Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.

In the current study, we have revisited this transition and embarked on a comprehensive, time-resolved experimental and mathematical approach to reveal and verify the kinetics and interplay between the different components of the DNA demethylation machinery and to unravel the mechanistic regulation of the main pathway responsible for genome-wide DNA demethylation.

In vivo, acquisition of pluripotency in both primordial germ cells (PGCs) and the early embryo coincides with genome-wide epigenetic reprogramming of histone modifications and DNA hypomethylation (). During the in vitro transition of ESCs from serum to 2i, the epigenome is also globally reprogrammed, with loss of H3K27me3 at repressed promoters, but only minor changes in H3K4me3 and H3K9me3 (). Recently, we and others showed that the genome of 2i ESCs is globally hypomethylated and as such similar to the cells in the ICM, whereas serum ESCs are hypermethylated (). To date, the molecular mechanisms regulating this conserved () genome-wide demethylation in the naive state are unclear.

Pluripotency describes the transient embryonic potential to form all embryonic germ layers and the germline, excluding the extra-embryonic tissues (). This state can be recapitulated in vitro in mouse embryonic stem cells (ESCs) derived from the inner cell mass (ICM), maintaining their pluripotent, self-renewing state and the ability to contribute to chimeric embryos (). Dictated by the culture conditions, mouse ESCs can adopt two interconvertible states resembling two slightly different in vivo developmental stages. While originally mouse ESCs were grown in serum/leukemia inhibitory factor (LIF)-containing media (“serum ESCs”), recently, serum-free culture conditions have been established that favor derivation and propagation of mouse ESCs in the absence of serum (). These conditions rely on specific inhibition of GSK3beta and Erk1/2 and downstream signaling by two small-molecule inhibitors (“2i ESCs”). 2i ESCs transcriptionally closely resemble cells from the ICM (), and serum ESCs tend to phenocopy cells from the early epiblast and show a greater heterogeneity and differential expression of pluripotency and differentiation factors, resulting in overt and spontaneous differentiation if not held back by LIF (). Accordingly, the state of 2i ESCs has been designated the “ground- or naive-state” of pluripotency, and cells grown in 2i are believed to be a much better representation of the cells from the ICM, compared to “primed” serum ESCs ().

Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis.

Finally, we asked how H3K9me2 levels were regulated during the serum-to-2i conversion. We quantified mRNA levels of key H3K9 methylases and demethylases by RNA-seq ( Figure S5 H) and protein levels by mass spectrometry ( Figure 5 G). Interestingly, we found that several H3K9 demethylases, including KDM3A, KDM3B, and KDM4A, were upregulated specifically at the protein level in 2i conditions. Conversely, a number of H3K9 methylases, such as EHMT1 (GLP) and EHMT2 (G9A), were downregulated.

Next, we generated RRBS libraries of Uhrf1and corresponding wild-type control ESCs during the serum-to-2i conversion ( Figure S5 C) to identify regions that are only maintained in the presence of UHRF1. Computational analyses of these and E14, Tet1/2/3 KO, and wild-type control lines identified a number of genomic regions resistant to DNA demethylation in wild-type cells, but not upon deletion of Uhrf1 ( Figure S5 D; blue cluster), indicating strong locus specific recruitment. These regions are enriched for H3K9me2 in 2i ESCs and for H3K9me3 in 2i and serum ESCs ( Figures S5 E and 5 F). We next measured 5mC levels over H3K9me2- ( Figure 5 F) and H3K9me3-enriched ( Figure S5 G) regions () and found that these regions retain high levels of DNA methylation in 2i ESCs in an UHRF1-dependent manner.

