The link between 5-hydroxymethylcytosine and DNA demethylation in early embryos.

EpigenomicsVol. 15, No. 6 EditorialOpen AccessOpen Access licenseThe link between 5-hydroxymethylcytosine and DNA demethylation in early embryosGerd P Pfeifer & Piroska E SzabóGerd P Pfeifer *Author for correspondence: E-mail Address: gerd.pfeifer@vai.orghttps://orcid.org/0000-0002-5080-9604Department of Epigenetics, Van Andel Institute, Grand Rapids, MI 49503, USASearch for more papers by this author & Piroska E Szabó https://orcid.org/0000-0001-9314-7009Department of Epigenetics, Van Andel Institute, Grand Rapids, MI 49503, USASearch for more papers by this authorPublished Online:16 May 2023https://doi.org/10.2217/epi-2023-0104AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: 5-hydroxymethylcytosineACE-seqepiblastgenome-wide DNA demethylationglobal reprogrammingH3K9me2Nlrp14 knockoutTET3Tet3 conditional knockoutzygoteAfter fertilization, mammalian genomes pass through a cycle of genome-wide DNA demethylation. A new study defines the prominent role of 5-methylcytosine (5mC) oxidation and the oxidized base 5-hydroxymethylcytosine (5hmC) in this process [1].Mammalian epigenomes undergo extensive global reprogramming during development (Figure 1). At the level of DNA methylation this remodeling occurs in two waves: a major phase of DNA demethylation immediately after fertilization, followed by a reacquisition of DNA methylation beginning at the blastocyst stage [2]. The second wave of global genome-wide DNA methylation remodeling occurs during development of primordial germ cells in the embryo, starting around 9 days after fertilization when loss of DNA methylation begins and continues through embryonic day 13.5 (E13.5). DNA methylation is then re-established and is almost complete by E18.5 in prospermatogonia but is delayed until after birth for oocyte development [2]. The biological meaning of these waves of methylation changes is unknown. It is thought that the demethylation cycles are essential for the soma–germline–soma transitions and resetting maternal and paternal genomic imprints, and for removing accumulated aberrations of the epigenome, thus preventing transgenerational inheritance of faulty information in the form of DNA methylation.The biological mechanisms leading to these extensive DNA demethylation events during development are beginning to be understood. Earlier work that used immunostaining methods showed that the demethylation events treat the paternally and maternally inherited genomes differently. After fertilization these changes primarily affect the more highly methylated paternal (sperm-derived) genome [3] and involve oxidation of the methyl group of 5mC by the 5mC dioxygenase TET3 [4–6]. TET enzymes in combination with replication-dependent passive demethylation remodel the epigenome in the germ cells [2,7,8]. The TET proteins produce 5hmC as their initial reaction product, which is readily observed in the paternal pronucleus of the fertilized oocyte [5]. However, there are open questions, such as the stability of the 5hmC mark, its potential biological meaning and the kinetics of the 5mC oxidation process. For example, it had remained unclear whether 5hmC is generated as an intermediate step during DNA demethylation or is formed following co-occurring de novo methylation events in zygotes [9,10].A new comprehensive study published in Nature Genetics earlier this year has provided the most detailed dataset on DNA hydroxymethylation and methylation in the zygote, in early embryos and in germ cells [1]. This work has also further established the mechanistic pathways involved [1]. In a tour de force study, Yan et al. characterized the genome-wide distribution of 5hmC at different stages of development in the maternally and paternally inherited chromosomes in wild-type and genetic model mice, such as Tet3 conditional knockout [11] and Nlrp14 knockout, thus providing clarification of mechanisms and the importance of this pathway in development [1].5hmC is a DNA base found at vastly different levels in various cell types. It is quite abundant in neuronal cells but in most somatic tissues its level is only a small fraction of that of 5mC [12]. Because of this, this DNA modification has been extremely difficult to map using high-throughput sequencing methods. Existing methods require highly active TET enzyme for in vitro oxidation of 5mC (TAB-sequencing) [13] or use chemical oxidation methods [14] which can cause DNA damage or may have an indirect readout. A breakthrough has been the development of a method called APOBEC-coupled epigenetic sequencing (ACE-seq) [15], a method which finally allows precise mapping of 5hmC even using limited numbers of cells. 