Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice
2012; Springer Nature; Volume: 32; Issue: 3 Linguagem: Inglês
10.1038/emboj.2012.331
ISSN1460-2075
AutoresSaya Kagiwada, Kazuki Kurimoto, Takayuki Hirota, Masashi Yamaji, Mitinori Saitou,
Tópico(s)Prenatal Screening and Diagnostics
ResumoArticle14 December 2012free access Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice Saya Kagiwada Saya Kagiwada Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Kazuki Kurimoto Kazuki Kurimoto Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Search for more papers by this author Takayuki Hirota Takayuki Hirota Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Search for more papers by this author Masashi Yamaji Masashi Yamaji Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Search for more papers by this author Mitinori Saitou Corresponding Author Mitinori Saitou Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan Search for more papers by this author Saya Kagiwada Saya Kagiwada Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Kazuki Kurimoto Kazuki Kurimoto Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Search for more papers by this author Takayuki Hirota Takayuki Hirota Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Search for more papers by this author Masashi Yamaji Masashi Yamaji Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Search for more papers by this author Mitinori Saitou Corresponding Author Mitinori Saitou Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan JST, ERATO, Kyoto, Japan Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan Search for more papers by this author Author Information Saya Kagiwada1, Kazuki Kurimoto1,2, Takayuki Hirota1,2, Masashi Yamaji1,2 and Mitinori Saitou 1,2,3,4 1Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan 2JST, ERATO, Kyoto, Japan 3Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan 4Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan *Corresponding author. Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. Tel.:+81 75 753 4335; Fax:+81 75 751 7286; E-mail: [email protected] The EMBO Journal (2013)32:340-353https://doi.org/10.1038/emboj.2012.331 There is a Have you seen? (February 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Genome-wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs) is a critical first step to creating a totipotent epigenome in the germ line. We show here that, contrary to the prevailing model emphasizing active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner largely consistent with replication-coupled passive DNA demethylation: PGCs erase imprints during their rapid cycling with little de novo or maintenance DNA methylation potential and no apparent major chromatin alterations. Our findings necessitate the re-evaluation of and provide novel insights into the mechanism of genome-wide DNA demethylation in PGCs. Introduction Primordial germ cells (PGCs) in mice, the precursors for both the spermatozoa and the oocytes, have long been known to undergo genome-wide epigenetic reprogramming (Surani et al, 2007; Sasaki and Matsui, 2008; Saitou et al, 2012). This includes, most notably, genome-wide DNA demethylation (Popp et al, 2010; Guibert et al, 2012), which leads to the erasure of parental imprints (Hajkova et al, 2002; Lee et al, 2002). Consequently, male and female PGCs acquire an equivalent naïve epigenome at around embryonic day (E) 13.5. They subsequently establish new sex-specific epigenetic modifications along with their progression into spermatogenic or oogenic pathways. Genome-wide epigenetic reprogramming in PGCs has therefore been considered essential as the first step for the creation of the totipotent epigenome in the germ cell lineage. However, despite its importance, the underlying mechanism for genome-wide DNA demethylation in PGCs remains elusive. DNA demethylation in animals can occur through either an active or a passive mechanism (Saitou et al, 2012). The active mechanism involves enzymes that directly modify 5-methylcytosine (5mC). Recent studies have revealed several potential pathways for active DNA demethylation. One such pathway involves AID (activation-induced cytidine deaminase) and APOBEC1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1), which can deaminate 5mC into thymine (T), and the resultant T/G (guanine) mismatch can be repaired by the base-excision repair (BER) pathway (Morgan et al, 2004). Another pathway involves TET (ten-eleven translocations) proteins (TET1, 2, 3), which oxidize 5mC into 5-hydroxymethylcytosine (5hmC) (Kriaucionis and Heintz, 2009; Tahiliani