Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis

We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1^kd,3a^−/−,3b^−/−]). Sequencing of 960 RRBS clones from Dnmt[1^kd,3a^−/−,3b^−/−] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1^kd,3a^−/−,3b^−/−] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.     

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

Objective

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.

Design

We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.

Results

Mean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).

Conclusions

LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.     

A Prospective Study of LINE-1DNA Methylation and Development of Adiposity in School-Age Children

Background

Repetitive element DNA methylation is related to prominent obesity-related chronic diseases including cancer and cardiovascular disease; yet, little is known of its relation with weight status. We examined associations of LINE-1 DNA methylation with changes in adiposity and linear growth in a longitudinal study of school-age children from Bogotá, Colombia.

Methods

We quantified methylation of LINE-1 elements from peripheral leukocytes of 553 children aged 5–12 years at baseline using pyrosequencing technology. Anthropometric characteristics were measured periodically for a median of 30 months. We estimated mean change in three age-and sex-standardized indicators of adiposity: body mass index (BMI)-for-age Z-score, waist circumference Z-score, and subscapular-to-triceps skinfold thickness ratio Z-score according to quartiles of LINE-1 methylation using mixed effects regression models. We also examined associations with height-for-age Z-score.

Results

There were non-linear, inverse relations of LINE-1 methylation with BMI-for-age Z-score and the skinfold thickness ratio Z-score. After adjustment for baseline age and socioeconomic status, boys in the lowest quartile of LINE-1 methylation experienced annual gains in BMI-for-age Z-score and skinfold thickness ratio Z-score that were 0.06 Z/year (P = 0.04) and 0.07 Z/year (P = 0.03), respectively, higher than those in the upper three quartiles. The relation of LINE-1 methylation and annual change in waist circumference followed a decreasing monotonic trend across the four quartiles (P trend = 0.02). DNA methylation was not related to any of the adiposity indicators in girls. There were no associations between LINE-1 methylation and linear growth in either sex.

Conclusions

Lower LINE-1 DNA methylation is related to development of adiposity in boys.     

Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation

Postreplicative maintenance of genomic methylation patterns was proposed to depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core component of the replication machinery. We investigated how the slow and discontinuous DNA methylation could be mechanistically linked with fast and processive DNA replication. Using photobleaching and quantitative live cell imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in living cells showed that this interaction accounts for a 2-fold increase in methylation efficiency. Expression of this mutant in mouse dnmt1^−/− embryonic stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with the replication machinery enhances methylation efficiency, but is not strictly required for maintaining global methylation. The transient nature of this interaction accommodates the different kinetics of DNA replication and methylation while contributing to faithful propagation of epigenetic information.     

Lsh is involved in de novo methylation of DNA

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.     

LINE-1 Hypomethylation in Blood and Tissue Samples as an Epigenetic Marker for Cancer Risk: A Systematic Review and Meta-Analysis

Objective

A systematic review and a meta-analysis were carried out in order to summarize the current published studies and to evaluate LINE-1 hypomethylation in blood and other tissues as an epigenetic marker for cancer risk.

Methods

A systematic literature search in the Medline database, using PubMed, was conducted for epidemiological studies, published before March 2014. The random-effects model was used to estimate weighted mean differences (MDs) with 95% Confidence Intervals (CIs). Furthermore, subgroup analyses were conducted by sample type (tissue or blood samples), cancer types, and by assays used to measure global DNA methylation levels. The Cochrane software package Review Manager 5.2 was used.

Results

A total of 19 unique articles on 6107 samples (2554 from cancer patients and 3553 control samples) were included in the meta-analysis. LINE-1 methylation levels were significantly lower in cancer patients than in controls (MD: −6.40, 95% CI: −7.71, −5.09; p<0.001). The significant difference in methylation levels was confirmed in tissue samples (MD −7.55; 95% CI: −9.14, −65.95; p<0.001), but not in blood samples (MD: −0.26, 95% CI: −0.69, 0.17; p = 0.23). LINE-1 methylation levels were significantly lower in colorectal and gastric cancer patients than in controls (MD: −8.33; 95% CI: −10.56, −6.10; p<0.001 and MD: −5.75; 95% CI: −7.75, −3.74; p<0.001) whereas, no significant difference was observed for hepatocellular cancer.

