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Lsh is involved in de novo methylation of DNA

2006; Springer Nature; Volume: 25; Issue: 2 Linguagem: Inglês

10.1038/sj.emboj.7600925

1460-2075

Heming Zhu, Theresa M. Geiman, Sichuan Xi, Qiong Jiang, Anja Schmidtmann, Taiping Chen, En Li, Kathrin Muegge,

Genetics and Neurodevelopmental Disorders

Article5 January 2006free access Lsh is involved in de novo methylation of DNA Heming Zhu Heming Zhu Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Theresa M Geiman Theresa M Geiman Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Sichuan Xi Sichuan Xi Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Qiong Jiang Qiong Jiang Laboratory of Molecular Immnuoregulation, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Anja Schmidtmann Anja Schmidtmann Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Taiping Chen Taiping Chen Epigenetics Program, Novartis Institute for Biomedical Research, Inc., Cambridge, MA, USA Search for more papers by this author En Li En Li Epigenetics Program, Novartis Institute for Biomedical Research, Inc., Cambridge, MA, USA Search for more papers by this author Kathrin Muegge Corresponding Author Kathrin Muegge Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Heming Zhu Heming Zhu Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Theresa M Geiman Theresa M Geiman Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Sichuan Xi Sichuan Xi Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Qiong Jiang Qiong Jiang Laboratory of Molecular Immnuoregulation, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Anja Schmidtmann Anja Schmidtmann Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Taiping Chen Taiping Chen Epigenetics Program, Novartis Institute for Biomedical Research, Inc., Cambridge, MA, USA Search for more papers by this author En Li En Li Epigenetics Program, Novartis Institute for Biomedical Research, Inc., Cambridge, MA, USA Search for more papers by this author Kathrin Muegge Corresponding Author Kathrin Muegge Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Author Information Heming Zhu1, Theresa M Geiman1,‡, Sichuan Xi1,‡, Qiong Jiang2, Anja Schmidtmann1, Taiping Chen3, En Li3 and Kathrin Muegge 1 1Laboratory of Cancer Prevention, SAIC-FCRDC, Basic Research Program, National Cancer Institute, Frederick, MD, USA 2Laboratory of Molecular Immnuoregulation, National Cancer Institute, Frederick, MD, USA 3Epigenetics Program, Novartis Institute for Biomedical Research, Inc., Cambridge, MA, USA ‡These authors contributed equally to the work *Corresponding author. Laboratory of Molecular Immunoregulation, SAIC, National Cancer Institute, BLDG 469, Room 243, Frederick, MD 21701-1201, USA. Tel.: +1 301 846 1386; Fax: +1 301 846 7077; E-mail: [email protected] The EMBO Journal (2006)25:335-345https://doi.org/10.1038/sj.emboj.7600925 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh−/− embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA. Introduction DNA methylation regulates a number of biological processes, including genomic imprinting, X chromosome inactivation, silencing of tumor suppressor genes, and repression of retroviral elements (Bird, 2002; Li, 2002). Loss of methylation in mice results in severe developmental defects and early embryonic lethality (Li et al, 1992; Okano et al, 1999; Dennis et al, 2001). A number of human inherited diseases linked to faulty methylation pathways and exhibiting abnormal development include Rett, ICF, and ATRX syndromes (Amir et al, 1999; Okano et al, 1999; Gibbons et al, 2000). Moreover, aberrant methylation patterns are thought to be involved in tumorigenesis (Jones and Baylin, 2002; Chen et al, 2004; Yu et al, 2005) causing genomic instability, abnormal imprinting, and deregulated expression of oncogenes or tumor suppressor genes. Two types of methylation pathways are functionally distinct: maintenance versus de novo methylation. During early mammalian embryogenesis, DNA methylation patterns are largely erased and re-established shortly after implantation in a wave of de novo methylation (Reik et al, 2001; Bird, 2002; Li, 2002). These newly established patterns are then thought to be faithfully copied after each round of replication onto the newly synthesized DNA strand. In contrast, genomic imprints, which are largely dependent on DNA methylation, are mostly established in germ cells and preserved throughout embryogenesis. Thus, maintenance activity is found in all somatic cells while the highest de novo methylation activity is found in embryonic cell lines, germ cells, or in postimplantation