et al, 2009). The resulting 5hmC can either be deaminated by AID or APOBEC1 and repaired by the BER pathway (Guo et al, 2011), or further oxidized into 5-formylcytosine (5fC) and then into 5-carboxylcytosine (5caC), which is removed and repaired by the BER pathway (He et al, 2011; Ito et al, 2011). In contrast, the passive mechanism involves replication-coupled dilution of 5mC in the absence or under the prevention of maintenance DNA methyltransferase (DNA methyltransferase 1: DNMT1) activity. There has been a prevailing view that the genome-wide DNA demethylation in PGCs occurs primarily via an active mechanism (Feng et al, 2010). This view is based on (1) the kinetics analysis of the DNA demethylation of the differentially methylated regions (DMRs) of several imprinted genes (Hajkova et al, 2002), (2) the apparently higher genome-wide DNA methylation in Aid (also known as Aicda)-deficient PGCs at E13.5 (Popp et al, 2010), and (3) the activation of the BER pathway and subsequent dramatic chromatin changes, including loss of linker histone H1 and loss of several histone modifications such as histone H3 lysine 27 tri-methylation (H3K27me3), perhaps through histone replacement involving NAP1/NAP1l1 (nucleosome assembly protein 1/nucleosome assembly protein 1 like 1) and HIRA (histone cell-cycle regulation homologue A) at the G2 phase of the cell cycle, which is specifically observed in PGCs at around E11.5 (Hajkova et al, 2008, 2010). However, all these observations include some ambiguity from a quantitative point of view (Saitou et al, 2012) (Results and Discussion), and definitive evidence for the involvement of an active mechanism in the genome-wide DNA demethylation in PGCs is still lacking. In order to provide a better resolution of the mechanism of genome-wide DNA demethylation in PGCs, we examined the expression and subcellular localization of key genes/proteins for DNA methylation/demethylation in PGCs, and re-visited the proliferation rate of PGCs, and the kinetics of imprint erasure and chromatin dynamics in PGCs. Strikingly, in contrast to the prevailing view, our data indicate that imprint erasure occurs in PGCs in a manner largely consistent with replication-coupled passive demethylation, necessitating the re-evaluation of the mechanism of genome-wide DNA demethylation in PGCs. Results Expression of genes involved in DNA methylation/demethylation in PGCs from E9.5 to E13.5 We have shown previously that PGCs repress genes encoding de novo DNA methyltransferases, Dnmt3a/3b, upon their specification (Yabuta et al, 2006; Kurimoto et al, 2008) and show low DNMT3A/3B at least until E12.5 (Seki et al, 2005). We also found that although PGCs express Dnmt1, they repress Uhrf1 (ubiquitin-like with PHD and ring finger domain 1), which encodes an essential factor to recruit DNMT1 to replication foci where maintenance DNA methylation takes place (Bostick et al, 2007; Sharif et al, 2007), at least at the mRNA level upon their specification (Kurimoto et al, 2008). We set out to obtain a more comprehensive view of the expression of genes involved in DNA methylation/demethylation during PGC development. We mated female mice (BDF1 background) with males bearing the Stella (also known as Dppa3/Pgc7)–EGFP transgenes (C57BL/6 background) (Payer et al, 2006; Seki et al, 2007), isolated Stella–EGFP-positive PGCs from embryos from E10.5 to E13.5 by fluorescence activated cell sorting (FACS), and purified mRNAs from these cells for microarray analysis (Materials and methods). We also analysed the gene expression data in the epiblasts at E5.75, in PGCs at E9.5, and, as a reference, during the in vitro PGC specification from embryonic stem cells (ESCs) (Hayashi et al, 2011). To validate the microarray analysis, we first looked at the expression of genes specific to PGCs: Blimp1 (also known as Prdm1) and Prdm14 were constantly detected in PGCs from E9.5 to E13.5, with a slight decrease in their expression at E13.5, Stella showed very high expression in PGCs from E9.5 to E13.5, and Mvh (mouse vasa homologue, also known as Ddx4) exhibited progressive upregulation from E9.5 to E13.5 (Figure 1). These data are consistent with previous findings (Tanaka et al, 2000; Chang et al, 2002; Saitou et al, 2002; Sato et al, 2002; Yamaji et al, 2008), corroborating the accuracy of our microarray analysis. We previously estimated that the expression levels of Blimp1 and Stella in PGCs were ∼100 and 1000 copies per cell, respectively (Yabuta et al, 2006; Kurimoto et al, 2008). Therefore, their arbitrary expression levels in the microarray analysis, roughly 1000 and 10 