Conclusions

The present meta-analysis adds new evidence to the growing literature on the role of LINE-1 hypomethylation in human cancer and demonstrates that LINE-1 methylation levels were significantly lower in cancer patients than in control samples, especially in certain cancer types. This result was confirmed in tissue samples, both fresh/frozen or FFPE specimens, but not in blood. Further studies are needed to better clarify the role of LINE-1 methylation in specific subgroups, considering both cancer and sample type, and the methods of measurement.     

The Prognostic Value of Global DNA Hypomethylation in Cancer: A Meta-Analysis

Background

Aberrant methylation of the global genome has been investigated as a prognostic indicator in various cancers, but the results are controversial and ambiguous.

Methods and Findings

This meta-analysis presents pooled estimates of the evidence to elucidate this issue. We searched the electronic databases: PubMed, Embase, ISI Web of Science and the Cochrane library (up to August 2013) to identify all of the relevant studies. The association between the level of surrogates'' indexes of genome-wide hypomethylation (LINE-1, Alu and Sat–α) and the overall survival (OS) of cancer patients was examined. In addition, the pooled hazard ratios (HRs) with their 95% confidence interval (95%CI) were calculated to estimate the influences through fixed-effects and random-effects model. Finally, twenty studies with total population of 5447 met the inclusion criteria. The results indicate that the summary HRs for the studies employing LINE-1, Alu, and Sat-α repetitive elements also show that the global DNA hypomethylation have significant desirable effects on the tumour prognostic value. The pooled HRs (and CIs) of LINE-1, Alu and Sat-α were 1.83 (1.38–2.44), 2.00 (1.16–3.45), and 2.92 (1.04–8.25), with a heterogeneity measure index of I² (and p-value) shows of 66.6% (p = 0.001), 57.1% (p = 0.053) and 68.2% (p = 0.076) respectively. The meta-regression and subgroup analysis indicated that the percentage of hypomethylated sample of cancer patients is one source of heterogeneity.

Conclusion

Our meta-analysis findings support the hypothesis that the global DNA hypomethylation is associated with a detrimental prognosis in tumour patients.     

5-Aza-2′-deoxycytidine Leads to Reduced Embryo Implantation and Reduced Expression of DNA Methyltransferases and Essential Endometrial Genes

Background

The DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation.

Methods and Principal Findings

Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type–specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5′ flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners.

Conclusions

The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.     

Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia

Tobacco smoking and reduced methylation of long interspersed element-1 (LINE-1) are crucial in oral carcinogenesis. 5′UTR of human LINE-1 sequence contains several CpG dinucleotides which are methylated in various proportions (0–100%). Methylation levels of many LINE-1s in cancer were reduced, hypomethylated. The hypomethylation of each LINE-1 locus can promote instability of genome and repress expression of a gene located on that same chromosome. This study investigated if cigarette smoking influences LINE-1 methylation of oral mucosal cells. The methylation of human LINE-1 in clinically normal oral mucosa of current smokers was compared to non-smokers. By using the combined bisulphite restriction analysis, each LINE-1 sequence was categorised into 4 patterns depending on the methylation status and location of the two 18-bp successive CpG from 5′ to 3′ including ^mC^mC, ^uC^uC, ^mC^uC and ^uC^mC. Of these, ^mC and ^uC represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of ^mC^mC and ^uC^uC were methylated and unmethylated, respectively. Nevertheless, some CpGs of each ^mC^uC or ^uC^mC allele were methylated. Imaging of the digestion products was used to generate %methylation value. No significant difference in the overall LINE-1 methylation level but the differences in percentages of some methylation patterns were discovered. The %^mC^mC and %^uC^uC increased, while the %^mC^uC decreased in current smokers (p = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower %^mC^uC still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The %^mC^uC also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered ^mC^uC to ^mC^mC and ^uC^uC forms, and changes ^uC^mC to ^uC^uC forms. In conclusion, smoking changes methylation levels of partial methylated LINE-1s and increased the number of hypo- and hypermethylated loci. These hypomethylated LINE-1s may possess carcinogenesis potential. Moreover, LINE-1 methylation patterns may be useful for monitoring oral carcinogenesis in smokers.     