To understand any locus-specific effects of the loss of H3K9me2, we performed H3K9me2 chromatin immunoprecipitation sequencing (ChIP-seq) analysis. While the overall pattern of H3K9me2 enrichment did not change during the serum-to-2i conversion, regions with low H3K9me2 enrichment further flattened out ( Figure S5 B). To correlate the presence of H3K9me2 with the levels of 5mC, we performed H3K9me2 chromatin immunoprecipitation followed by bisulfite sequencing (ChIP-BS-seq) (). The 5mC levels in serum ESCs as well as 2i ESCs were significantly increased in the H3K9me2-bound DNA when compared to corresponding input samples ( Figure 5 D). We then divided the genome into regions with high or low H3K9me2 enrichment and found a significant increase in the 5mC levels at H3K9me2-high regions ( Figure 5 E).

The drastic impairment of recruitment of UHRF1 to replication sites was surprising, as the levels of UHRF1 were reduced albeit still detectable in 2i conditions. The recruitment of UHRF1 depends on the recognition of both hemimethylated CpGs via its SRA domain and of methylated lysine 9 on histone 3 (H3K9) through its Tudor domain (). Loss of H3K9 methyltransferase G9a results in loss of global DNA methylation (), and similarly, it has been concluded that loss of H3K9 methylation, in particular H3K9me2, is an additional key step leading to genome-wide demethylation in PGCs (). H3K9me3 localization is relatively unchanged between serum and 2i ESCs and mainly enriched at repetitive elements (), but the importance of loss of H3K9me2 is not clear (). We found a significant reduction in the levels of H3K9me2 upon serum-to-2i conversion (∼2-fold reduction) ( Figures 5 A, 5B , and S5 A). Interestingly, loss of H3K9me2 was rapid, and we detected a weak correlation by IF between the global levels of H3K9me2 and DNA methylation in serum ESCs ( Figure 5 C), in line with the notion that H3K9me2-dependent recruitment of UHRF1 plays a key role in DNA methylation maintenance in ESCs, possibly explaining the drastic impairment of recruitment of UHRF1 to the replication foci.

(G) Relative protein levels of known H3K9 modifiers detected by protein mass spectrometry (label-free quantification [LFQ]) at different time points during serum-to-2i conversion. Levels are normalized to serum ESCs.

(F) Levels of 5mC, determined by RRBS at H3K9me2 enriched regions during the serum-2i transition in E14, Uhrf1 WT, and Uhrf1 KO ESCs.

(D) 5mC levels in H3K9me2-bound DNA and corresponding input samples from serum and LT 2i ESCs, measured by H3K9me2-ChIP-BS-seq.

(C) Correlation of the quantified signals of H3K9me2 and 5mC in immunofluorescence staining of serum, 24-hr 2i, and LT 2i ESCs. The dashed line represents the linear regression between H3K9me2 and 5mC signals in serum ESCs.

The rapid, though not complete, loss of UHRF1 protein in 2i conditions prompted us to ask whether recruitment of the DNA maintenance methylation machinery to the replication forks was impaired. Previous IF experiments in cells lacking Uhrf1 proved that the presence of UHRF1 is absolutely required for recruitment of DNMT1 to replication foci (). We labeled replication foci by incubating serum or 2i ESCs for 8 min prior to fixation with 5-ethynyl-2′-deoxyuridine (EdU), which is incorporated into newly synthesized DNA. Co-staining for UHRF1 and EdU allowed us to count the number of cells with EdU-positive foci for co-localization of UHRF1 with replication foci. While UHRF1 co-localizes with EdU-positive replication foci in the majority of cells in serum conditions, this number is significantly reduced under 2i conditions ( Figure 4 F). The recruitment of UHRF1 to EdU-positive replication foci is further reduced with time in 2i. These results suggest that DNA demethylation in the serum-to-2i conversion is a consequence of impaired DNA methylation maintenance through impaired recruitment of DNMT1 by UHRF1 to the replication fork.

