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• W2005756834 abstract "•Paternal DNA methylome remains stable until midblastula stage•Maternal methylome resets to a pattern similar to sperm by midblastula stage•Maternal DNA undergoes passive DNA demethylation and de novo methylation•Inheritance of the sperm DNA methylome facilitates embryogenesis 5-methylcytosine is a major epigenetic modification that is sometimes called “the fifth nucleotide.” However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo’s methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos. 5-methylcytosine is a major epigenetic modification that is sometimes called “the fifth nucleotide.” However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo’s methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos. Epigenetic modifications such as DNA methylation and histone modifications play critical roles during embryogenesis (Li et al., 1992Li E. Bestor T.H. Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3216) Google Scholar; Okano et al., 1999Okano M. Bell D.W. Haber D.A. Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4486) Google Scholar). However, knowledge of how much epigenetic information in gametes can be transferred to the offspring is limited. Recent studies show that epigenetic modifications from gametes in general are cleared and reestablished after fertilization (Blewitt et al., 2006Blewitt M.E. Vickaryous N.K. Paldi A. Koseki H. Whitelaw E. Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice.PLoS Genet. 2006; 2: e49Crossref PubMed Scopus (198) Google Scholar; Daxinger and Whitelaw, 2010Daxinger L. Whitelaw E. Transgenerational epigenetic inheritance: more questions than answers.Genome Res. 2010; 20: 1623-1628Crossref PubMed Scopus (199) Google Scholar, Daxinger and Whitelaw, 2012Daxinger L. Whitelaw E. Understanding transgenerational epigenetic inheritance via the gametes in mammals.Nat. Rev. Genet. 2012; 13: 153-162Crossref PubMed Scopus (308) Google Scholar; Feng et al., 2010bFeng S. Jacobsen S.E. Reik W. Epigenetic reprogramming in plant and animal development.Science. 2010; 330: 622-627Crossref PubMed Scopus (875) Google Scholar; Henderson and Jacobsen, 2007Henderson I.R. Jacobsen S.E. Epigenetic inheritance in plants.Nature. 2007; 447: 418-424Crossref PubMed Scopus (634) Google Scholar; Wu and Zhang, 2010Wu S.C. Zhang Y. Active DNA demethylation: many roads lead to Rome.Nat. Rev. Mol. Cell Biol. 2010; 11: 607-620Crossref PubMed Scopus (839) Google Scholar) except that a number of loci in some model organisms are resistant to the clearing (Arteaga-Vazquez and Chandler, 2010Arteaga-Vazquez M.A. Chandler V.L. Paramutation in maize: RNA mediated trans-generational gene silencing.Curr. Opin. Genet. Dev. 2010; 20: 156-163Crossref PubMed Scopus (101) Google Scholar; Cavalli and Paro, 1998Cavalli G. Paro R. The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis.Cell. 1998; 93: 505-518Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar; Morgan et al., 1999Morgan H.D. Sutherland H.G. Martin D.I. Whitelaw E. Epigenetic inheritance at the agouti locus in the mouse.Nat. Genet. 1999; 23: 314-318Crossref PubMed Scopus (1101) Google Scholar). However, this theory lacks evidence in the form of high-resolution epigenetic maps in oocytes, sperm, and early embryos. DNA methylation is one major epigenetic modification that is crucial for the development and differentiation of various cell types in an organism (Li et al., 1992Li E. Bestor T.H. Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3216) Google Scholar; Okano et al., 1999Okano M. Bell D.W. Haber D.A. Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4486) Google Scholar). In mammals, DNA demethylation occurs in the whole-genome level after fertilization, but not in some loci, such as intracisternal A particle (IAP) (Daxinger and Whitelaw, 2010Daxinger L. Whitelaw E. Transgenerational epigenetic inheritance: more questions than answers.Genome Res. 2010; 20: 1623-1628Crossref PubMed Scopus (199) Google Scholar; Wu and Zhang, 2010Wu S.C. Zhang Y. Active DNA demethylation: many roads lead to Rome.Nat. Rev. Mol. Cell Biol. 2010; 11: 607-620Crossref PubMed Scopus (839) Google Scholar). To further understand how offspring obtain DNA methylation information from parents, reduced representation bisulfite sequencing (RRBS) was used to achieve the most comprehensive genome-scale methylomes in mouse gametes and prespecified embryos (Smallwood and Kelsey, 2012Smallwood S.A. Kelsey G. De novo DNA methylation: a germ cell perspective.Trends Genet. 