Coordinate regulation of DNA methyltransferase expression during oogenesis

Background  

Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of Dnmt3a and Dnmt3b, as well as a regulator of DNA methylation, Dnmt3L, in isolated female germ cells.     

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1o^mat−/− mouse embryos born to Dnmt1^Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1^Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.     

PGC7, H3K9me2 and Tet3: regulators of DNA methylation in zygotes

In zygotes, a global loss of DNA methylation occurs selectively in the paternal pronucleus before the first cell division, concomitantly with the appearance of modified forms of 5-methylcytosine. The adjacent maternal pronucleus and certain paternally-imprinted loci are protected from this process. Nakamura et al. recently clarified the molecular mechanism involved: PGC7/Stella/Dppa3 binds to dimethylated histone 3 lysine 9 (H3K9me2), thereby blocking the activity of the Tet3 methylcytosine oxidase in the maternal genome as well as at certain imprinted loci in the paternal genome.DNA methylation is a crucial epigenetic modification that regulates imprinting (differential silencing of maternal or paternal alleles) and repression of retrotransposons and other parasitic DNA, as well as possibly X-chromosome inactivation and cellular differentiation. DNA methylation needs to be faithfully maintained throughout the life cycle, since loss of DNA methylation can result in gene dosage problems, dysregulation of gene expression, and genomic instability due to retrotransposon reactivation¹. Nevertheless, genome-wide loss of DNA methylation has been observed during germ cell development² and in the paternal pronucleus soon after fertilization³.For almost a decade, the global decrease of DNA methylation observed in the paternal genome within a few hours of fertilization was ascribed to an “active”, replication-independent process³. The maternal pronucleus is spared and instead undergoes “passive”, replication-dependent demethylation during early embryogenesis, arising from inhibition of the DNA maintenance methyltransferase Dnmt1 (Dnmt1 is normally recruited to newly-replicated DNA because of the high affinity of its obligate partner, UHRF1, for hemi-methylated DNA strands, which are produced from symmetrically-methylated CpG dinucleotides as a result of DNA replication). The basis for active and passive demethylation of the paternal and maternal genomes remained a mystery until proteins of the TET family – TET1, TET2 and TET3 in humans – were discovered to be Fe(II)- and 2-oxoglutarate-dependent enzymes capable of oxidizing 5-methylcytosine (5mC) in DNA^4,5,6. TET enzymes serially convert 5mC into 5-hydroxymethyl-cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC)^5,7,8.With the generation of specific antibodies to 5hmC, it became clear that the supposed “active demethylation” of the paternal pronucleus in mouse zygote after fertilization was due to the inability of anti-5mC antibodies to recognize 5hmC and other 5mC oxidation products^9,10. The enzyme responsible for 5mC oxidation was shown to be Tet3, which unlike Tet1 and Tet2 is highly expressed in mouse oocytes and zygotes. RNAi-mediated depletion of Tet3 decreased the staining of the paternal pronucleus with 5hmC, suggesting that immediately after fertilization, Tet3 in the zygote selectively oxidizes 5mC in the paternal genome to 5hmC^9,10.How is the maternal pronucleus protected from Tet3 activity? Nakamura et al.¹¹ previously showed that zygotes lacking PGC7/Stella/Dppa3 lose asymmetric regulation of DNA methylation, instead showing global loss of 5mC staining in both paternal and maternal pronuclei. This was correlated with hypomethylation at several maternally-imprinted loci (Peg1, Peg3, Peg10) in PGC7-deficient zygotes, as judged by bisulfite sequencing. Further, certain paternally-imprinted loci (H19, Rasgrf1), which are normally protected from global loss of methylation in the paternal genome, also became hypomethylated in PGC7-deficient zygotes. These data suggested that PGC7 protects the maternal genome, as well as certain paternally imprinted loci, from loss of 5mC.In their recent publication, Nakamura et al.