000, can be considered to correspond to 100 and 1000 copies per cell, respectively (Figure 1). Figure 1.Expression of key genes for DNA methylation/demethylation during PGC development in vivo and in vitro. Expression levels of housekeeping genes, genes specific to PGCs, and key genes involved/potentially implicated in DNA methylation/demethylation during PGC development in vivo (green plots: E5.75 epiblasts, E9.5, E10.5, and E11.5 PGCs of mixed sexes, and E12.5 and E13.5 male PGCs; red plots: E12.5 and E13.5 female PGCs) and in vitro (blue plots: male ESCs, male epiblast-like cells (EpiLCs) at day 2 of induction, and male PGC-like cells (PGCLCs) at day 6 of induction) as determined by microarray analysis. We used E9.5–E11.5 PGCs of mixed sexes because gene expression of PGCs is indistinguishable until E11.5 in both sexes (Jameson et al, 2012). Expression data of E5.75 epiblasts, E9.5 PGCs, ESCs, day 2 male EpiLCs and day 6 male PGCLCs were obtained from our previous publication (Hayashi et al, 2011) (GEO database accession number GSE30056). The vertical axis represents arbitrary expression levels determined by microarray analysis. The average values with s.d.'s (two independent experiments) are indicated. Download figure Download PowerPoint We next looked at the expression of Dnmts and Uhrf1. We found that Dnmt1 shows strong expression (roughly 300–400 copies per cell) in PGCs from E9.5 to E13.5, whereas Uhrf1 is acutely downregulated in PGCs and shows consistently low expression (around or <10 copies per cell) from E9.5 to E13.5 (Figure 1). Dnmt3a, 3b, and 3l were also downregulated and were present in consistently low quantities (around or <10 copies per cell) in PGCs from E9.5 to E13.5 (Figure 1). Combined with our previous findings (Seki et al, 2005; Yabuta et al, 2006; Kurimoto et al, 2008), these data demonstrate that the key genes implicated in maintenance and de novo DNA methylation are transcriptionally repressed in PGCs from their specification up until E13.5. We next looked at the expression of genes implicated in active DNA demethylation. Tet1 showed consistent expression in PGCs from E9.5 to E13.5 (around 100 copies per cell), whereas the expression of Tet2 was consistently low (around or <10 copies per cell) and Tet3 was undetectable (Figure 1). Aid and Apobec1 were also undetectable or expressed at very low levels (around or <10 copies per cell) in PGCs from E9.5 to E13.5 (Figure 1). Thymine DNA glycosylase (Tdg), which can remove T from T/G mismatches and also 5caC from 5caC/G pairs for BER (Gallinari and Jiricny, 1996; He et al, 2011), showed relatively high expression in early PGCs (∼around 100–200 copies per cell at E9.5) and was slightly downregulated thereafter ( 0.4 μm, relative staining intensity more than twice that of the nucleoplasm). As a control, we stained ESCs, which showed that DNMT1 localizes at PCNA-positive replication foci at the mid–late S phase (Figure 2C). As shown in Figures 2C and D, ∼90% of the gonadal somatic cells at the mid–late S phase from E10.5 to E13.5 exhibited localization of DNMT1 at the PCNA-positive replication foci, indicating that, in these cells, maintenance DNA methylation occurs in a replication-coupled manner, at least at late replicating heterochromatic foci (including DAPI-positive peri-centromeric heterochromatin) and most likely in other regions of the genome. In sharp contrast, only ∼10–20% and ∼30% of the PGCs at the mid–late S phase showed localization of DNMT1 at replication foci at E10.5–E11.5 and at E12.5–E13.5, respectively (Figures 2C and D). These findings suggest the possibility that a majority of PGCs fail to undergo/are inefficient for replication-coupled maintenance DNA methylation, at least at late replicating heterochromatic foci and presumably also in other regions of the genome, especially at E10.5 and E11.5, and hence erase their DNA methylation via replication-coupled passive DNA demethylation. Constant and rapid proliferation of PGCs after E9.5 Our previous study showed that a majority (∼60%) of PGCs migrating into the hindgut (E7.75–E8.75) are arrested at the G2 phase of the cell cycle, and when they come out into the mesentery after E9.5, they start rapid proliferation (Seki et al, 2007). To further explore the possibility of replication-coupled passive DNA demethylation in PGCs, we next set out to determine the cell-cycle distribution and proliferation of PGCs after E9.5. We mated females (ICR background) with males bearing the Stella–EGFP transgenes (C57BL/6 background), and injected BrdU into pregnant females at six different time points (E9.5, E10.5, E11.25, E11.5, E11.75, and E12.5). We isolated embryos and stained the dissociated embryonic cells containing PGCs for their BrdU incorporation and DNA contents, which were analysed by FACS. As shown in Figures 3A and B, Stella–EGFP-positive PGCs at E9.5 incorporated relatively little BrdU and ∼38, ∼30, and ∼32% of these cells were in the G1, S, and G2/M phases, respectively. In contrast, PGCs at E10.5 incorporated a high amount of BrdU and ∼23, ∼61, and ∼16% of these cells were in the G1, S, and G2/M phases. After E10.5, PGCs constantly incorporated a high amount of BrdU and ∼55% of these cells were in the S phase at least until E12.5 (Figures 3A and B). We did not observe a clear G2 arrest of PGCs around E11.5 (Figures 3A and B). Combined with our previous analysis (Seki et al, 2007), these findings indicate that some of the PGCs at E9.5 are still in the hindgut endoderm and are arrested at the G2 phase; and thereafter, the PGCs exhibit rapid proliferation at a relatively constant rate at least until E12.5. The Stella–EGFP-negative somatic cells, especially those of the genital ridges and mesonephros after E10.5, showed little BrdU incorporation and hence slow proliferation (Figures 3A and B). Figure 3.Cell-cycle state and proliferation of PGCs. (A) Representative plots for the cell-cycle state of PGCs (top) and somatic cells (bottom) after E9.5 as analysed by FACS. The vertical axis represents BrdU incorporation and the horizontal axis represents DNA content. Cells in S, G2/M, and G1 phase are shown in purple, blue, and red, respectively, along with the percentage of each. Representative data from three independent experiments are shown. (B) Plots showing the percentage of PGCs (green; E12.5 female PGCs are shown in red) and somatic cells (blue; E12.5 female somatic cells are shown in pink) after E9.5 in the G1, S, and G2/M phase. The average values from three independent experiments are shown with s.d.'s. (C) Plots showing PGC proliferation. The average values from three independent experiments are shown with s.d.'s. The regression line indicates that the doubling time of PGCs from E9.5 to E12.5 is ∼12.6 h. Download figure Download PowerPoint Based on the number of Stella–EGFP-positive PGCs analysed on FACS, we calculated the number of PGCs per embryo from E9.5 to E12.5 (see Materials and methods). Although the calculated number of PGCs per embryo at each stage varied to a certain extent (Table I), which was also the case in previous studies (Tam and Snow, 1981; Seki et al, 2007), the average number of PGCs at each stage showed a constant exponential increase (Figure 3C), indicating that PGCs proliferate at a constant rate from E9.5 to E12.5. From the slope of the regression line of the cell number plots, the doubling time of PGCs after E9.5 was calculated as ∼12.6 h, indicating that PGCs divide twice per day from E9.5 to E12.5. Table 1. Number of PGCs from E9.5 to E12.5 Experimental number Number of PGCs per embryo E9.5 E10.5 E11.5 E12.5 male #1 360 762 2027 8938 #2 170 410 3813 13 965 #3 100 688 1966 9030 Average 210 620 2602 10 644 Kinetics of imprint erasure in PGCs Our analysis has so far shown that during the relevant period for the genome-wide DNA demethylation, PGCs possess little maintenance and de novo DNA methylation potential, and divide more rapidly than previously thought (twice a day; doubling time, ∼12.6 h). We next determined the precise kinetics of the demethylation of the DMRs of imprinted genes by bisulphite sequence analysis. In this analysis, we consider it critical to discriminate the parental alleles, since the original methylation level in a cell is 50% and the bisulphite sequence analysis involves, prior to sequencing, many cycles of PCR amplification and random selection of the amplified clones, which can create ∼10–20% biases/fluctuations relatively easily and lead to misinterpretation of the results. For the discrimination of the parental alleles by single-nucleotide polymorphisms, we crossed C57BL/6 Stella–EGFP females with JF1 males (Koide et al, 1998), isolated PGCs from E10.5 to E13.5 (male PGCs for E12.5 and E13.5), and examined the methylation of the gametic DMRs of one paternally methylated imprinted gene (H19) and five maternally methylated imprinted genes (Nnat (also known as Peg5), Peg10, Snrpn, Peg3, and Kcnq1ot1 (also known as Lit1)) in these cells (Lee et al, 2002; Kobayashi et al, 2006; Tomizawa et al, 2011). The paternally methylated gene and the maternally methylated genes essentially lack DNA methylation of the DMRs on the maternal and paternal alleles, respectively, at all stages