2012; 28: 33-42Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar; Smith et al., 2012Smith Z.D. Chan M.M. Mikkelsen T.S. Gu H. Gnirke A. Regev A. Meissner A. A unique regulatory phase of DNA methylation in the early mammalian embryo.Nature. 2012; 484: 339-344Crossref PubMed Scopus (731) Google Scholar), which explored the unique regulatory phase of DNA methylation in early mammalian embryos. Unfortunately, the RRBS method covers only 5% of the genome for the comparative analysis (Ball et al., 2009Ball M.P. Li J.B. Gao Y. Lee J.H. LeProust E.M. Park I.H. Xie B. Daley G.Q. Church G.M. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells.Nat. Biotechnol. 2009; 27: 361-368Crossref PubMed Scopus (795) Google Scholar; Harris et al., 2010Harris R.A. Wang T. Coarfa C. Nagarajan R.P. Hong C. Downey S.L. Johnson B.E. Fouse S.D. Delaney A. Zhao Y. et al.Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.Nat. Biotechnol. 2010; 28: 1097-1105Crossref PubMed Scopus (542) Google Scholar; Smith et al., 2012Smith Z.D. Chan M.M. Mikkelsen T.S. Gu H. Gnirke A. Regev A. Meissner A. A unique regulatory phase of DNA methylation in the early mammalian embryo.Nature. 2012; 484: 339-344Crossref PubMed Scopus (731) Google Scholar). The limited genome coverage in oocyte and early embryos prevents a full understanding of how much DNA methylation information is inherited and how it is transferred from sperm and oocyte to progenies, respectively. Moreover, the limited understanding extends to the biological purposes and outcomes of DNA methylation inheritance from gametes. Although the genome-wide DNA demethylation is a hallmark of mammalian embryogenesis, a controversy surrounds the question of whether such a phenomenon is general for all vertebrates. Some studies report the absence of genome-wide demethylation in zebrafish (Danio rerio) and Xenopus (Macleod et al., 1999Macleod D. Clark V.H. Bird A. Absence of genome-wide changes in DNA methylation during development of the zebrafish.Nat. Genet. 1999; 23: 139-140Crossref PubMed Scopus (111) Google Scholar; Veenstra and Wolffe, 2001Veenstra G.J. Wolffe A.P. Constitutive genomic methylation during embryonic development of Xenopus.Biochim. Biophys. Acta. 2001; 1521: 39-44Crossref PubMed Scopus (36) Google Scholar), but others argue for the existence of genome-wide demethylation in zebrafish embryos (MacKay et al., 2007MacKay A.B. Mhanni A.A. McGowan R.A. Krone P.H. Immunological detection of changes in genomic DNA methylation during early zebrafish development.Genome. 2007; 50: 778-785Crossref PubMed Scopus (40) Google Scholar; Mhanni and McGowan, 2004Mhanni A.A. McGowan R.A. Global changes in genomic methylation levels during early development of the zebrafish embryo.Dev. Genes Evol. 2004; 214: 412-417Crossref PubMed Scopus (98) Google Scholar). Here, we chose zebrafish as the model to measure DNA methylomes at single-base resolution in gametes and early embryos. We reveal that zebrafish inherit the DNA methylome from sperm. The zebrafish is a common model organism for vertebrate developmental studies. The annotated zebrafish genome is about 1.4 giga (G) bases, including 24.2 million CpG sites. Genetic polymorphisms (SNPs) would potentially interrupt the calling of methylation status of cytosines. Therefore, we performed whole-genome resequencing of the Tübingen (TU) strain (depth 22-fold) used in this study and identified about 11 million SNPs between our TU strain and the reference genome (Zv9, UCSC). Indeed, 1.2 million CpG sites are disrupted by SNPs in the TU genome. These sites were therefore excluded from further analyses. To explore how progenies inherit the DNA methylation information from parents, we collected both sperm and oocytes, as well as cleavage-stage embryos at the 16-cell, 32-cell, and 64-cell stages, the early-blastula stage at 128 cell, the midblastula stage (MBT) at 1,000 cell (or 1k cell), the gastrula stage at the germ ring, and testis from inbred TU strain. We generated single-base resolution methylomes in these samples with MethylC-seq (Lister et al., 2009Lister R. Pelizzola M. Dowen R.H. Hawkins R.D. Hon G. Tonti-Filippini J. Nery J.R. Lee L. Ye Z. Ngo Q.M. et al.Human DNA methylomes at base resolution show widespread epigenomic differences.Nature. 