5hmC bases are first glycosylated using a β-glucosyltransferase enzyme. This base modification protects 5hmC from deamination by APOBEC enzymes. The cytosine (C) and 5mC bases stay unprotected and are deaminated to uracil or thymine, respectively, by the deaminase. After PCR and sequencing, only the initial 5hmC bases will be read as a C base in the sequencing data. Yan et al. utilized ACE-seq throughout their study to obtain the following insights.They isolated male and female pronuclei from zygotes, early cleavage-stage embryos and primordial germ cells (PGCs) from different stages of embryonic development. Paternal and maternal genomes were distinguished by sequence polymorphisms. Yan et al. observed a sharp increase of 5hmC in the paternal pronucleus, as expected, but only a small increase in the female pronucleus (Figure 1). To determine which genomic positions depended on TET3 for 5hmC generation, a maternal Tet3 knockout model was used. The data showed that almost all 5hmC generated in the paternal (and maternal) genome depended on TET3. A smaller fraction of these TET3-dependent 5hmC loci also required DNA replication, in particular in the maternal genome. As the authors suggest, the dependency on replication may be caused by the need to dilute interfering histone modifications (e.g., H3K9me2) that block TET3 activity [16]. What remains somewhat of a mystery is the fate of the 5hmC generated in the paternal pronucleus. Does 5mC demethylation require additional enzymatic oxidation steps of TET3 to produce 5-formylcytosine (5fC) followed by 5-carboxylcytosine (5caC)? These modifications occur at extremely low levels in any cell type, either because they are rarely produced or because they are quickly removed by base excision repair [17]. In this regard, it is interesting to note that thymine DNA glycosylase, which initiates repair of 5fC and 5caC [17], is not expressed in zygotes, unlike TET3, which is highly expressed. Furthermore, mice with germline knockout of Tdg produce embryos with normal levels of DNA demethylation which proceed through early development [11]. This could mean there is an uncharacterized pathway that removes 5caC from DNA, perhaps by decarboxylation. Alternatively, only a small portion of 5hmC is further oxidized and 5hmC (plus the low amounts of 5fC and 5caC) is simply diluted by successive cell divisions going from the two-cell embryo to the blastocyst stage. Other reports have concluded that demethylated CpG sites arise independently of passive dilution from gametes to four-cell embryos [18]. However, the gradually diminishing levels of 5hmC during these stages, as reported by Yan et al. [1], are consistent with the first scenario and with earlier studies that used immunostaining for 5hmC [5,19].The authors followed the 5hmC patterns from the zygote pronuclei in the paternally and maternally inherited chromosomes. They found TET3-dependent hyper-hydroxymethylated regions (hyper-hmDMRs) that were specific to the male pronucleus and the paternal allele in two-cell embryos. On the other hand, hyper-hmDMRs detected in the female pronucleus were not unique to the maternally inherited chromosome of 5hmC-enriched regions in the paternal genome coincided with regions of DNA demethylation (i.e., conversion of 5mC to C), clearly establishing a link between 5mC oxidized sites and loss, but not gain, of DNA methylation at this time of development. Whereas 5hmC was not targeted to de novo methylated sites during global demethylation in the zygote, it was targeted there in the E6.5 epiblast during global de novo methylation. In PGCs, a loss of 5hmC was detected at sites that lost 5mC at the time of global demethylation, but 5hmC was targeted to de novo methylated sites in the fetal male germ cells at the time of global remethylation.Interestingly, 5hmC remained stable at many (∼10,000) male pronuclear hyper-hmDMRs even beyond preimplantation development. This suggests that 5hmC may have some importance other than as a demethylation intermediate in the early embryo. Motif analysis showed that 5hmC loci were enriched in the male pronuclei (and predominantly the paternal allele) in the two-cell embryo for binding sites of transcription factors RUNX2, ENFIL3, THRB, EBF1 and ATF. In E6.5 epiblasts, when an increase of 5hmC was observed, motifs of transcription factors active during gastrulation or organogenesis (OTX2, POU5F1, NANOG, ZIC2/3, T and MYC) showed enrichment in both parental alleles [1]. 5hmC was also mapped in in vitro culture model embryos which resemble E6.5 natural embryos [20]. Some of the hmDMRs detected in the E6.5 epiblast at developmental genes had less 5hmC in these embryos. These genes had reduced levels of expression, which may explain the reduced developmental potential of the embryos. In E12.5 PGCs, both parental alleles carried 5hmC at POU5F1, NANOG, SOX2 and PRDM14 sites. Fetal germ cells showed 5hmC enrichment at ZFX, ZBTB33, PRDM9 and TCF21 sites. It is likely that TET enzymes are preferentially recruited in the early embryo by transcription factors, as can also be observed during activation of lineage-specific enhancers [21,22].The genomic localization of 5hmC in the embryo was analyzed with respect to chromatin marks reported earlier. 5hmC was enriched at CpG islands and promoters, together with H3K4me3. In addition, a large fraction of the 5hmC sites were localized to gene bodies, together with the H3K36me3 histone mark. A depletion of H3K9me2 was observed at the majority of TET3-dependent hmDMRs in the maternal pronucleus, consistent with a protective role of this modification against TET3-mediated oxidation [16].Parental allele-specific DNA methylation at germline differentially methylated regions (gDMRs) determines the imprinted expression of a relatively small number of imprinted genes with importance to development. Sperm- or oocyte-specific imprints are set up in the male and female germlines. After fertilization, the imprints are maintained in the paternally or maternally inherited chromosomes in somatic cells. Imprints are erased between generations, and oxidation of 5mC is involved in this process. Yan and colleagues found that paternally inherited gDMRs have high levels of 5hmC in the E6.5 epiblast, which includes the newly forming primordial germ cells, and in E9.5 male PGCs [1]. Female PGCs have 5hmC at maternally inherited gDMRs at E11.5. What comes as a surprise is that gDMRs are not free of 5hmC after fertilization, when methylation is thought to be strictly maintained at those sequences. 