Significance Naïve embryonic stem cells are characterized by genome-wide low levels of cytosine methylation, a property that may be intrinsic to their function. We found that retinol/retinoic acid (vitamin A) and ascorbate (vitamin C) synergistically diminish DNA methylation levels and in doing so enhance the generation of naïve pluripotent stem cells. This is achieved by two complementary mechanisms. Retinol increases cellular levels of TET proteins (which oxidize DNA methylation), whereas ascorbate affords them greater activity by reducing cellular Fe3+ to Fe2+. This mechanistic insight is relevant for the production of induced pluripotent stem cells used in regenerative medicine, and contributes to our understanding of how the genome is connected to extrinsic and intrinsic signals.

Abstract Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.

Epigenetic modification is a mechanism used to stably enforce and maintain gene expression patterns between different cell types. Cytosine methylation is perhaps the most intensively studied of these modifications. A low level of cytosine methylation (<30% of CpG dinucleotides) is one of the few features that distinguish the most basal stem cells of the body—naïve embryonic stem cells (nESCs)—from stem cells primed for differentiation (1⇓⇓⇓–5) or committed to somatic lineages (70–85% CpG methylation) (6, 7). Importantly, inhibitors of the DNA methylation maintenance machinery can accelerate the reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) (8, 9). These features suggest that reduced DNA methylation, either globally or at specific genomic regions, is a fundamental property of naïve pluripotency (Fig. 1A).

Fig. 1. TET protein catalytic activity in vitro is rescued by the addition of Fe2+, ascorbate, or other antioxidants. (A) Major loss of DNA methylation, globally and at gene promoters, is associated with naïve pluripotent cells. (B) Demethylation during reprogramming is affected by the activity of the Fe2+-dependent TET hydroxylases, which create 5hmC and other oxidized derivatives (5fC and 5caC). However, what regulates TET levels is largely unknown, and the mechanism by which factors such as ascorbate affect TET-mediated oxidation are unclear. (C) Global levels of 5mC and 5hmC (%) in nESCs following 72 h of supplementation with 50 μg/mL ascorbate (± SD; n = 3). (D) Kinetics of TET1-CD–mediated 5hmC production when supplemented with 100 μM iron (Fe2+ or Fe3+) and 1 mM ascorbate (corresponds to 172.12 μg/mL). (E, Upper) Relative activity of TET1-CD when supplemented with 100 μM iron (Fe2+ or Fe3+) and various antioxidants. (E, Lower) The same experiment repeated, with the antioxidant and iron mix preincubated for 30 min before the addition of TET1-CD (± SD; n = 4). NR represents reaction conditions without added reducing agents. (F) Relative activity of TET1-CD at various Fe2+ concentrations encompassing those seen in cellular contexts (0.2–1.5 μM, indicated by a red box). The mean apparent dissociation constant (K d ) for this reaction was determined to be 0.41 ± 0.05 µM (n = 2).

A key pathway of active DNA demethylation involves the ten-eleven translocation (TET) protein family. These Fe2+- and oxoglutarate-dependent enzymes remove methylated cytosine (5mC) by converting it to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives (10⇓⇓–13) (Fig. 1B). TET proteins can contribute to locus- specific demethylation in nESCs (1, 14), and their depletion reduces the expression of pluripotency genes and increases methylation at their promoters (12, 15). Furthermore, forced expression of TET1 and TET2 dramatically enhances iPSC reprogramming in a catalytically dependent manner (16⇓–18). Nevertheless, the molecular signals that control TET activity in nESCs, and how they can be manipulated during reprogramming, are poorly characterized. For example, although ascorbate (vitamin C) is known to enhance 5hmC production in a TET-dependent manner (19⇓⇓⇓–23), the mechanism by which this occurs is unclear (Fig. 1B).

