medstrand et al, 2002), constituting ∼37% of the mouse genome (Mouse Genome Sequencing Consortium, 2002). A subset of these elements are transcriptionally competent, placing a significant mutational load on their hosts (Maksakova et al, 2006). To minimize the likelihood of retrotransposition, a number of pathways that function at the transcriptional or post-transcriptional stages of the replicative cycle have evolved to inhibit the expression of these parasitic elements. DNA methylation for example, has an important function in transcriptional silencing of retrotransposons in mammalian cells (Li et al, 1992; Yoder et al, 1997; Walsh et al, 1998), as illustrated by the high level of expression of the intracisternal A particle (IAP) endogenous retrovirus (ERV) in mouse embryos deficient in the DNA methyltransferase (DNMT), Dnmt1 (Walsh et al, 1998). DNA methylation also has a critical function in transcriptional silencing of repetitive elements and their relics in filamentous fungi and plants (Goyon et al, 1996; Lindroth et al, 2001; Zhou et al, 2001), substantiating the importance of this epigenetic mark in suppressing transposable elements in distantly related eukaryotes. Repetitive elements in eukaryotes are also marked by specific covalent histone modifications (Bernstein et al, 2007). Methylation of lysine 9 of the histone H3 tail (H3K9) in particular, has an important function in silencing of these elements in yeast (Nakayama et al, 2001), filamentous fungi (Tamaru and Selker, 2001), plants (Jackson et al, 2002) and animals (Martens et al, 2005). Recent genome-wide studies reveal that ERVs are marked by H3K9 dimethylation (H3K9me2) and/or H3K9 trimethylation (H3K9me3) in murine cells (Peters et al, 2003; Martens et al, 2005; Mikkelsen et al, 2007); however, the specific histone methyltransferases (HMTases) responsible have not been identified. Intriguingly, the H3K9 HMTase DIM-5 is required for CpG methylation in Neurospora (Tamaru and Selker, 2001) and the H3K9 HMTase KRYPTONITE is required for CpNpG methylation in Arabidopsis (Jackson et al, 2002), suggesting the existence of an evolutionarily conserved silencing pathway in which H3K9 methylation promotes de novo DNA methylation of repetitive elements (Freitag and Selker, 2005; Stancheva, 2005). However, the role, if any, that H3K9 methylation has in DNA methylation of retrotransposons in mammalian cells has not been systematically addressed. Five HMTases in the ‘Suv39’ subfamily of SET (Suv39, Enhancer of Zeste, Trithorax) domain-containing proteins with H3K9 catalytic activity, including Suv39h1 and the closely related Suv39h2, G9a and the closely related GLP/EuHMTase1 and SETDB1/Eset, have been characterized in mammalian cells. On the basis of its sequence similarity to SETDB1, the sixth Suv39 family member, SETDB2/CLLD8, is also likely to have specificity for H3K9 (Mabuchi et al, 2001; Kouzarides, 2007). Although Suv39h1 and Suv39h2 double-negative (Suv39h1/2−/−) embryonic stem (ES) cells show a dramatic reduction in H3K9me3 and DNA methylation at major satellite repeats, IAP elements show no reduction in H3K9 methylation (Peters et al, 2003; Martens et al, 2005; Mikkelsen et al, 2007) and murine leukaemia virus (MLV) ERVs show no reduction in DNA methylation (Lehnertz et al, 2003) in these cells. Taken together, these results indicate that Suv39h1 and Suv39h2 do not have a major function in H3K9 methylation or DNA methylation of LTR retrotransposons in mammalian cells. In contrast to the Suv39h HMTases, G9a and GLP/Eu-HMTase1, which form a heteromeric complex in vivo, are widely dispersed in the euchromatic compartment and deletion of either leads to a dramatic decrease in H3K9me1 and H3K9me2 in ES cells (Tachibana et al, 2002, 2005). A recent analysis revealed that ∼300–400 genes show altered expression in G9a−/− cells (Sampath et al, 2007) and several studies have shown that G9a regulates the expression and/or DNA methylation status of specific genes (Feldman et al, 2006; Ikegami et al, 2007). However, experiments aimed at determining whether G9a influences the expression and/or DNA methylation states of interspersed repetitive elements have not been reported. Here, we investigated the function that G9a and GLP have in DNA methylation and silencing of potentially active ERVs and non-LTR retrotransposons. We show that DNA methylation of these elements, and a subset of non-repetitive sequences including CpG-rich promoters, is reduced in G9a−/− cells and that Dnmt3a recruitment to retrotransposons is decreased in these cells. However, H3K9me3 enrichment and HP1α binding are unaltered, demonstrating that an alternative H3K9 HMTase marks ERVs and that H3K9 methylation per se is not sufficient to promote DNA methylation of these elements. In support of this model, we show that the introduction of two different G9a transgenes that lack catalytic activity into G9a−/− cells partially ‘rescues’ the observed DNA methylation defect, indicating that G9a promotes DNA methylation of retrotransposons independent of its catalytic activity. Results G9a is required for DNA methylation of retrotransposons To establish whether