2009; 462: 315-322Crossref PubMed Scopus (3352) Google Scholar). The average genomic depth among these nine samples was 13-fold per strand (Table 1). We did not observe significant methylation at non-CpG sites in any stage of embryos (data not shown). Therefore, all subsequent analyses were focused on the CpG sites.Table 1Summary of Shotgun Bisulfite SequencingSampleStageGenome DepthsBS Conversion RateNumber of CG (1×)CG (1×) CoveredNumber of CG (5×)CG (5×) CoveredSpermgamete3199.53%22.0 M95.18%20.6 M89.25%Egg3599.73%22.1 M95.93%21.0 M90.89%16 cellcleavage1999.44%21.6 M93.76%18.9 M82.07%32 cell3299.89%22.1 M95.69%20.8 M90.19%64 cell1999.34%20.8 M90.19%17.8 M77.25%128 cellblastula2299.51%21.9 M95.00%19.9 M86.14%1,000 cell3899.53%22.0 M95.45%20.8 M90.15%Germ ringgastrula2099.57%21.7 M94.09%19.3 M83.41%Testisorgan2199.44%22.0 M95.27%20.1 M87.03%Paired reads were mapped uniquely to the reference genome (Zv9, UCSC) by Bismark. Number of CpG (1×) or number of CpG (5×) indicate total number of CpG sites mapped at least one read or five reads. Covered indicates proportion of mapped CpG sites over total CpG sites in genome. “M” indicates million. Open table in a new tab Paired reads were mapped uniquely to the reference genome (Zv9, UCSC) by Bismark. Number of CpG (1×) or number of CpG (5×) indicate total number of CpG sites mapped at least one read or five reads. Covered indicates proportion of mapped CpG sites over total CpG sites in genome. “M” indicates million. First, we analyzed the genomic features of the zebrafish methylome and calculated the methylation level of each CpG site across the genome. In this study, we considered only CpG sites that were covered at least ten times. In all samples, the majority of CpG sites are either highly methylated (methylation level between 75% and 100%) or unmethylated (less than 25%) (Figure S1A available online) (Lister et al., 2009Lister R. Pelizzola M. Dowen R.H. Hawkins R.D. Hon G. Tonti-Filippini J. Nery J.R. Lee L. Ye Z. Ngo Q.M. et al.Human DNA methylomes at base resolution show widespread epigenomic differences.Nature. 2009; 462: 315-322Crossref PubMed Scopus (3352) Google Scholar; Molaro et al., 2011Molaro A. Hodges E. Fang F. Song Q. McCombie W.R. Hannon G.J. Smith A.D. Sperm methylation profiles reveal features of epigenetic inheritance and evolution in primates.Cell. 2011; 146: 1029-1041Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Then, we checked the methylation features in various functional elements such as promoters, exons, and CpG islands (CGI). The results show that the unmethylated CpG sites are highly enriched in promoters and CGIs (Figure S1B). In general, the pattern of DNA methylation in functional regulatory elements in zebrafish is similar to that observed in mammals (Lister et al., 2009Lister R. Pelizzola M. Dowen R.H. Hawkins R.D. Hon G. Tonti-Filippini J. Nery J.R. Lee L. Ye Z. Ngo Q.M. et al.Human DNA methylomes at base resolution show widespread epigenomic differences.Nature. 2009; 462: 315-322Crossref PubMed Scopus (3352) Google Scholar; Molaro et al., 2011Molaro A. Hodges E. Fang F. Song Q. McCombie W.R. Hannon G.J. Smith A.D. Sperm methylation profiles reveal features of epigenetic inheritance and evolution in primates.Cell. 2011; 146: 1029-1041Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Next, we were interested in the dynamic change in the DNA methylome in zebrafish gametes and early embryos. We plotted the average DNA methylation level across the whole genome in gametes and early embryos. Surprisingly, the methylation level of oocytes is lowest among all examined samples, whereas sperm shows the highest methylation level (Figure 1A). Oocytes (average methylation level, 80%) are globally hypomethylated compared to sperm (91%). Interestingly, after fertilization, the methylation level appears to be stable in early cleavage stages of embryos compared to the predicted level in zygote, the mean level of sperm and oocyte. From 32 cell onward, the methylation level increases gradually to achieve a comparable level to sperm upon MBT (Figure 1A). For each pair of consecutive stages, we compared the methylation level of each CpG site and called it as differentially methylated CpG if the difference in methylation level exceeded 0.2 with p value < 0.05 according to Fisher’s exact test. About 14% of CpG sites are differentially methylated CpGs between sperm and oocyte genome wide (Figure 1B). The methylation levels of most CpG sites are stable during the transitions of consecutive stages (Figure 1B), but we identified significant changes between oocyte and 1,000-cell embryos (Figure 1C). The methylomes of oocyte and sperm are significantly different (Figure 