¹² elegantly extended these findings to address the mechanism involved. Based on the fact that a major difference between maternal and paternal genomes is that the maternal genome contains histones, whereas the DNA of the entering sperm is tightly packaged with protamine, they asked whether PGC7 recognizes specific histone marks. Indeed, the maternal genome harbors considerable levels of the histone mark H3K9me2¹¹, leading them to examine whether PGC7 distinguishes maternal and paternal genomes by recognizing H3K9me2 in the maternal genome. Using wild-type (WT) ES cells and ES cells deficient in the G9a lysine methyltransferase which generates H3K9me2 mark, they showed that PGC7 associated loosely with nucleosomes and chromatin lacking H3K9me2, but tightly if H3K9me2 was present. The binding was recapitulated using recombinant bacterially-expressed PGC7 and histone tail peptides, indicating a direct interaction of PGC7 with the H3K9me2 mark. In agreement, genomic loci enriched with H3K9me2 recruited PGC7 as judged by chromatin immunoprecipitation (ChIP), but this recruitment was abrogated in G9a-deficient ES cells. These data indicated that PGC7 targets genomic regions occupied by nucleosomes containing H3K9me2 (Figure 1); an interesting extension would be to ask whether loss of maternal G9a also results in 5hmC conversion in the maternal pronucleus in zygotes.Open in a separate windowFigure 1Schematic view of paternal (left) and maternal (right) genomes soon after fertilization. Paternal and maternal pronuclei are indicated with immunostaining results in the boxes. PGC7 binds H3K9me2 in the maternal pronucleus and at certain paternally-imprinted loci (H19, Rasgrf1) in the paternal pronucleus, thereby potentially regulating chromatin organization to interfere with Tet3 accessibility.Next, Nakamura et al.¹² tested by immunocytochemistry whether PGC7 in zygotes also required H3K9me2. It is known that H3K9me2 staining is concentrated in the maternal but not the paternal pronucleus¹³. Using conventional staining methods in which the cells are first fixed and then permeabilized to allow antibodies to enter the cell, the authors observed in their earlier study that PGC7 bound to both pronuclei¹¹. Remarkably, by simply reversing the order of the fixation and permeabilization steps – permeabilizing first to allow the loss of loosely bound proteins by dissociation, then fixing and staining – they found that PGC7 associated much more tightly with the maternal pronucleus that bears H3K9me2 mark. Injection of mRNA encoding Jhdm2a (an H3K9me1/ me2-specific demethylase) into zygotes eliminated staining for H3K9me2 as well as PGC7 in the maternal pronucleus, and concomitantly caused loss of 5mC and acquisition of 5hmC. Taken together, these data strongly suggested that PGC7 was selectively recruited to the maternal pronucleus through binding H3K9me2, and that this binding protected zygotic maternal DNA from oxidation of 5mC to 5hmC and beyond (Figure 1).These findings led Nakamura et al. to investigate how PGC7 controls Tet3 activity in zygotes. They showed (in cells that were permeabilized before fixation and immunocytochemistry) that Tet3 was tightly associated only with the paternal pronucleus in WT zygotes, but was present in both pronuclei in PGC7-deficient zygotes. When PGC7 was prevented from binding to the maternal pronucleus by injection of Jhdm2a mRNA, Tet3 became tightly associated with both pronuclei. In other words, loss of PGC7 or loss of H3K9me2 that recruits PGC7 had the same effect – eliminating selective association of Tet3 with the paternal genome. The implication is that PGC7 – which preferentially binds the maternal genome – somehow promotes the selective binding of Tet3 to the paternal genome, thus permitting rapid 5mC oxidation in paternal but not maternal DNA (Figure 1).PGC7 is a small protein (150 amino acids (aa) in the mouse, 159 aa in humans) whose sequence is only moderately conserved. Nakamura et al.