To confirm whether the regulation of UHRF1 at the protein level was also relevant in human ESCs, we assessed mRNA and protein levels in conventional and naive () human ESCs by immunofluorescence (IF). Similar to our observations in mouse ESCs, UHRF1 and DNMT1 mRNA levels are not changed between the two states ( Figure S4 F), but UHRF1 protein levels were significantly reduced (∼2-fold reduced) in the naive cells ( Figure S4 G), pointing to a conserved regulation of UHRF1 in naive ESCs.

In primordial germ cells, maintenance methylation was reported to be impaired in part by nuclear exclusion of UHRF1 (). We analyzed the subcellular localization of UHRF1 and DNMT1 in ESCs grown in serum and 2i ( Figure 4 A) and did not detect any nuclear exclusion of either UHRF1 or DNMT1 in ESCs. However, we observed a reduction in the signal intensity of UHRF1 protein in the 2i samples. Our initial transcriptomic analysis of the cells undergoing serum-to-2i transition ( Figure 1 B) did show that the expression of Dnmt3a and Dnmt3b, together with the catalytically inactive regulatory isoform Dnmt3l, was substantially reduced in 2i, while Uhrf1 mRNA levels were unchanged. In contrast, UHRF1 protein levels were significantly reduced (∼3-fold by western blot; 2-fold by quantitative mass spectrometry) in 2i ESCs ( Figures 4 B–4D), as were the levels of the de novo DNMTs ( Figure S4 A). We found heterogeneous expression of UHRF1 in serum and 2i ESCs ( Figure 4 C), which did not correlate with the expression of the pluripotency marker NANOG ( Figure S4 B) but can be attributed to the cell-cycle-dependent regulation of UHRF1 (). To further confirm the observation that UHRF1 is regulated at the protein level, we generated an ESC line with constitutive overexpression of an UHRF1-GFP fusion protein. Similar to our observations on endogenous UHRF1, fluorescence-activated cell sorting (FACS) analysis of the UHRF1-GFP cell line showed that UHRF1-GFP was expressed in serum ESCs but was rapidly lost upon serum-to-2i conversion ( Figures 4 E and S4 C), while mRNA levels of endogenous Uhrf1 and exogenous Uhrf1-Gfp remained stable ( Figure S4 D). Interestingly, we also observed a rapid increase of UHRF1-GFP protein levels upon transfer back to serum growth conditions ( Figure S4 E), suggesting that the changed signaling environment in 2i specifically affects UHRF1 protein stability.

(F) Percentage of replication foci with co-localization of UHRF1 in serum, 24-hr 2i and LT 2i ESCs. Images from serum ESCs showing examples for the two quantified states; arrows indicate EdU+ replication foci.

(E) FACS analysis of UHRF1-GFP fusion protein in ESCs during serum-to-2i conversion. Histograms show the GFP signal intensity at different time points during the conversion. The black arrow depicts the threshold used to quantify the percentage of GFP+ cells, shown in the line graph. Error bars indicate mean ± SD from three biological replicates.

(D) Protein levels of UHRF1 detected by protein mass spectrometry (label-free quantification [LFQ]) at different time points during serum-to-2i conversion, relative to serum ESCs. Error bars indicate mean ± SD from two biological replicates.

(B) Western blot analysis for UHRF1 protein in serum and LT 2i ESCs. Relative quantifications of the signal intensities of the bands on the western blot are shown in the bar chart.

(A) Cellular localization of UHRF1 (green) and DNMT1 (red) in mouse PGCs (E11.5), serum ESCs, 24-hr 2i ESCs, and long-term (LT) 2i ESCs. DAPI (blue) labels the nucleus. The RGB profiles show the signal intensity for each pixel along the horizontal yellow line.

Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry.

The mathematical model had predicted that maintenance methylation is significantly impaired and a major driver of the DNA demethylation observed ( Figure 1 F). Subsequently, we confirmed the rapid demethylation upon deletion of Dnmt1 or Uhrf1 ( Figure 2 B) and also showed that loss of de novo methylation does not explain the demethylation dynamics observed ( Figures 2 C and 2D). In order to understand the mechanistic regulation of this impairment, we focused on the individual components of the maintenance methylation machinery, in particular on the role of Uhrf1.