embryos. Several DNA cytosine methyltransferases have been identified in mammalian cells (Chen and Li, 2004; Goll and Bestor, 2005). Dnmt1 is primarily responsible for maintenance methylation since Dnmt1 shows high affinity for hemimethylated substrates and is present at replication forks via its association with PCNA (Leonhardt et al, 1992; Chuang et al, 1997; Okano et al, 1998; Pradhan et al, 1999). De novo methylation activity is primarily dependent on Dnmt3a and Dnmt3b, two partially redundant Dnmt family members (Okano et al, 1999). Dnmt3a plays an additional crucial role in de novo methylation of imprinted sites in germ cells together with Dnmt3L, a Dnmt family member lacking catalytic activity (Hata et al, 2000; Bourc'his and Bestor, 2004; Kaneda et al, 2004). Although the enzymes responsible for methylation patterns have been identified, the precise molecular mechanisms including cofactors that lead to recruitment and efficient targeting of the enzymatic machinery to their appropriate sites are unknown. We have previously reported that Lsh controls genomic methylation patterns in mice (Dennis et al, 2001, Muegge, 2005). Lsh belongs to the SNF2 family of proteins (Jarvis et al, 1996; Geiman et al, 1998), whose members participate in chromatin remodeling (Fyodorov and Kadonaga, 2001; Langst and Becker, 2004). Targeted disruption of Lsh in mice leads to developmental defects and early lethality (Geiman and Muegge, 2000; Dennis et al, 2001; Geiman et al, 2001; Fan et al, 2003; Sun et al, 2004). Lsh controls normal heterochromatin structure and function in mice, and upon deletion a number of epigenetic modifications are perturbed. For example, Lsh-deficient cells show genome wide CpG hypomethylation, altered histone H3 methylation, and increased acetylation levels for histone H3 and histone H4 (Dennis et al, 2001; Yan et al, 2003a, 2003b; Huang et al, 2004, Sun et al, 2004). Since epigenetic modifications are closely linked, it remains unclear which one is the initial epigenetic modification targeted by Lsh. In this report we attempt to determine the functional role of Lsh in either maintenance or de novo methylation, and to characterize the role of Lsh in the establishment of epigenetic modifications. We provide evidence that Lsh is required for de novo methylation of DNA and that it is directly involved in the methylation process. Results Lsh is required for methylation of episomal DNA in MEF cells Lsh is a global regulator of DNA methylation in mice. To investigate further the functional role of Lsh in methylation, we used an episomal vector system (Figure 1A) that allows for the discrimination between de novo and maintenance of genomic methylation. The episomal vector can be transfected into mammalian cells to serve as a target for de novo methylation (Hsieh, 1999). The presence of novel CpG methylated sites can be determined using methylation-sensitive PCR once the episomal construct is recovered and completely digested with a methylation-sensitive restriction enzyme such as HpaII. Figure 1A illustrates the design of PCR primers to amplify regions of the episomal construct, pCEP4. Primer pair P5/6 surrounds multiple HpaII sites and can detect successful methylation. In contrast, the product of primer pair P1/P2 contains no HpaII sites, thus serving as an internal control for the amount of recovered episomal DNA. Primer pair P3/P4 amplifies a fragment that contains multiple DpnI sites and therefore is used as control to indicate successful replication in mammalian cells (DpnI can only digest bacterial Dam methylated DNA, and thus fails to cleave DNA that has replicated in mammalian cells). Figure 1.Lsh is required for methylation of an episomal vector in MEF cells. (A) Map of the episomal vector pCEP4 illustrating the location of HpaII/MspI and DpnI sites as well as the position of the primers used for methylation-sensitive PCR analysis. Primer pairs P5/P6 are designed for detection of methylation, P3/P4 for detection of successful replication, and P1/P2 as an internal control. The length of the expected PCR fragment is indicated in base pairs (bp). (B) Western blot analysis using nuclear extracts derived from Lsh−/− and Lsh+/+ embryonal fibroblasts (MEF) and specific antibodies against Lsh, Dnmt3a, Dnmt3b and PCNA (serving as control). (C) Methylation-sensitive PCR (upper panel). Episomal DNA derived from stably transfected Lsh−/− and Lsh +/+ MEF cells was digested with DpnI and then either with the methylation-sensitive enzyme HpaII (H) or the methylation independent enzyme MspI (M) followed by PCR analysis with indicated