examined (Supplementary Figure S1), indicating successful discrimination of the parental alleles in our experiments. Our analysis revealed that, first, many DMRs analysed (those of H19, Nnat, Snrpn, Peg3, and Kcnq1ot1) (Figure 4A) exhibited a substantial erasure of imprints (∼30–∼50% erasure) as early as E10.5 (Figure 4B), indicating that in most imprinted genes, erasure of imprints commences prior to the PGCs’ entry into the genital ridges, most likely in migrating PGCs. Second, the demethylation rates of all the DMRs examined did not exceed those expected from the replication-coupled passive demethylation, considering that the doubling time of PGCs after E9.5 was ∼12.6 h (Figures 4B and C). Third, the demethylation rates of the DMRs examined were heterogeneous and the demethylation of the DMR of one maternally imprinted gene, Peg10, was especially slow and occurred in a similar fashion to that of intracisternal A particles (IAPs) (Figures 4B and C). Figure 4.Imprint erasure in PGCs. (A) The location and size of the DMRs (red bars) of one paternally (H19) and five maternally (Peg3, Nnat, Peg10, Snrpn, Kcnq1ot1) imprinted genes analysed in this study are shown. The location and size of the region analysed for the methylation of IAP are also shown (a red bar). The horizontal black bars represent genomic sequences and the black boxes on the bars represent exons of the genes. The arrows indicate the transcription start sites. The DMRs or a portion of the LTR of IAP analysed (red bars) are shown as enlarged bars with the locations of CpG dinucleotides (small horizontal bars). (B) The methylation states in PGCs from E10.5 to E13.5 (male PGCs at E12.5 and E13.5) of the DMRs of the methylated alleles of one paternally (H19) and five maternally (Peg3, Nnat, Peg10, Snrpn, Kcnq1ot1) imprinted genes and of the promoter/long terminal repeat (LTR) of IAPs (a mass population of essentially all IAPs) determined by bisulphite sequence. White and black circles represent un-methylated and methylated cytosine, respectively. The per cent methylation for each DMR/promoter at each stage is indicated. (C) Kinetics of demethylation of the DMRs/promoter analysed. The vertical axis represents the relative methylation rate of each DMR/promoter against the methylation rate of each DMR/promoter at E10.5 shown in a logarithmic scale. The horizontal axis represents the developmental stage. The demethylation kinetics in PGCs expected from purely passive demethylation with no maintenance or de novo methylation when the doubling time of PGCs is ∼12.6 h is shown by a diagonal bold brown line. Note that the demethylation rates of the DMRs/promoter analysed are slower than or close to that expected from the purely passive demethylation. Download figure Download PowerPoint These findings indicate that the erasure of gametic imprints initiates in migrating PGCs and proceeds at a rate that can be explained largely by replication-coupled passive demethylation, with gene-dependent partial resistances against demethylation. No apparent major chromatin changes in PGCs at around E11.5 Previous studies have documented that PGCs at around E11.5 are arrested at the G2 phase and undergo dynamic changes in the chromatin state, including nuclear enlargement, loss of chromocentres (intensively DAPI-stained foci), loss of linker histone H1, and loss of many histone modifications, including H3K27me3 (Hajkova et al, 2008, 2010). These changes have been proposed to reflect the genome-wide histone replacement involving NAP1 and HIRA following the genome-wide DNA repair for the genome-wide active DNA demethylation in PGCs (Hajkova et al, 2008, 2010). Since we did not detect the G2 arrest of PGCs at around E11.5 (Figure 3) and our data support the possibility of passive DNA demethylation in PGCs, we next examined whether the reported dynamic changes of the chromatin state could be observed in PGCs in the present study. First, we looked at the chromocentres in PGCs. From E9.5 to E12.5, PGCs consistently showed relatively weak DAPI staining throughout their nuclei, with less eminent chromocentres: The chromocentres in PGCs were difficult to observe and smaller in size than those in somatic cells at all the stages examined (Figure 5A and see also Figures 6A and C). We counted the number of detectable chromocentres (major axis >0.4 μm, relative staining intensity more than twice that of the nucleoplasm) in PGCs, but we did not find a specific change of morphology/distribution of chromocentres in PGCs at around E11.5 (Figure 5B). We estimated the
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