Finally, we analyzed the methylome in Tet1/2/3 KO cells. Using k-means clustering on average DNA methylation over 500-bp tiles across the genome, we found a cluster of tiles (n = 3,770) that maintains methylation in Tet1/2/3 KO cells, but not in the corresponding wild-type control cells ( Figure 3 D). These regions have higher 5mC levels in the Tet1/2/3 KO cells, but we could not identify any significant functional enrichment associated with them ( Table S2 ).

Having established that exclusively in the presence of vitC TET proteins contribute to global loss of 5mC in the serum-to-2i conversion, we asked whether TET-dependent hydroxymethylation is essential for locus specific demethylation. We performed TAB-seq in parallel to WGBS on the same DNA during the early phase of the time course in the presence or absence of vitC ( Figure S3 B) as well as RRBS from Tet1/2/3 KO cells and corresponding wild-type control ESCs during different time points of the serum-to-2i conversion ( Figure S3 C). These datasets confirmed that TET-dependent hydroxymethylation does not significantly contribute to global demethylation, unless their activity was enhanced by vitC (∼3-fold increase in 5hmC in the first 32 hr). We analyzed the distribution of 5mC and 5hmC over functionally distinct genomic regions with 5hmC enrichment in serum ( Figure 3 C) and stratified promoters into high-, intermediate-, and low-CpG-density promoters (HCP, ICP, and LCP, respectively) (). The majority of HCPs (n = 3,121) are very low methylated in serum, and only a small subgroup are intermediate (n = 143) or intermediate-low (n = 563) methylated, containing some germ cell-specific genes (e.g., Dazl, Prdm14, and Dppa3) ( Figure S3 D). In the absence of vitC, we observe only minor if any change in 5mC/5hmC levels over any of the three classes of promoters. In the presence of vitC, however, conversion of 5mC to 5hmC is already apparent as early as 4 hr, and the speed of 5mC loss correlates with CpG density and is different for each class. The kinetics of 5mC to 5hmC conversion and subsequent loss of 5hmC (73% loss) is fastest at HCPs, while ICPs show less dramatic changes (32% loss), with substantial loss of 5hmC being apparent only later. At LCPs, 5hmC slowly accumulates over the time period investigated. Enhancers, defined as elements overlapping H3K4me1, H3K27ac, and DNaseI hypersensitivity and excluding transcription start sites (TSS) (±2 kb) in E14 serum ESCs, show 10%–40% 5mC levels in serum ESCs, in line with previous studies showing that enhancers have low abundance of 5mC () and follow kinetics similar to ICPs. The largest part of the genome behaves similar to LCPs; conversion to 5hmC and subsequent erasure is very slow ( Figure 3 C).

The increased levels of 5hmC during the serum-to-2i conversion as well as publications showing further TET-dependent hypomethylation in vitamin C (vitC)-treated 2i ESCs () raised the possibility that TET proteins could contribute to the observed demethylation dynamics. We determined the 5mC and 5hmC levels of ESCs at several time points during the serum-to-2i conversion in the presence or absence of vitC ( Figure 3 A) and observed a significant increase in 5hmC upon vitC treatment, which resulted in an accelerated rate of demethylation and further global hypomethylation. To confirm that this effect was TET dependent, we measured the levels of 5mC and 5hmC in Tet1/2 KO cells in the presence or absence of vitC ( Figure 3 B), showing that the increased rate of demethylation (and hydroxymethylation) is dependent on the activity of TET proteins. Using the values of pand pfrom E14 ESCs ( Figure 1 F), we estimated an ∼4-fold increase of active demethylation (p) in the presence of vitC. Mathematical modeling of the dynamics of 5hmC accurately predicts the observed 5hmC levels during the serum-to-2i conversion in the presence of vitC ( Figure S3 A).