¹² showed that the binding of PGC7 to H3K9me2 required the N-terminal half of PGC7, whereas its ability to exclude Tet3 from the maternal pronucleus required the C-terminal half. It is unclear how Tet3 exclusion is mediated. One possibility is that the C-terminal region of PGC7 sterically excludes Tet3 from binding, either to DNA or to a chromatin mark; another is that the C-terminal region of PGC7 is capable of altering chromatin configuration to prevent the binding of Tet3 to chromatin. In support of the latter hypothesis, the rate with which micrococcal nuclease (MNase) digested high-molecular weight chromatin was significantly slower in WT ES cells in which PGC7 was present, compared to PGC7^−/− and G9a^−/− ES cells in which PGC7 was either absent or not recruited to DNA because of the loss of H3K9me2 mark. In contrast, DNA methylation did not alter the chromatin association of PGC7 or its ability to protect high-molecular weight chromatin from MNase digestion, as shown by using Dnmt1^−/−Dnmt3a^−/−Dnmt3b^−/− triple knockout ES cells that completely lack DNA methylation.How does PGC7 protect paternally-imprinted loci from Tet3-mediated 5mC oxidation? Although the haploid sperm genome is mostly packaged with protamine, a genome-wide analysis revealed that 4% of the genome of mature human sperm bears nucleosomes located at developmental and imprinted genes¹⁴. Nakamura et al.¹² found that among paternally-imprinted differentially methylated regions (DMRs), the H19 and Rasgrf1 DMRs contained H3K9me2 whereas the Meg3 DMR did not, consistent with their previous finding that in PGC7-deficient zygotes, the H19 and Rasgrf1 DMRs were hypomethylated but the Meg3 DMR was unaffected¹¹. Therefore, PGC7 may be recruited to paternally-imprinted loci through H3K9me2-containing nucleosomes that pre-exist in the sperm haploid genome upon fertilization. Alternatively, Nakamura et al. point out that protamine in the sperm is replaced soon after fertilization by the histone H3.3 variant, which in somatic cells does not bear H3K9me2 mark.In conclusion, Nakamura et al.¹² demonstrate unambiguously that PGC7 specifically binds to H3K9me2 in the maternal genome in zygotes, where its global occupancy excludes Tet3 and inhibits Tet3-mediated 5mC oxidation. This novel finding provides new insights into the global alterations of DNA methylation status that occur during early embryogenesis. Follow-up questions abound. First, can PGC7 protect other methylated loci such as transposable elements and the X-chromosome? It would be interesting to assess H3K9me2 at these loci. Second, how does the N-terminal half of PGC7 recognize H3K9me2? Structural characterization of this interaction may elucidate a novel epigenetic “reader” domain specific for H3K9me2. Third, PGC7 is a marker for cells of the inner cell mass, and is co-expressed with Tet1 and Tet2 rather than Tet3 in ESCs¹⁵. Does PGC7 also antagonize Tet1 and Tet2 and protect imprinted loci in ESCs? Fourth, how does PGC7 inhibit the access of Tet3 to chromatin? Considering that PGC7 is small and is not equipped with known enzymatic domains, it is likely that PGC-interacting proteins, rather than PGC7 itself, function to regulate chromatin status. Fifth, how is Tet3 recruited to paternal chromatin – are there specific histone or other epigenetic marks that facilitate Tet3 recruitment? Finally, while technically challenging, it seems imperative to identify the target genes of PGC7 and Tet3, by profiling the genomic location of 5hmC and other 5mC oxidation products in the paternal and maternal genomes of zygotes from WT, Tet3-deficient and PGC7-deficient mice.