G9a is required for DNA methylation of ERVs, genomic DNA isolated from TT2 wild-type (wt) and G9a−/− ES cells (Tachibana et al, 2002) (Supplementary Figure S1) was analysed by Southern blotting using the methylation-sensitive restriction enzyme HpaII and probes specific for IAP and MLV ERVs, of which there are ∼1200 and ∼60 copies in the mouse genome, respectively (Figure 1A and B). Genomic DNA samples isolated from Dnmt1−/− (Lei et al, 1996), Suv39h1/2−/− (Peters et al, 2001) and the wt parent ES cell lines from which they were derived were analysed in parallel. A dramatic reduction in DNA methylation of both ERVs was detected in the G9a−/− line relative to the wt control. At the resolution of Southern blot analysis, this reduction in methylation is not distinguishable from that observed for the Dnmt1−/− ES line, or TT2 genomic DNA digested with the methylation-insensitive isoschizomer MspI. 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). Download figure Download PowerPoint To obtain a more accurate measure of the methylation status of these elements, high-resolution bisulphite sequencing analysis was conducted using primers specific for the CpG-rich 5′LTR regions of IAP and MLV elements. A >2.5-fold decrease in the mean number of mCpGs per molecule sequenced was detected in the G9a−/− line relative to the wt parent line (Figure 1D and E), with a subset of sequenced molecules in the G9a−/− line showing almost complete loss of methylation in these regions. A decrease in DNA methylation across the LTR and downstream regions of the potentially active class II ERV MusD (Mager and Freeman, 2000), of which there are ∼90 full-length copies in the mouse genome, was also detected in the G9a−/− line (Supplementary Figure S2). All three of these LTR retrotransposons show an even more severe DNA methylation defect in Dnmt1−/− cells (Figure 1D and E; Supplementary Figure S2). Consistent with the observations of Chen et al (2003), early passage Dnmt3a/b−/− cells (Okano et al, 1999) show a significantly more severe DNA methylation defect for MLV elements than IAP elements. Interestingly, whereas IAP elements show a less severe defect in Dnmt3a/b−/− cells than in G9a−/− cells, the reverse is true of MLV repeats. Thus, although it is clear that G9a is required for DNA methylation of distantly related ERVs in murine ES cells, the degree of demethylation is distinct from that observed for Dnmt1−/− or Dnmt3a/b−/− cells. In contrast, no reduction in DNA methylation of MLV or IAP elements was detected in Suv39h1/2−/− cells (Figure 1A and B), consistent with a previous report showing that Suv39h1 and Suv39h2 are not required for DNA methylation of MLV (Lehnertz et al, 2003). DNA methylation of LINE1 (L1) elements, non-LTR retrotransposons that comprise ∼20% of the mouse genome (Mouse Genome Sequencing Consortium, 2002), also depends on the presence of both Dnmt1 and Dnmt3a and/or Dnmt3b in ES cells (Liang et al, 2002). To determine whether G9a has a function in DNA methylation of this class of interspersed repeats, Southern blot analysis was conducted with a probe that spans the promoter region of the L1Md-A2 subfamily of L1 elements. A significant decrease in DNA methylation of L1 elements is also apparent in G9a−/− cells, although this defect is not as severe as that detected in the DNMT mutant lines (Figure 1C). Taken together, these observations indicate that G9a influences DNA methylation of both LTR and non-LTR retrotransposons in ES cells. To determine whether G9a is also required for DNA methylation of tandem repeats in ES cells, Southern blot analysis using a probe specific for major satellite repeats (present at approximately 700 000 copies per cell) was conducted using the methylation-sensitive restriction enzyme HpyCH4IV (Supplementary Figure S3). Consistent with a previous report showing that Suv39h1 and Suv39h2 are required for methylation of major satellite repeats (Lehnertz et al, 2003), a dramatic reduction in DNA methylation of major satellite repeats was detected in Suv39h1/2−/− cells. Unexpectedly, a dramatic reduction in DNA methylation of this class of repeats was also detected in the G9a−/− line, revealing that G9a is required for DNA methylation of pericentromeric heterochromatin as well. Introduction of a G9a transgene rescues the DNA methylation defect observed in G9a−/− cells Reintroduction of Dnmt3a, Dnmt3a2 (the predominant isoform of Dnmt3a in ES cells; Chen et al (2002)) or Dnmt3b1 into Dnmt3a/b−/− ES cells restores DNA methylation of MLV and IAP elements (Chen et al, 2003), indicating that the de novo DNMTs are capable of reestablishing DNA methylation patterns in these cells. To determine whether reintroduction of G9a is capable of reversing the DNA methylation defect observed in G9a−/− cells, the methylation status of these elements was also analysed in a G9a−/− line stably expressing a wt G9a transgene (G9a−/−Tg) (Tachibana et al, 2002) at a level similar to that of the endogenous protein (Supplementary Figure S1). Strikingly, the DNA methylation state of MLV, IAP, L1 (Figure 1) and MusD (Supplementary Figure S2) retrotransposons and major satellite