primers. Replication of the episomal vector was confirmed using DpnI (lower panel). DpnI cuts only DNA that has been methylated in bacteria by the dam methylase. The replicated episomal DNA in mouse cells should be DpnI resistant. For adjustment of input undigested DNA (Un) was used before digestion. (D) Episomal DNA was derived as in C. and subjected to real time-PCR analysis using methylation-sensitive primer pair P5/P6 (upper panel) or the internal control P1/P2 (lower panel). Download figure Download PowerPoint The episomal construct pCEP4 was stably transfected into MEFs derived from Lsh−/− or Lsh+/+ embryos. Since the episomal construct serves as a target for de novo methylation by Dnmt3a and Dnmt3b, we first examined protein expression levels by Western blot analysis using nuclear extracts of stably transfected cell lines. As expected, Lsh protein levels were not detectable in Lsh−/− MEFs (Figure 1B). In contrast, DNA methyltransferases Dnmt3a and 3b were both expressed equally well in the presence or absence of Lsh, suggesting a comparable de novo methylation activity in both cell lines. To examine the methylation status of the recovered episome, methylation-sensitive PCR analysis was performed as shown in Figure 1C. After quantification of recovered episomal DNA to adjust for equal input using primers P5/P6 (undigested samples), the DNA was digested with the methylation-sensitive restriction enzyme HpaII. The successful amplification, using P5/P6 primers, of Lsh+/+ derived DNA after treatment with HpaII indicated the presence of methylated CpG sites. Digestion with MspI (which cleaves DNA independent of methylation) served as control. In contrast, the P5/P6 PCR fragment was not detectable using DNA derived from Lsh−/− MEFs, suggesting an impaired gain of methylation in the absence of Lsh. To ensure that equal amounts of DNA were indeed present in wild type and Lsh−/− samples after digestion (since digestion and further handling can lead to unavoidable loss of DNA), the control primers P1/P2 were used which do not surround HpaII sites (Figure 1C). Using these internal control primers, the amplification was indistinguishable between Lsh wild type and Lsh−/− DNA. In order to quantify the appearance of methylation in the episome sequence, real-time PCR was performed. As shown in Figure 1D, the use of the methylation-sensitive primer set P5/P6 revealed a significant difference of methylation comparing wild type and Lsh−/− samples, whereas the internal control primers confirmed equal amounts of DNA after digestion. In an attempt to quantify methylation levels, a standard curve for PCR amplifications using known concentrations of the episomal DNA was performed and used to calculate the copy numbers using primers P5/P6 before and after digestion (Supplementary Figure 1). Wild-type samples were completely methylated at the examined sites of pCEP4. In contrast, Lsh−/− samples were only methylated about 1% by this calculation. These observations demonstrated that Lsh is essential for the acquisition of methylation on episomal DNA in MEFs. Lsh does not play a role in maintenance of methylation in MEF cells Gain of methylation on the episomal vector pCEP4 requires de novo methylation activity as well as the ability to maintain newly acquired methylation patterns. To differentiate between these two processes, we tested the ability of Lsh−/− cells to maintain methylation on previously in vitro methylated DNA. The episomal vector pCEP4 was treated with SssI methyltansferase and the degree of methylation was determined by digestion with the methylation-sensitive restriction enzyme, HpaII. Resistance to HpaII digestion indicated the successful methylation of the vector (Figure 2A). Figure 2.Lsh does not play a role in the maintenance of methylation in MEF cells. (A) Ethidium bromide stain of in vitro methylated episomal vector pCEP4 that has been digested with HpaII (H) and MspI (M). Resistance to HpaII digestion indicates that the episome has been fully methylated. (B) Methylation-sensitive PCR. Methylated episomal DNA was stably transfected into Lsh−/− and Lsh+/+ MEF cells and the recovered DNA was first digested with DpnI. Then the DNA was either digested with the methylation-sensitive enzyme HpaII (H) or the methylation independent enzyme MspI (M) followed by PCR analysis with the indicated primers as shown in Figure 1A. (C) Real-time PCR analysis. Episomal DNA was derived as described in (B). and subjected to real time-PCR analysis using the methylation-sensitive primer pair P5/P6 (upper panel) or the internal control primer pair P1/P2 (lower panel). (D) The