(D) Average methylation levels over 500-bp tiles in Tet1/2/3 KO cells and corresponding control cells (Tet WT). Tet-dependent regions were identified using k-means clustering.

(C) Distribution of 5mC (top) and 5hmC levels (bottom) during the first 32 hr of reprogramming from serum (gray) to 2i (green) or 2i+vitC (orange) measured by WGBS and TAB-seq over high-CpG promoters (HCP), intermediate-CpG promoters (ICP), low-CpG promoters (LCP) and enhancers. For each class of genomic element, only the subset having 5hmC enrichment in serum (T = 0 hr) was considered. The horizontal line within the box plots represents the median.

(B) Percentage of 5mC (top) and 5hmC (bottom) as measured by LC-MS in Tet1/2 KO during serum-to-2i transition in the absence (gray line) or presence of vitC (green line). Error bars indicate mean ± SD from three biological replicates.

(A) Percentage of 5mC (top) and 5hmC (bottom) as measured by LC-MS in E14 during serum-to-2i transition in the absence (blue line) or presence of vitC (red line). Error bars indicate mean ± SD from three biological replicates.

All mutant ESCs tested displayed global loss of 5mC upon serum-to-2i conversion. We specifically compared the rate of demethylation in ESCs lacking either Dnmt1 or Uhrf1 with control ESCs and observed an increased rate of demethylation ( Figure 2 B), showing that loss of DNA methylation maintenance results in increased demethylation rates. This supports the prediction from the mathematical model (dotted lines in the colored boxes) and implicates a failure of DNA methylation maintenance in 2i ESCs, albeit not a complete loss. Next, we compared the demethylation kinetics in ESCs lacking Dnmt3a and Dnmt3b. Interestingly, deletion of Dnmt3a/b in serum grown ESCs results in only a marginal decrease in the genomic level of 5mC ( Figure 2 C), and the kinetics of DNA demethylation are unaltered in the serum-to-2i conversion ( Figure 2 D), showing that loss of de novo methylation is not responsible for global loss of DNA methylation. Finally, we assessed the contribution of enzymes involved in active demethylation pathways in the serum-to-2i conversion. As predicted by the model, ESCs lacking Tet1/2/3, Tdg, or Aicda showed strikingly similar demethylation dynamics to their wild-type control counterparts ( Figures 2 E, S2 A, and S2B). Since TET-driven oxidation has been previously suggested as a potential driver of demethylation in the serum-to-2i conversion, we confirmed the loss of 5hmC in Tet1/2/3 knockout (KO) cells ( Figure 2 F). This shows that the TET enzymes are actively oxidizing 5mC during the serum-to-2i conversion but are neither sufficient nor necessary for global DNA demethylation.

To validate and fully understand the contribution of the individual DNA methylation and demethylation enzymes in the genome-wide epigenetic reprogramming that characterizes the transition from serum to 2i ESCs, we examined the dynamics of this loss of methylation in mouse embryonic stem cells in which one or more of the components of the DNA methylation machinery had been deleted. To this end, we determined the DNA methylation state at several time points in serum and during the transition from serum to 2i ESCs ( Figure 2 A) with inducible deletion of Dnmt1 (Dnmt1); Uhrf1 (Uhrf1); Dnmt3a/b (Dnmt3a× Dnmt3b) or constitutive deletion of Aicda (Aicda); Tdg (Tdg); Tet1/2 (Tet1× Tet2); Tet1/2/3 (Tet1× Tet2× Tet3) and in corresponding wild-type control ESCs and compared these to the predictions of our model.

(F) Levels of 5hmC in Tet1/2/3 KO and controls during the serum-to-2i transition measured by mass spectrometry. Error bars indicate mean ± SD from three biological replicates.