repeats (Supplementary Figure S3) in the G9a−/−Tg resembles that of the original wt parent line (TT2) rather than the G9a−/− line from which they were directly derived. These observations indicate that loss of DNA methylation in G9a−/− ES cells is not an irreversible process and that reintroduction of G9a is sufficient for the reestablishment of DNA methylation in G9a-deficient ES cells. GLP is required for DNA methylation of retrotransposons As G9a forms a complex with the closely related HMTase GLP, and both are required for the deposition of the H3K9me2 mark (Tachibana et al, 2005), we next determined whether GLP is also required for DNA methylation of retrotransposons. Genomic DNA isolated from wt TT2 and GLP−/− ES cells (Tachibana et al, 2005) was analysed by Southern blotting as above, using probes specific for IAP, MLV (Supplementary Figure S4) and L1 elements (data not shown). A significant DNA methylation defect is apparent for all three elements in the GLP−/− line as well, with IAP elements showing the most dramatic decrease. Furthermore, introduction of a wt GLP transgene into the GLP−/− line (generating the GLP−/−Tg line (see Supplementary Figure S1; Tachibana et al, 2005) rescues the IAP DNA methylation defect and partially rescues the MLV methylation defect, revealing that DNA methylation can be reestablished on reintroduction of this HMTase as well. Consistent with these results, bisulphite sequencing analysis of polytrophic MLV elements reveals an ∼40% reduction in DNA methylation across the 5′LTR in the GLP−/− line relative to the wt control, and a partial rescue of this methylation defect in the GLP−/−Tg line (Supplementary Figure S4). Thus, both G9a and GLP have a function in DNA methylation of retrotransposons in ES cells. DNA methylation at non-repetitive genomic regions is reduced in G9a−/− cells To determine whether this DNA methylation defect extends to non-repetitive elements in the genome, we carried out MeDIP (Weber et al, 2005) on genomic DNA isolated from TT2, G9a−/− and G9a−/−Tg ES cells and analysed the methylation status of 11 single-copy genomic regions, including 9 CpG-rich promoters shown previously to be methylated in ES cells (Mohn et al, 2008) (Figure 2A). Strikingly, all of the regions that are highly methylated in the TT2 line show a significant decrease in the G9a−/− line, including the germline-specific gene Mage-a2, shown previously to be aberrantly expressed in G9a−/− cells (Tachibana et al, 2002). As for the repetitive elements, DNA methylation is increased at most of these regions in the G9a−/−Tg line. The DNA methylation defect was confirmed through bisulphite sequencing of the Dazl and Tuba3 promoter regions, both of which show an ∼40% reduction in DNA methylation in the G9a−/− line (Figure 2B). The degree of demethylation across the Dazl promoter is similar to that observed in Dnmt1−/− and Dnmt3a/b−/− ES cells. In contrast, the degree of demethylation across the Tuba3 promoter in G9a−/− cells more closely resembles that observed in the Dnmt3a/b−/− line. Thus, although DNA methylation of CpG-rich promoter regions is also dependent on G9a, the degree to which G9a influences DNA methylation state depends on the genomic context. 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. Download figure Download PowerPoint DNMT expression is not dramatically altered in G9a−/− cells The observed DNA methylation defect prompted us to address whether Dnmt1, Dnmt3a, Dnmt3b or DNMT-like (Dnmt3L) are downregulated in G9a−/− ES cells. Quantitative RT–PCR analysis did not reveal a significant difference in mRNA levels of any of the DNMT family members in these lines (Figure 3A). Similarly, quantitative western blot analyses revealed a 2.5-fold) than the parent line from which they were derived. Although not as dramatic as the methylation defect observed in the Dnmt3a/b−/−-deficient cell line, this observation indicates that G9a is required for efficient de novo methylation in ES cells. G9a is not required for transcriptional silencing of retrotransposons As Dnmt1 was previously shown to be required for silencing of IAP elements in embryos (Walsh et al, 1998), we next determined whether the defect in DNA methylation of retrotransposons in G9a−/− cells is associated with aberrant expression of these potentially active endogenous elements. Expression of MLV and LINE1 elements was not detected above background levels in wt, G9a−/−, Dnmt1−/− or Dnmt3a/b−/− lines by northern blotting (Figure 5A and data not shown). In contrast, IAP elements of each subtype (I, IΔ1 and II) (Kuff and Lueders, 1988) (Figure 5B) and MusD elements (Supplementary Figure S5) are expressed at a significantly higher level in the Dnmt1−/− line than the G9a−/− line, relative to the parent lines from which they were derived. Although not as dramatic as that observed in the Dnmt1−/− line, aberrant IAP expression was also observed in the Dnmt3a/b−/− line by RT–PCR (Figure 5C). As the G9a and DNMT deletions were generated in ES cells of differing genetic backgrounds, it is not possible to attribute the differences in ERV expression exclusively to the genes deleted. Never