relative copy numbers for real time-PCR products of HpaII digested episomal DNA were calculated based on the standard curve equation. Results of Figure 1 are represented as 'de novo' and results from Figure 2 as 'maintenance' methylation. Download figure Download PowerPoint The methylated episomal DNA was stably transfected into Lsh−/− and Lsh+/+ MEFs and methylation-sensitive PCR performed after recovery of the episome. Episomal DNA derived from wild-type MEFs showed the expected PCR fragment using primer pair P5/P6 after HpaII digestion, indicating the presence of methylated sites on the episome (Figure 2B). Using episomal DNA derived from Lsh−/− MEFs, a comparable PCR amplification was observed suggesting a similar degree of methylation in the absence of Lsh (Figure 2B). These results were further confirmed using real-time PCR analysis. As shown in Figure 2C, there were no detectable differences in the amplification using primer pair P5/6 or the internal controls P1/P2 when comparing wild type and Lsh deficient cells indicating a similar ability to preserve methylation patterns. As summarized in Figure 2D, the calculated copy numbers which were amplified from premethylated plasmids were indistinguishable between Lsh+/+ and Lsh−/− cells. Thus, methylation patterns are faithfully maintained in the absence of Lsh, while the acquisition of a novel methylation mark depends on Lsh. We therefore conclude that Lsh plays a role in de novo methylation rather than in maintenance methylation. Lsh is required for Dnmt3a or Dnmt3b mediated silencing of a retroviral transgene De novo methylation is thought to be conferred by either Dnmt3a or Dnmt3b activity in mice (Hsieh, 1999; Okano et al, 1999). In order to understand whether Lsh is involved in the methylation process mediated by either DNA methyltransferase, we stably overexpressed both proteins in Lsh−/− and Lsh+/+ MEFs. By Western blot analysis, an increase in Dnmt3a or Dnmt3b proteins over endogenous methyltransferase levels was observed using nuclear extracts from Lsh−/− MEFs and Lsh +/+ MEF controls (Figure 3A). To investigate the silencing function caused by methylation, the retroviral reporter vector pMSCV-hGFP was used (kind gift of Dr Jonathan Keller, NCI, Frederick). Upon methylation, expression of the green fluorescence protein (GFP) is suppressed and can be monitored by either fluorescence microscopy or FACS analysis. After infection of Lsh−/− and Lsh+/+ MEFs with the retroviral vector pMSCV-hGFP, GFP expression was monitored by microscopy (Figure 3B). Lsh wild-type cells that were either stably transfected with Dnmt3a, Dnmt3b, or both showed a decrease in GFP expression within 8 days after infection. In contrast, Lsh deficient cells stably expressing Dnmt3a, Dnmt3b, or both were unable to significantly reduce GFP expression. Quantitative measurement of fluorescence expression by FACS analysis revealed a reduction of GFP intensity of about 25–30% when comparing wild-type cells with Lsh−/− MEFs, suggesting an impairment of reporter silencing in the absence of Lsh (Figure 3C). In contrast, untransfected MEFs (Un) that did not overexpress the Dnmt3 proteins were not able to silence the integrated reporter gene and thus, GFP expression levels were indistinguishable between wild type and Lsh deficient cells (Figure 3C). Lsh, therefore, plays an important role in Dnmt3a or Dnmt3b mediated silencing of retrovirus directed protein expression. Figure 3.Lsh is required for silencing of Dnmt3a or Dnmt3b mediated silencing of a retroviral transgene. (A) Western blot analysis using nuclear extracts derived from Lsh−/− and Lsh+/+ mouse embryonal fibroblasts stably expressing Dnmt3a, Dnmt3b, Dnmt3a/Dnmt3b, or untransfected (Un) MEFs. For detection, specific antibodies were used against Dnmt3a, Dnmt3b, or PCNA as control. (B) Fluorescence analysis. Lsh+/+ and Lsh−/− MEFs that were stably expressing Dnmt3a, Dnmt3b, or Dnmt3a/Dnmt3b were infected with pMSCV-hGFP and examined after 8 days for GFP expression using a fluorescence microscope. (C) FACS analysis. The GFP intensity of Lsh+/+ and Lsh−/− MEF cells expressing Dnmt3a, Dnmt3b, Dnmt3a/Dnmt3b, or untransfected (Un) was measured by FACS analysis 8 days after retroviral infection. The difference in fluorescence intensity was expressed as GFP ratio of Lsh+/+ over Lsh−/−. (D) Map of the retroviral vector pMSCV-hGFP indicating the location of HpaII/MspI sites and the position of the primers used for methylation-sensitive PCR analysis. Primer pair P9/P10 detects methylation within the GFP region. Primer pair P7/P8 serves as internal control. Primer pair P11/P12 