Here we report that ascorbate enhances 5hmC production and potentiates TET catalysis, not as a cofactor as reported previously, but rather by reduction of Fe3+ to Fe2+, making it available for participation in the TET enzyme catalytic center. Retinol, the most common form of vitamin A in the body, is chemically unrelated to ascorbate but similarly enhances the production of iPSCs (24). We discovered that it also increases 5hmC production and DNA demethylation in a TET-dependent manner. This is achieved not by an effect on enzymatic activity, but rather through increased TET2 and TET3 expression. We show that increased TET2 mRNA is dependent on an evolutionary conserved retinoic acid (RA) receptor element (RARE) in the first intron of its underlying gene. Finally, given the overlapping effects of retinol and ascorbate on 5hmC production and DNA demethylation, we tested their effects on the reprogramming of primed cells to naïve pluripotency. We found synergistic effects between these two vitamins in a manner predicted by their interdependent effects on epigenetic memory erasure.

Discussion DNA methylation erasure in the germ line and during experimental reprogramming are closely linked to the acquistition of pluripotency. TET-mediated production of 5hmC can enhance DNA demethylation (reviewed in ref. 6); however, the mechanistic details involved with this process and what can be done to manipulate it are much less understood. Here we show that DNA demethylation and steady-state 5hmC levels can be enhanced in nESCs by various retinoid forms and ascorbate through distinct mechanisms (Fig. 5). Fig. 5. Retinol and ascorbate enhance DNA demethylation, 5hmC production, and pluripotent stem cell reprogramming by synergistic mechanisms. The RARE in the first intron of Tet2 allows increased expression of TET2 mRNA on stimulation of RA signaling (by retinol, retinyl acetate, or RA itself) and enhanced binding of the RAR (brown enzyme). In contrast, ascorbate increases the active iron (Fe2+, green circles) required for the TET catalytic center by reduction from Fe3+ (red circles). Together, retinol and ascorbate additively enhance 5hmC production, resulting in greater removal of methylation from DNA. The enhancing effect of ascobate and retinol on naïve pluripotent stem cell reprogramming is greater than the sum of their individual effects. We find that ascorbate supports TET activity not as an essential cofactor, but rather by reduction of nonenzyme-bound Fe3+ to Fe2+. Unlike C-P4H, which is reliant on ascorbate for its activity, TET does not undergo uncoupled reactions that destroy its activity in the absence of substrate (Fig. S1G). Moreover, under conditions of sufficient Fe2+ and neutral pH, ascorbate does not enhance TET function (Fig. 1D). When faced with insufficient Fe2+ (and excess Fe3+), ascorbate dramatically rescues TET activity, but other reducing agents can do this as well, provided that sufficient incubation time is provided (Fig. 1E). Although these results challenge the findings reported in the literature (19, 21⇓–23), they are in complete agreement with the recent finding that redox-active quinones stimulate TET activity in cell culture through reduction of enzyme-free Fe3+ to Fe2+ (42). Taken together, these results imply that TET proteins are inherently sensitive to labile iron concentrations in the cell, an idea supported by the fact that the dissociation constant of Fe2+ and TET1-CD overlaps with the physiological range of labile iron in the cell (Fig. 1F). In contrast to ascorbate, we found that retinol and RA do not have any effect on TET enzyme efficiency in cell culture (Fig. 2D) or in vitro (Fig. 1E), but instead enhance DNA demethylation and increase 5hmC by activating TET2 and TET3 expression (Fig. 3). We also found that human nESCs grown in media with vitamin A supplementation (i.e., retinyl acetate; Fig. 2B) have more genomic 5hmC than those grown without it (Fig. S5), supporting our original observations in mouse nESCs (Fig. 2C) and implying that this is a conserved regulatory effect. TET2 activation has been previously associated with RA treatment in embryonal carcinoma cells (43); however, the nature of this association is unknown. Given that TET2 responds to retinol and RA stimulation within 8 h in a manner dependent on a eutherian-conserved IR0-type RARE within its first intron (Fig. 3), we conclude that Tet2 is a direct target of RA signaling. Fig. S5. Retinol enhances 5hmC in human naïve ESCs. Immunofluorescence using antibodies specific to 5mC (red) and 5hmC (green) on human naïve ESCs grown with retinol (VitA+) and without retinol (VitA−). When ascorbate and retinol are supplemented in combination, RA signaling will increase TET protein levels and ascorbate will potentiate its activity (Fig. 5). A prediction arising from this scenario is that the combined effect of retinol and ascorbate should be greater than the sum of their individual effects, owing to complementary effects on shared cellular components. Indeed, ascorbate treatment essentially “sensitizes” EpiSCs to lower levels of retinol (Fig. 4C), which suggests that the improvement in reprogramming by both small molecules is affecting the same pathway, the nexus of which is 5hmC. Our results demonstrate that intermediate levels of RA signaling enhance the reprogramming of EpiSCs to naïve pluripotency (Fig. 4). Supplemented retinol above 6.25 ng/mL suppresses reprogramming in a dose-dependent manner, an effect that we speculate is due to the well-described ability of RA to stimulate differentiation. Interestingly, retinol levels in the serum of mice (44) and humans (45) is higher than the range that we tested; thus, adult levels of RA signaling may form one potential barrier by which somatic cells are protected from spontaneous de-differentation. A previous study showed that optimum levels of retinoid stimulation are critical for the reprogramming of EpiSCs to iPSCs, and that RA signaling strongly affects the reprogramming of mouse embryonic fibroblasts (24). That study further showed that small-molecule antagonists of RA signaling attenuate β-catenin and activate Wnt signaling in EpiSCs, thus providing a likely mechanism for how RA enhances reprogramming. We suggest that in addition to this effect, RA signaling enhances reprogramming by directly activating TET2 expression, a known reprogramming factor (16, 18). In summary, our work provides mechanistic insight into how TET proteins remove epigenetic information during reprogramming to naïve pluripotency, and how this process can be manipulated through the use of retinol and ascorbate. Nevertheless, the significance of our work is not limited to the elucidation of these fundamental processes and their application to iPSC reprogramming. For example, our finding that TET activity is sensitive to physiological changes in iron suggests that TET may represent a conduit through which alterations in this ion are signaled to the genome. Moreover, the observation that RA signaling enhances TET2 expression could be relevant for the treatment of certain cancers. TET2 is a well-described tumor suppressor that is regularly mutated in a number of hematopoietic malignancies (46). Acute promyelocytic leukemia (APL) is a form of myeloid malignancy characterized by PML-RARα translocation and sensitivity to RA treatment, such that RA used in combination with arsenic trioxide can provide a 5-year event-free survival rate of >90% (47, 48), a dramatic improvement for what was once considered the deadliest form of acute leukemia. Nevertheless, a significant number of patients are resistant to RA treatment. A recent analysis found that 4.5% of patients with APL have mutations in TET2, and that a mutation in this and other epigenetic modifiers is a significant indicator of poor disease outcome (49). Our work provides a potential mechanistic explanation for RA insensitivity in patients with APL with TET2 mutations, and if proven in further experimentation, could affect the management of this disease.