5hmC is detected at maternally inherited gDMRs in the female pronucleus and also at paternally inherited gDMRs in the male pronucleus and two-cell embryos. 5hmC in the male pronucleus at paternally inherited DMRs is TET3-dependent. This brings the question of what role, if any, TET3 has in 5mC maintenance at gDMRs. It will be interesting to find out how gDMRs regain high 5mC levels after accumulating some 5hmC.Demethylation of the zygote genome not only requires extensive activity of TET3 but is also associated with a need to prevent remethylation of the genome by DNA methyltransferases. DNMT1, the DNA methyltransferase chiefly responsible for maintenance of DNA methylation (in addition to its ability of de novo DNA methylation), is excluded from the oocyte and zygote pronuclei [23]. The mechanism for retention of UHRF1, a cofactor of DNMT1, and to some extent DNMT1 itself in the cytoplasm involves the protein PGC7 (also known as STELLA or DPPA3). Loss of maternal PGC7 leads to hypermethylation of the zygotic genome [23]. In their new report, Yan et al. identified a new pathway for sequestering DNMT1 and UHRF1 in the cytoplasm at the zygote-to-two-cell stage and preventing DNA hypermethylation [1]. This pathway is also required for female fertility in the mouse. It was known that maternal effect mutations of genes encoding NOD-like receptors with a pyrin domain (NLRP2, NLRP5 and NLRP7) can lead to imprinting defects and DNA methylation abnormalities in humans [24]. Yan and colleagues found that among family members, Nlrp5 and Nlrp14 were highly expressed in mouse oocytes and zygotes, and their expression declined during cleavage stages [1]. Using a novel Nlrp14 knockout mouse, they found that embryos with maternal Nlrp14 deficiency could not proceed beyond the two-cell stage. These cells had abnormal nuclear localization of DNMT1 and increased 5mC in the paternal genome. By what mechanism does NLRP14 protein retain DNMT1 and its cofactor UHRF1 in the cytoplasm? While a role of NLRP14 has been described in spermatogenesis [25], it is currently unclear how this protein, and perhaps other members of this family, participate in excluding DNMT1 from the pronucleus/nucleus. Some family members (including NLRP2, NLRP5 and NLRP7) are components of the subcortical maternal complex (SCMC) [26,27]. Defects in the SCMC have been linked to human imprinting disorders [28]. Interestingly, knockout of Nlrp2 in mice leads to a detachment of DNMT1 away from its subcortical localization in oocytes to display a more diffuse localization in the cytoplasm, but it still shows weak staining in the nucleus [26]. It could be speculated that NLRP14 plays a similar role in the mouse SCMC by retaining DNMT1 and perhaps UHRF1 as strongly localized at the subcortical region.In addition to DNMT1 mislocalization and elevated 5mC levels, the maternal Nlrp14 mutant embryos also exhibited a reduced TET3-mediated 5mC oxidation, via unknown mechanisms. Regions that normally undergo TET3-dependent demethylation in the zygote showed reduced 5hmC in the two-cell embryo, especially in the paternal allele. DMRs with increased 5mC showed reduced 5hmC. This model suggests that enhanced de novo methylation by DNMT1 is not coupled to de novo formation of 5hmC by TET3 in zygotes. These embryos had a defect in zygotic genome activation from both parental chromosome sets, and the decay of maternal transcripts was also defective. Such misexpressions provide a possible explanation for the failed early development .Figure 1. Dynamic changes of 5-methylcytosine and 5-hydroxymethylcytosine during early mouse development.(A) 5hmC. (B) 5mC + 5hmC. The scales for 5hmC and the sum of 5mC and 5hmC are shown schematically based on data from reference [1] for 5hmC and references [18,29,30] for 5mC + 5hmC.5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; FGC: Female germ cells; MGC: Male germ cells.In summary, the new study has provided much-needed details about the intricate mechanisms of DNA methylation remodeling in early embryos. 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Cell Stem Cell 15(4), 459–471 (2014).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 15, No. 6 STAY CONNECTED Metrics History Received 21 March 2023 Accepted 24 April 2023 Published online 16 May 2023 Published in print March 2023 Information© 2023 Future Medicine LtdKeywords5-hydroxymethylcytosineACE-seqepiblastgenome-wide DNA demethylationglobal reprogrammingH3K9me2Nlrp14 knockoutTET3Tet3 conditional knockoutzygoteFinancial & competing interests disclosureResearch in the authors’ labs is supported by NIH grants AR079174 (to G Pfeifer) and GM143308 (to P Szabó). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download