detects methylation in the 5′-LTR and the adjacent region. The length of the expected PCR fragments is indicated in base pairs (bp). Download figure Download PowerPoint Lsh functionally cooperates with de novo methylation mediated by either Dnmt3a or Dnmt3b Since Lsh plays an important role in the downregulation of GFP expression, we tested whether CpG hypermethylation was directly involved in gene silencing of the retroviral transgene using methylation-sensitive PCR. For detection of methylation, primers were designed around HpaII/MspI sites in the GFP gene (P9/P10) and the 5′-LTR region (P11/P12) of the vector (Figure 3D). The control primers P7/P8 did not surround HpaII sites thus serving as internal control. After the undigested genomic DNA was quantified to adjust for equal input using primers P11/12 (Figure 4A), restriction enzyme digestion with the methylation-sensitive enzyme HpaII was performed. Lsh wild-type samples were successfully amplified and generated PCR fragments in the 5′LTR (primer P11/P12) and the GFP coding region (P9/P10), suggesting methylation within the amplified regions (Figure 4A). In contrast, Lsh−/− samples did not generate the expected PCR fragments indicating reduced methylation (Figure 4A). This decrease of methylation occurred in Dnmt3a, Dnmt3b, or Dnmt3a/b overexpressing Lsh−/− MEFs, suggesting that Lsh is required for methylation induced by either Dnmt3 protein. As control, to ensure equal amounts of DNA after digestion, the internal methylation independent primers were used (P7/P8). Distinct methylation levels when comparing wild type and Lsh−/− samples were further confirmed using real-time PCR (Figure 4B–D). Whereas the use of primer pair P7/P8 verified equal loading of DNA after digestion, use of primer pair P11/12 indicated significant differences in methylation at the 5′LTR region in wild type versus Lsh deficient samples. Without overexpression of the Dnmt3 proteins, Lsh+/+ and Lsh−/− samples were indistinguishable since methylation was undetectable (data not shown). In an attempt to quantify methylation levels, a standard curve for PCR amplifications using known concentrations of the template was performed and used to calculate the copy numbers using primers P11/12 before and after digestion (Supplementary Figure 2). Although the frequency of having all four sites methylated in wild-type cells was low, the deletion of Lsh revealed consistently a 10-fold reduction in Dnmt3a, Dnmt3b or Dnmt3a,b transfected cells. Possibly, the low methylation efficiency in the retroviral transgene in comparison to the full methylation of the episomal vector was due to distinct retroviral target sequences, the integration of the transgene or a difference in the experimental time frame. Taken together, the difference in CpG methylation levels between wild type and Lsh deficient cells correlated with the difference in silencing of GFP expression. These results suggest, therefore, that Lsh cooperates with either Dnmt3a or Dnm3b for de novo methylation. Figure 4.Lsh functionally cooperates with de novo methylation mediated by either Dnmt3a or Dnmt3b. (A) Methylation-sensitive PCR. At 8 days after the retroviral infection, genomic DNA from Lsh−/− and Lsh+/+ MEF cells stably expressing Dnmt3a, Dnmt3b, or Dnmt3a/Dnmt3b was extracted, digested with HpaII (H) or MspI (M) and subjected to PCR with the indicated primer pairs. For adjustment of DNA undigested DNA (Un) was used before digestion. (B–D). Real-time PCR analysis of HpaII digested DNA using primer pair P11/P12. Control primers P7/P8 are used in the lower panel of the graphs. Download figure Download PowerPoint Silencing of Lsh in embryonal stem cells results in loss of de novo methylation Embryonal stem (ES) cells are used for de novo methylation assays since they are rich in endogenous Dnmt3a/b proteins in contrast to somatic cells and readily methylate newly integrated retroviral DNA (Okano et al, 1999; Chen et al, 2002). To further investigate the molecular mechanism of Lsh in methylation, we established ES cell lines with low levels of the Lsh protein using RNA interference. Stably expressing Lsh hairpin siRNA in ES cell lines (siLsh#1 and siLsh#2) significantly decreased Lsh protein levels by more than 90% as shown by Western blot analysis, whereas Dnmt3a and Dnmt3b levels remained unchanged (Figure 5A). Using Southern blot analysis, genomic DNA was examined for DNA hypomethylation at minor satellite repeat sequences (Figure 5B). Whereas control ES cells were fully methylated at these repeats, low levels of Lsh protein were associated with a small degree of