Methods Our methodology is described in detail in SI Methods. In brief, stem cell culture was performed according to standard techniques as reported previously (1, 16), with modifications as described in Tables S2 and S3. Mass spectrometry analysis of nucleosides was performed as described previously (50). An ELISA-based plate assay was used to quantify the 5hmC produced by a TET1 catalytic domain protein (TET1-CD) in vitro. EpiSC reprogramming was performed as described previously (39), with modifications as outlined in SI Methods. Table S2. Cell lines used in this study Table S3. Media and additives used in this study

SI Methods Cell Lines and Culturing Conditions. All ESC and EpiSC lines used in this study are listed in Table S2, and respective culture conditions and media compositions are listed in Table S3. All cells were grown on gelatin-coated plates without feeders except for EpiSCs, which were grown on fibronectin coated plates. Nucleic Acid Extraction, Manipulation, and qRT-PCR. Total nucleic acid was extracted from cell pellets using the Agencourt RNAdvance Cell v2 Kit (Beckman Coulter, catalog no. A47942) according to the manufacturer’s instructions. Total nucleic acid was DNase-treated using the Ambion TURBO DNA-free Kit (Thermo Fisher Scientific, catalog no. AM1907), from which 500–2000 ng of RNA was converted into cDNA using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, catalog no. K1622). qRT-PCR was performed on a Bio-Rad 384-well machine using the primers and reaction conditions listed in Table S4. Cycle number differences related to input RNA level were normalized using a standard curve and two reference genes (Atp5b and Hsp90ab1). Table S4. Primers used in this study Reprogramming and TET-TKO Rescue Experiments. EpiSCs were transfected using FuGENE reagent (E2311; Promega) with 1 μg of PB-flox-Klf4-Pgk-Hygro, 1 μg PB-flox-Empty-IRES-Blast, and 2 μg of PBase expression vector pCAGPBase54 (16). Dual hygromycin and blasticidin selection was applied to transfectants for a minimum of 10 d. Stable transgene expression was confirmed by qRT-PCR. Here 20,000 stable transfectant cells were seeded per well in a 12-well plate in standard EpiSC conditions (N2B27 + FGF2 + Activin A). After 24 h, the medium was switched to N2B27 + 2i/LIF. Puromycin selection for an Oct4-GFP-IRES-Puro reporter transgene was applied from day 6 of 2i/LIF treatment. Image tiles covering entire wells were recorded using both the transmitted and GFP channels (±VitA), or only the GFP channel (retinol series) using a BD Pathway 855 microscope (BD Biosciences). Mosaic images were created using on-board CALIPER software. GFP-positive colonies were counted manually from GFP images, taking into consideration both signal morphology and intensity. 2i/LIF conditioned TET-TKO cells were transfected with 1 μg of PB-flox-HsaTET1-IRES-Blast, PB-flox-HsaTET2-IRES-Blast (16), or an empty vector control according to the foregoing protocol. Single colonies of stable transfectants were selected for 10 d and then subjected to retinol treatment for 72 h. TET2 RARE Deletion. The Tet2 RARE element was deleted in E14 ESCs using CRISPR/Cas9. ESCs were transfected with 1 µg of pSpCas9(BB)-2A-GFP (PX458) (Addgene plasmid 48138) (51) expressing Cas9 and GFP and a plasmid expressing the guide RNA (GTCATGACCCGGTCAGAGCCAGG), targeting the Tet2 RARE (position 210–232; minus strand). Single cells expressing high levels of GFP after 48 h were sorted into 96-well plates, expanded, screened for deletions of the Tet2 RARE, and the expression of Tet2 after retinol treatment for 72 h was assayed. Mass Spectrometry. Here 500 ng of genomic DNA was incubated with DNA Degradase Plus (Zymo Research) at 37 °C for 4 h. Analysis of global 5mC and 5hmC levels was carried out with an AB Sciex Triple Quad 6500 mass spectrometer as described previously (48). Results are expressed as percentage of the total cytosines. For retinoid mass spectrometry, all manipulations of samples were carried out below 10 °C with exposure to light kept to a minimum. Media samples were diluted with acetonitrile (3 volumes), incubated at 4 °C for 30 min, and then centrifuged