1A). But there is no significant difference between sperm and 1,000-cell embryo (Figure 1B). To investigate the potential similarities of the methylation landscape of sperm and 1,000-cell embryo, we began our analysis from oocyte-specific methylated sites (mCpG percentage ≥ 75% in oocyte and ≤ 25% in sperm) and oocyte-specific unmethylated sites (mCpG percentage ≥ 75% in sperm and ≤ 25% in oocyte). We plotted their dynamic changes across all examined stages of embryos. The methylation levels of both oocyte-specific unmethylated sites and oocyte-specific methylated sites become intermediately methylated in 16-cell embryos, which is consistent with the knowledge that the average methylation level in early embryos (16-cell embryos) is close to the mean value of oocyte and sperm (Figure 1A). Interestingly, those sites exhibit a gradual change to the similar levels seen in sperm upon MBT stage (Figures 2A and 2B ). Similar results were also observed in the other differentially methylated sites between sperm and oocyte (Figures S2A and S2B). Sites with similar methylation levels between gametes do not exhibit significant changes during embryogenesis (Figures 2C, 2D, and S2C).Figure S2Zebrafish Progenies Reset to Sperm Methylome Pattern upon Specification to MBT Stage, Related to Figure 2Show full caption(A and B) Dynamic pattern of DNA methylation level for differentially methylated sites between gametes across early embryogenesis. Highly methylated in sperm and intermediate methylated in oocyte means methylation level ≥ 0.75 in sperm, and 0.25 < methylation level < 0.75 in oocyte. Unmethylated in sperm and intermediate methylated in oocyte means methylation level ≤ 0.25 in sperm and 0.75 > methylation level > 0.25 in oocyte.(C) Dynamic pattern of DNA methylation level for sites with similar intermediate methylation level in both gametes across early embryogenesis.(D) Graphical representation of methylation level of one CGI in gametes, 32-cell and 1,000-cell embryos. Green bar highlights the position of CGI. Line vertical height indicates the methylation level.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Dynamic pattern of DNA methylation level for differentially methylated sites between gametes across early embryogenesis. Highly methylated in sperm and intermediate methylated in oocyte means methylation level ≥ 0.75 in sperm, and 0.25 < methylation level < 0.75 in oocyte. Unmethylated in sperm and intermediate methylated in oocyte means methylation level ≤ 0.25 in sperm and 0.75 > methylation level > 0.25 in oocyte. (C) Dynamic pattern of DNA methylation level for sites with similar intermediate methylation level in both gametes across early embryogenesis. (D) Graphical representation of methylation level of one CGI in gametes, 32-cell and 1,000-cell embryos. Green bar highlights the position of CGI. Line vertical height indicates the methylation level. Analyses of the dynamics of CGIs further support this finding. CGIs are known as important functional genomic regions in the regulation of gene expression. The DNA methylation state of CGIs is also reprogrammed to the sperm methylation state upon MBT specification (Figures 2E, 2F, and S2D). Further analysis shows that genome-wide correlation of methylomes between sperm and early embryos becomes increasingly higher upon specification to the MBT stage (Figure 2G). In contrast, the change of correlation between methylomes of oocytes and early embryos presents an entirely reversed trend (Figure 2G). Additionally, six oocyte-specific methylated regions and ten oocyte-specific unmethylated regions were independently validated by bisulfite-sequencing PCR, which provided further proof that all the examined loci reset to sperm methylation state upon specification to MBT stage (Table S1). In summary, evidence of CpG sites, CGIs, and whole-genome correlation all support the idea that sperm defines the DNA methylome of early embryos upon specification to MBT. Upon specification to MBT, differentially methylated sites between sperm and oocyte are reset gradually to sperm methylation pattern, whereas from MBT onward, the methylome is further programmed (Figures 2A, 2B, 2E, and 2G) and becomes more and more different from sperm. But all the reprogramming to the somatic cell is based on the sperm methylation pattern, which is limited to specific regions. As a result, the methylome of somatic cells is still close to the sperm pattern. These data suggest that MBT is a transitional stage during DNA methylation reprogramming in zebrafish early embryogenesis. Next, we were interested in how the early embryo methylome becomes similar to sperm methylome upon the MBT stage. Our previous results show that there is no genome-wide DNA demethylation between gametes and early cleavage stages of embryos but that a significant number of sites are demethylated during early embryogenesis (Figure 1C). Previous reports show that the AID/Gadd45 enzyme mediates DNA demethylation in zebrafish (Rai et al., 2008Rai K. Huggins I.J. James S.R. Karpf A.R. Jones D.A. Cairns B.R. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45.Cell. 2008; 135: 1201-1212Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar). But, AID/Gadd45 has no activity before 4 hr postfertilization (Rai et al., 2008Rai K. Huggins I.J. James S.R. Karpf A.R. Jones D.A. Cairns B.R. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45.Cell. 2008; 135: 1201-1212Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar). Given that oocyte-specific methylated regions become totally unmethylated by the MBT stage (around 3 hr postfertilization), we chose not to pursue the potential role of AID/Gadd45 further in this work. Several recent studies have suggested that 5hmC may be involved in paternal DNA demethylation after fertilization (Inoue and Zhang, 2011Inoue A. Zhang Y. Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos.Science. 2011; 334: 194Crossref PubMed Scopus (393) Google Scholar; Iqbal et al., 2011Iqbal K. Jin S.G. Pfeifer G.P. Szabó P.E. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine.Proc. Natl. Acad. Sci. USA. 2011; 108: 3642-3647Crossref PubMed Scopus (550) Google Scholar). To explore whether 5hmC mediates DNA demethylation in zebrafish early embryos, we used a glycosylation-mediated enrichment method (Robertson et al., 2012Robertson A.B. Dahl J.A. Ougland R. Klungland A. Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads.Nat. Protoc. 2012; 7: 340-350Crossref PubMed Scopus (50) Google Scholar) to detect the genomic distribution of 5hmC in sperm, 2-cell, and 16-cell embryos. Our data show a very limited number of 5hmC-enriched regions (Table S2), none of which are associated with DNA demethylation regions, thereby suggesting that 5hmC does not mediate DNA demethylation in early embryos. To further confirm this result, we measured 5hmC in 32-cell embryos with single-nucleotide resolution using Tet-assisted bisulfite sequencing (TAB-seq) (Yu et al., 2012Yu M. Hon G.C. Szulwach K.E. Song C.X. Zhang L. Kim A. Li X. Dai Q. Shen Y. Park B. et al.Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.Cell. 2012; 149: 1368-1380Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). The data demonstrate a small number of 5hmC sites and no 5hmC enriched regions in 32-cell embryos (Table S3). Additionally, 5hmC is not detectable by immunofluorescence staining in zebrafish early embryos (Figure S3). Taken together, 5hmC does not mediate the DNA demethylation in zebrafish early embryos. Sperm and oocyte methylome are significantly different, but embryos reset to a methylome very similar to sperm upon MBT. To understand how differentially methylated regions (DMRs) between oocyte and sperm reprogram to the same state, we tracked the dynamic changes of oocyte-specific unmethylated loci. In this analysis, we picked up the paired reads covering at least four consecutive oocyte-specific unmethylated sites (mCpG percentage ≥ 75% in sperm and ≤ 25% in oocyte) from each stage and then calculated the methylation level of these reads. As expected, almost all paired reads in oocyte-specific unmethylated regions are highly methylated in sperm (Figure 3A, first panel) and are unmethylated in oocytes (Figure 3A, second panel). While in the cleavage 16-cell embryos, about half of the paired reads are unmethylated, and the other half are highly methylated (Figure 3A, third panel). This result is consistent with the previous finding that the average methylation level in early embryos (16-cell embryos) is close to the mean value of oocyte and sperm (Figure 1A). Similar findings were also revealed for paired reads, which covered at least four oocyte-specific methylated sites (mCpG percentage ≤ 25% in sperm and ≥ 75% in oocyte) in the 16-cell stage (Figure 3B first, second, and third panels). Two representative loci are shown in Figures 3C and S4A. Additionally, results of bisulfite-sequencing PCR show that the DNA methylation levels of both oocyte-specific methylated loci and unmethylated loci in 8-cell embryos are around 50% (Table S1). These results indicate that the DNA methylomes of 8-cell and 16-cell embryos result from the addition of sperm and oocyte methylome, thus suggesting that paternal DNA may maintain sperm methylome and maternal DNA may maintain oocyte methylome.Figure S4Oocyte Methylation Landscape Is Gradually Discarded during Early Embryogenesis, Related to Figure 3Show full caption(A) Dynamic changes of DNA methylation for a representative locus inchr19:12,944,645-12,944,716. Open circles represent unmethylated CpGs, and filled circles represent methylated CpGs.