(E) Levels of 5mC in Tet1/2/3 KO and controls during the serum-to-2i transition measured by mass spectrometry. The colored box shows the overlay between the mathematical model prediction (dotted line) with p 3 = 0% and the normalized measured 5mC data. Error bars indicate mean ± SD from three biological replicates.

(D) Levels of 5mC in inducible Dnmt3a/b KO during the serum-to-2i transition. The colored box shows the overlay between the mathematical model prediction (dotted line) with p 1 = 0% and the normalized measured 5mC data. Error bars indicate mean ± SD from three biological replicates.

(C) Levels of 5mC in inducible Dnmt3a/b KO after induction of deletion in serum media measured by mass spectrometry. The colored box shows the overlay between the mathematical model prediction (dotted line) with p 1 = 0% and the normalized measured 5mC data. p 2 ≈ 90% since the cells were maintained in serum. Error bars indicate mean ± SD from three biological replicates.

(B) Levels of 5mC in Uhrf1 and Dnmt1 KO ESCs during the serum-to-2i transition, measured by mass spectrometry. The colored box shows the overlay between the mathematical model prediction (dotted line) with p 2 = 0% and the normalized measured 5mC data. Error bars indicate mean ± SD from three biological replicates.

(A) Schematic representation of the experimental setup. Mutants for candidate genes related to global demethylation in the serum-to-2i conversion were initially grown in serum medium. At day 0, culture medium was switched to 2i. Cells were harvested after indicated times. DNA was extracted and analyzed for 5mC and 5hmC levels by LC-MS and/or RRBS.

To dissect the role and relative contribution of the three pathways and the various regulatory factors involved, we used mathematical modeling to predict DNA demethylation throughout the time course. Since the first population-epigenetic models for DNA methylation dynamics were published (), several studies were undertaken to improve the predictions by using different approaches and incorporating new biological concepts into the models (). Due to the lack of adequate experimental data describing DNA methylation changes genome-wide, previous descriptive and predictive models could not be fuelled with accurate input values and precise estimates of the parameters. To overcome this obstacle and to obtain accurate input values, we performed genome-wide hairpin bisulfite sequencing () and combined these with our other sequencing datasets. We calculated the percentages of fully methylated CpG dyads (mCpG/GpCm), hemi-methylated CpG dyads (mCpG/GpC), and unmethylated CpG dyads (CpG/GpC) ( Figure S1 D; Table S1 ) as well as the levels of hydroxymethylated CpGs from TAB-seq data and hairpin bisulfite sequencing. These input values along with the global 5mC values from LC-MS were used to estimate the following parameters, which are directly proportional to the enzyme abundance and/or activity and reflect the amount of substrate that is converted to the product: p, a dynamic proportionality value for de novo methylation; p, a proportionality constant for maintenance methylation; and p, a proportionality constant for active demethylation (hydroxymethylation). Through several iterations of fitting the mathematical model to the 5mC data, we were able to estimate the values of the constants for the serum-to-2i transition in E14 ESCs with the lowest minimum mean square error (MMSE) ( Figure 1 E). The model also takes into account that the rate of de novo methylation (p) changes gradually ( Figure S1 E). Using these values, the model recapitulates 5mC dynamics and predicts the dynamics of 5hmC in ESCs with excellent approximation (R= 0.99) ( Figure 1 F, blue panel, dotted line). p, p, and preflect the individual activity and overall contribution of the three pathways to the DNA methylation dynamics observed and predicts that maintenance methylation is significantly impaired and a major driver of the DNA demethylation observed ( Figure 1 F).

To further understand the kinetics of the transition from serum to 2i ESCs, we determined their DNA methylation state at several time points. First, we quantified global levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) by liquid chromatography followed by mass spectrometry (LC-MS) ( Figure 1 C) as well as by reduced representation bisulfite sequencing (RRBS) ( Figure 1 D), whole-genome bisulfite sequencing (WGBS), and TET-assisted bisulfite sequencing (TAB-seq) ( Figures S1 B and S1C). In line with our previous studies (), DNA demethylation rapidly ensued after medium replacement (∼32 hr; about two rounds of replication) and thereafter continued gradually, reaching a steady-state level after 14 days ( Figure 1 C). A moderate increase in 5hmC levels was observed up to 72 hr, suggesting the presence of TET activity.