at 14,000 × g for 5 min. The supernatants were diluted with water (2 volumes), then further diluted with 50% aqueous acetonitrile as required immediately before analysis. Samples were analyzed by LC-MS on a Q-Exactive Plus high-resolution mass spectrometer (Thermo Fisher Scientific) coupled, via a Proxeon nanoelectrospray ion source, to a Dionex UltiMate 3000 Nano LC System. LC separation was achieved isocratically with a solvent of 0.1% formic acid in 90% aqueous acetonitrile, at a flow rate of 800 nL/min, on a reversed-phase column packed with ReproSil-Pur C18AQ (250 × 0.1 mm; 3-µm particles). Because both retinol (Sigma-Aldrich; R2625) and retinyl acetate (Sigma-Aldrich; R7882) fragment spontaneously during electrospray ionization to generate the C 20 H 29 + ion (monoisotopic m/z, 269.2264), both compounds were quantified from extracted ion chromatograms of this ion in high-resolution (140,000 nominal) SIM scans, recorded over the m/z range of 264.2–274.2. For confirmation of identity, MS2 spectra were also recorded, with an isolation window of 1 Thomson unit and a normalized collision energy of 30 V. Cloning, Expression, and Purification of Recombinant mTET1-CD. The genes coding for the catalytic domains of murine TET1, TET2, and TET3 proteins were amplified from pFastBac-mTet1, -mTet2, and -mTet3 full-length constructs (generously provided by Yi Zhang, Howard Hughes Medical Institute, Boston) and cloned in pET28a (+) vector. For TET2 and TET3, the variable regions found between two parts of dioxygenase domains were substituted by a 15-aa flexible linker. Recombinant TET-CD proteins were expressed in Escherichia coli strain BL21 (DE3) CodonPlus-RIL at 20 °C overnight (12–13 h) in LB medium after the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside. The cells were harvested and resuspended in sonication/wash buffer containing 50 mM Hepes pH 8.0, 30 mM imidazole, 500 mM NaCl, 10 mM α-ketoglutarate, and 10% glycerol, after which the cells were lysed by sonication. The crude lysate was clarified by centrifugation, and the protein was purified using Ni-NTA affinity chromatography (Genaxxon). The proteins were eluted with wash buffer supplemented with 250 mM imidazole and dialyzed against buffer containing 50 mM Hepes pH 8.0, 300 mM NaCl, 10 mM α-ketoglutarate, and 10% glycerol for 3 h. After dialysis, the proteins were aliquoted and stored at −80 °C for further use. ELISA-Based Plate Assay for the Detection of 5hmC. The ELISA plate assay for measuring TET activity was performed in 96-well white high-binding plates (Lumitrac 600; Greiner), precoated overnight with 100 µL containing 1.3 µg of avidin (Sigma-Aldrich) dissolved in 0.1 M sodium bicarbonate (pH 9.6). At the start of the assay, the wells were washed five times with PBST-500 wash buffer (PBS supplemented with 0.01% Tween 20 and 0.5 M NaCl) and then filled with 100 µL of 0.05 M NaOH stop solution. DNA oxidation reaction mixtures (20 µL) were prepared in microcentrifuge tubes and comprised 0.5 µM methylated substrate DNA (86-mer) and 1.5 µM recombinant murine TET-CD in buffer containing 50 mM Hepes pH 6.8, 1 mM ascorbate, 10 or 100 µM Fe2+ or Fe3+ (FeCl 3 ) and 1 mM α-ketoglutarate. The reactions were started by addition of the enzyme, and 2-µL samples were taken from the reaction mixture at respective time points. The samples were added to the corresponding well of a microtiter plate containing stop solution and incubated on a rotating platform for at least 20 min. Then the wells were washed five times with PBST-500 wash buffer and blocked with 300 µL of 2% BSA dissolved in PBST for 1 h on a rotating platform. Subsequently, the wells were washed five times with 300 µL of PBST-500 per well, and 100 µL of primary antibody [anti-5hmC; 1:10,000 (ActiveMotif) diluted in 2% BSA in PBST solution] was added to each well. The plate was incubated for a1 h on a rotating platform, followed by five washes with 300 µL of PBST-500 wash buffer. Next, 100 µL of secondary antibody [anti-rabbit conjugated with HRP (GE Healthcare); 1:6,000 dilution in 2% BSA/PBST] was added to each well, followed by incubation for 1 h. The wells were washed