(B–G) All of 6 HOX genes clusters regions progressively reset to sperm methylation state. Line vertical height indicates the methylation level.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Dynamic changes of DNA methylation for a representative locus inchr19:12,944,645-12,944,716. Open circles represent unmethylated CpGs, and filled circles represent methylated CpGs. (B–G) All of 6 HOX genes clusters regions progressively reset to sperm methylation state. Line vertical height indicates the methylation level. To validate this, we tracked DNA methylation states of paternal and maternal DNA copies according to SNPs from crossed zebrafish, TU strain female with Tupfel long fin (TL) strain male. About two million homogenous SNPs were identified between TL and TU strains. The bisulfite-sequencing PCR method was used to examine the methylation states of 22 DMRs (Table S4). All of these loci contain SNPs, which can distinguish maternal DNA from paternal DNA. As expected, paternal DNA copies maintain the sperm methylation state, and maternal DNA copies maintain the oocyte methylation state in the 16-cell embryos. A representative locus is presented in Figure 3D. Collectively, our data demonstrate that paternal DNA copies maintain the sperm methylome and maternal DNA copies maintain the oocyte methylome, indicating that both paternal and maternal DNA use a DNA maintenance system to maintain the methylome until the 16-cell stage. Our mRNA expression data also demonstrate that maintenance methyltransferase DNMT1 is highly expressed across all of these early cleavage stages (Table S5), which is consistent with a previous report (Rai et al., 2006Rai K. Nadauld L.D. Chidester S. Manos E.J. James S.R. Karpf A.R. Cairns B.R. Jones D.A. Zebra fish Dnmt1 and Suv39h1 regulate organ-specific terminal differentiation during development.Mol. Cell. Biol. 2006; 26: 7077-7085Crossref PubMed Scopus (131) Google Scholar). Our data show that no whole-genome DNA demethylation occurs after zebrafish fertilization, a finding that goes against previous reports that have demonstrated whole-genome DNA demethylation (MacKay et al., 2007MacKay A.B. Mhanni A.A. McGowan R.A. Krone P.H. Immunological detection of changes in genomic DNA methylation during early zebrafish development.Genome. 2007; 50: 778-785Crossref PubMed Scopus (40) Google Scholar; Mhanni and McGowan, 2004Mhanni A.A. McGowan R.A. Global changes in genomic methylation levels during early development of the zebrafish embryo.Dev. Genes Evol. 2004; 214: 412-417Crossref PubMed Scopus (98) Google Scholar). Next, we were interested in how the DNA methylome is established after the 16-cell stage. Similar to the 16-cell stage, the majority of paired-reads are still either highly methylated or unmethylated in later stages (Figures 3A and 3B). Notably, both oocyte-specific unmethylated reads (Figure 3A) and oocyte-specific methylated reads (Figure 3B) genome wide gradually decrease after the 16-cell stage, and the majority of them are changed to the sperm methylation state upon MBT. For an oocyte-specific methylated locus, the proportion of the methylated reads also gradually decreases, and the locus becomes the sperm unmethylated state in 1,000-cell stage (Figure 3C). This result indicates that demethylation of this oocyte-specific highly methylated locus occurs through passive demethylation. For an oocyte-specific unmethylated locus, the proportion of the unmethylated reads also gradually decreases, and the locus is methylated in the 1,000-cell stage (Figure S4A), indicating that de novo methylation occurs in this locus. Our messenger RNA sequencing (mRNA-seq) results show that de novo methyltransferases DNMT3, 4, 5, and 7 (ma" @default.
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• W2005756834 title "Sperm, but Not Oocyte, DNA Methylome Is Inherited by Zebrafish Early Embryos" @default.
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