hypomethylation after several passages in vitro. The delay in hypomethylation mimicked a similar time course generated by conditional deletion of Dnmt3b in ES cells, which suggests ongoing de novo methylation in ES cells at minor satellite sequences (Chen et al, 2003). In contrast to embryonal stem cells, 3T3 fibroblasts represent somatic cells that have generally lower levels of Dnmt3a or Dnmt3b proteins and de novo methylation activity (Chen et al, 2002). Silencing of Lsh in 3T3 cells using the same siLsh vector had no detectable effect on CpG methylation at minor satellite sequences (Supplementary Figure 3A,B) consistent with the idea of low or absent de novo methylation activity at pericentric sequences in somatic cells (Chen et al, 2002). To determine the effect of Lsh reduction on silencing of retroviral genes, siLsh ES cells were infected with the retroviral vector pMSCV-hGFP that controls expression of the green fluorescence protein (GFP). Within 48–72 h after infection, GFP mRNA levels were notably reduced in wild-type ES cells in contrast to siLsh ES cells, which maintained GFP mRNA levels as shown by RT–PCR analysis (Figure 5C). FACS analysis revealed a decrease in GFP protein of about 80% when comparing control ES cells to Lsh deficient ES cells, suggesting that successful silencing of a retroviral vector requires the presence of Lsh (Figure 5D). Using methylation-sensitive PCR analysis, the genomic DNA derived from control ES cells 72 h after infection demonstrated HpaII resistance indicating the acquisition of methylation (Figure 5E). In contrast, siLsh ES cells showed no detectable CpG methylation at the GFP gene (P9/P10) or the 5′-LTR (P11/P12) regions as demonstrated by sensitivity to HpaII digestion. Real-time PCR analysis confirmed the differences in methylation (P11/P12) seen in the presence (control ES) or absence of Lsh (siLsh) (Figure 5F). In contrast, the control PCR reactions (P7/P8) remained indistinguishable comparing control or siLsh derived DNA. In summary, the use of retroviral target sequences in Lsh deficient embryonic stem (ES) cells confirms a role for Lsh in de novo methylation. Figure 5.Silencing of Lsh in embryonic stem cells results in loss of de novo methylation. (A) Western analysis: nuclear extracts from two ES cell lines (#1 and #2 both received the same construct) stably expressing a silencing vector for Lsh (siLsh) were examined by Western analysis using specific antibodies against Lsh, Dnmt3a, Dnmt3b, Chd4 or PCNA as control. A scrambled sequence serves as the siRNA vector control. (B) Southern blot analysis: genomic DNA derived from two siLsh ES cell lines, an ES cell control, Lsh−/− MEFs, and Lsh+/+ MEF cells was digested with HpaII (H) or MspI (M), blotted, and probed for minor satellite sequence using the probe MR150. (C) RT–PCR analysis: siLsh ES cells were infected with the retroviral vector pMSCV-hGFP and after the indicated time points (24, 48, 72 h) RNA was extracted, reverse transcribed and analyzed by PCR for expression of GFP. β-Actin serves as a control. (D) FACS analysis: 72 h after retroviral infection of siLsh and control ES cells GFP expression was measured by FACS analysis. (E) Methylation-sensitive PCR: genomic DNA derived from the siLsh and control ES cells 3 days after retroviral infection was digested with HpaII or MspI and subjected to PCR analysis with the indicated primer pairs. (F) Real time-PCR analysis: as in (E), HpaII digested genomic DNA was subjected to real time-PCR using P11/P12. The right panel shows the internal control reaction with primers P7/P8. Download figure Download PowerPoint Lsh is involved in de novo methylation of endogenous genes Since Lsh participates in de novo methylation of episomal constructs and retroviral transgenes, we also wanted to test whether Lsh deletion in ES cells effects de novo methylation of endogenous genes such as the Oct-4 gene (Gidekel and Bergman, 2002). Using methylation-sensitive PCR at two distinct sites in the promoter region of the Oct-4 gene (Figure 6A), we had previously noticed that Lsh−/− MEFs have decreased methylation levels at site 2 in comparison with Lsh+/+ (Figure 6B) but show a similar degree of methylation at site 1 (data not shown). This suggested that Lsh played a role in methylation at selected sites of the Oct-4 gene. It has been reported that differentiation of ES cells in vitro is accompanied by CpG methylation at the endogenous Oct-4 gene (Lee et al, 2004). Using siLsh ES cells and control ES cells, we compared methylation levels at site 1 and site 2 before (0 day) and after differentiation (6 days) using treatmen