again five times with 300 µL per well of PBST-500. Finally, 100 µL of the ECL substrate solution (Pierce ECL Plus) was added, and luminescence was measured for 5 s in each well using an EnSpire microtiter plate reader (PerkinElmer). To check the effect of various reducing agents on Tet activity, the reaction mixtures containing 10 or 100 µM Fe2+ or Fe3+ ions were supplemented with reducing agents, and the reaction was started by adding the enzyme either directly or after a 30-min preincubation. Beta-mercaptoethanol, DTT, tris(2-carboxyethyl)phosphine, l-glutathione, d-iso ascorbate, N-acetyl l-cysteine, and sodium l-ascorbate were used at a concentration of 1 mM in the reaction. Hydroquinone was used at 50 µM, potassium iodide was used at 10 µM, and retinol and RA were used at 0.2 µM and 100 µM. Preparation of Biotinylated Substrate. The biotinylated substrate (86-mers) used in the assay was amplified from a pUC19 vector by qRT-PCR using oligos (DpnII_pUC19_f: 5′- GAG TAA ACT TGG TCT GAC AGT TAC CA -3′ and DpnII_pUC19_r: 5′- CAA CTA TGG ATG AAC GAA ATA GAC AGA T -3′) with forward primer carrying biotin molecules. The substrate- containing modified cytosines (5mC) were generated using 5mdCTP (New England Biolabs) instead of dCTP in the amplification reaction. Fe2+ Binding Assay. To investigate the Tet1-CD and Fe2+ binding affinity, we assayed the enzyme activity at different Fe2+ concentrations. The kinetic data were plotted in Microsoft Excel, and the initial reaction rates were extracted using linear regression. The reaction rates were fitted using a least squares fitting procedure to a bimolecular binding equilibrium model. To account for prebound Fe2+, which copurifies with the enzyme from bacteria, a variable representing the concentration of prebound Fe2+ was added to the concentration of supplemented Fe2+. Quantification of Ferrous Iron. The concentration of ferrous iron present in the reaction mixtures was quantified with the ferrozine method (52). The reaction mixture containing 10 µM ammonium iron (II) sulfate hexahydrate, 50 mM HEPES (at either pH 6.8 or 8.8) was prepared and incubated at 37 °C. At defined time points, 500-µL aliquots were removed from the reaction mixture and added to the tube containing 100 µL of 500 µM ferrozine and 100 µL of 2 M ammonium acetate at pH 6.0 to quench potential further oxidation of ferrous iron during the subsequent incubation step. The samples were kept at room temperature for 20 min to ensure full color development, and their absorbance was measured at 562 nm in 10-mm glass cuvettes using a Hitachi U-2810 spectrophotometer. Samples containing 50 mM HEPES buffer (at either pH 6.8 or 8.8) without ammonium iron (II) sulfate hexahydrate served as blanks. The data were plotted in Microsoft Excel and fitted with a single exponential function. A standard curve prepared with known concentrations of ferrous iron was used to quantify the observed Fe2+ concentrations in the samples. Bioinformatics. ChIP datasets (32) (GSM482749 and GSM482749) were analyzed using SeqMonk and custom R scripts. RAREs were discovered within the Tet family sequence by a text search and homology alignment. Regions orthologous to the Tet2 IR0-type RARE were retrieved from 35 eutherian mammals using Ensembl release 77 (www.ensembl.org). The eutherian consensus logo was created using Weblogo3 (weblogo.threeplusone.com). Immunofluorescence, Microscopy, and Image Analysis. Staining of methylated and hydroxymethylated DNA was performed as described previously (1) with modifications. Cells were fixed in 2% paraformaldehyde for 30 min. After permeabilization by PBS + 0.5% Triton X-100 for 1 h, fixed cells were incubated in 2 N HCl for 30 min at 37 °C, washed in PBS-Tween, and then blocked overnight. Cells were incubated in 1:500 anti-5mC (Eurogentec, BI-MECY) and 1:1,000 anti-5hmC (Active Motif, 39769). Secondary antibodies (Alexa Fluor) were diluted 1:1,000 and incubated for 1 h. Nuclei were stained with YOYO1 (Molecular Probes). Single optical sections were captured with a Zeiss LSM780 microscope (63× oil-immersion objective), and the images were pseudocolored using Adobe Photoshop.