DNA demethylation dynamics can be attributed to three major pathways (): (1) maintenance DNA methylation or replication dependent passive dilution, (2) de novo DNA methylation, and (3) active DNA demethylation, primarily via DNA hydroxymethylation ( Figure 1 A). During the conversion from serum to 2i ESCs, the maintenance methylation components Dnmt1 and Uhrf1, the ten-eleven translocation (TET) enzymes (Tet1, Tet2, and, at low levels, Tet3), Aicda, and Tdg, which have all been implicated in active DNA demethylation, are expressed at similar transcriptional levels. In contrast, the de novo methylases Dnmt3a/b and their regulator Dnmt3l are suppressed in the 2i state ( Figures 1 B and S1 A).

(F) Overlay of the mathematical model predictions (dotted line) with real measurements (red line) obtained from LC-MS in E14 during serum-to-2i conversion. The table summarizes the results of the mathematical modeling, showing the values estimated for p 1 , p 2 , and p 3 in the serum steady state and in 2i. p 1 , proportion of unmethylated CpGs that become hemi-methylated per average cell division; p 2 , proportion of hemi-methylated CpGs that become fully methylated per average cell division; and p 3 , proportion of methylated CpGs that become hydroxymethylated per average cell division.

(E) Graphical representation of the mathematical modeling. The minimum mean square error (MMSE) is plotted against different values for de novo methylation (p 1 ) and maintenance methylation (p 2 ).

(D) Percentage of 5mC methylation as measured by RRBS in E14 during serum-to-2i conversion. Horizontal bars represent the median values.

(C) Percentage of 5mC (top) and 5hmC (bottom) as measured by LC-MS in E14 during serum-to-2i conversion. Error bars indicate mean ± SD from three biological replicates.

(B) Expression levels of genes involved in the DNA methylation machinery during serum-to-2i transition. Error bars indicate mean ± SD from three biological replicates.

(A) Schematic representation of cytosine methylation/demethylation cycle. Different forms of modified CpG dyads and the corresponding processes are indicated.

Discussion

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Surani M.A. A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development. In addition to the reduction of UHRF1 protein levels, the signaling changes imposed by 2i in ESCs (MAPK and GSK3 signaling inhibition) elicit a decrease in the levels of H3K9me2, thereby synergistically driving genome-wide DNA demethylation, while allowing methylation maintenance of specific genomic loci. This regulation of H3K9me2 is also evident in PGCs, with apparent loss of H3K9me2 in mouse () and human PGCs (). We found that regions with high levels of H3K9me2 in naive ESCs are also characterized by higher level of DNA methylation ( Figure 5 F) and regions resistant to DNA demethylation in the presence of UHRF1 show significant enrichment of H3K9me2 ( Figure S5 E). We propose that in the face of global demethylation, locus-specific enrichment of H3K9me2/3 allows DNA methylation maintenance at specific regions via recruitment of UHRF1. Similarly, while TET enzymes were dispensable for global epigenetic reprogramming, TET-dependent demethylation was evident at a limited number of specific loci. WGBS and TAB-seq experiments revealed that active demethylation is restricted to a small proportion of the genome, predominantly including active promoters and enhancers.

Taken together, loss of DNA methylation in ESCs, PGCs, and preimplantation embryos is the result of the concerted regulation of two pathways, namely reduction of UHRF1 protein levels and global loss of H3K9me2, thereby impairing recruitment of DNMT1 to the replication fork. This elegant mechanism facilitates the maintenance of methylation at specific loci while the genome is simultaneously demethylated on a global scale.