Acknowledgments We thank Austin Smith, Guoliang Xu, and Jose Silva for providing human ESCs, TET triple-KO ESCs, and OEC-2 EpiSCs, respectively. We also thank Felix Krueger and Simon Andrews for bioinformatic help and Simon Walker for assistance with imaging. This work was funded by the Wellcome Trust (senior investigators W.R. and S.B.; 095645/Z/11/Z and 099232/z/12/z, respectively), the Biotechnology and Biological Sciences Research Council (BB/K010867/1 to W.R.), the Medical Research Council, the European Union EpiGeneSys Network of Excellence, the European Union BLUEPRINT Consortium, the Human Frontier Science Program (T.H.), the Swiss National Science Foundation/Novartis (F.v.M.), and the German Research Foundation (Grant Deutsche Forschungsgemeinschaft SPP1784, to T.P.J).

Footnotes Author contributions: T.A.H., F.v.M., M.R., G.F., D.O., T.P.J., and W.R. designed research; T.A.H., F.v.M., M.R., M.B., G.F., D.O., F.S., and T.P.J. performed research; T.A.H., F.v.M., M.R., M.B., G.F., D.O., F.S., and T.P.J. contributed new reagents/analytic tools; T.A.H., F.v.M., M.R., M.B., G.F., D.O., F.S., S.B., T.P.J., and W.R. analyzed data; and T.A.H., F.v.M., and T.P.J. wrote the paper.

Conflict of interest statement: W.R. and S.B. serve as consultants for Cambridge Epigenetix Ltd.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1608679113/-/DCSupplemental.