Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b
- Kevin T. Ebata†1,
- Kathryn Mesh†1,
- Shichong Liu2,
- Misha Bilenky3,
- Alexander Fekete4,
- Michael G. Acker4,
- Martin Hirst3, 5,
- Benjamin A. Garcia2 and
- Miguel Ramalho-Santos1Email author
© The Author(s) 2017
Received: 2 April 2017
Accepted: 3 July 2017
Published: 12 July 2017
Histone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes.
We report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in naïve ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 h and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes at specific loci. Lastly, we document Kdm3a/b are partially required for the upregulation of germline genes by vitamin C.
These results reveal a specific role for vitamin C in histone demethylation in ES cells and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.
Epigenetic information encoded in chromatin is crucial for cell identity and differentiation . One major layer of chromatin-level regulation is histone methylation. In particular, histone lysine residues can be modified by mono-methylation (me1), dimethylation (me2), or tri-methylation (me3). Histone methyltransferases deposit methyl groups, which can then be recognized by reader proteins that modulate gene expression . Depending on the reader proteins recruited to specific methylated histone residues, the corresponding genes may be repressed or activated. For example, H3K4me3 is associated with the recruitment of factors that promote gene activation, whereas H3K9me2/3 is associated with recruitment of repressive factors. In turn, histone methyl groups are removed by histone demethylases, most of which belong to the family of Fe(II)- and 2-oxoglutarate-dependent dioxygenases [2, 3]. The balance of the activity of histone methylases and demethylases determines the overall histone methylation patterns within a cell. Shifts in this balance underlie the extensive remodeling of histone methylation patterns that occurs during development.
The essential nutrient vitamin C has historically been described as a co-factor of collagen prolyl hydroxylases, the prototypical members of the family of Fe(II)- and 2-oxoglutarate-dependent dioxygenases, by recycling Fe(III) to Fe(II) . The more recent discoveries that enzymes involved in DNA and histone demethylation are also Fe(II)- and 2-oxoglutarate-dependent dioxygenases  raises the interesting possibility that epigenetic programs may be modulated by diet . In support of this notion, we and others reported that vitamin C enhances the activity of Tet enzymes, which are also Fe(II)- and 2-oxoglutarate-dependent dioxygenases, leading to widespread DNA demethylation and a blastocyst-like state in Embryonic Stem (ES) cells [7, 8].
Several studies have explored DNA methylation and histone mark patterning in ES cells during the transition from formative or primed (hypermethylated) to naïve (hypomethylated) state [9–12]. This transition can be modeled in vitro by switching ES cells maintained in serum containing media to a serum-free media supplemented with GSK3β and ERK1/2 inhibitors (2i) [9, 10]. In the presence of 2i, ES cells show a gradual global decrease in DNA methylation and concurrent reduction of H3K9me2 while maintaining H3K9me3 [11, 12]. Additionally, the distribution of H3K27me3 is globally redistributed with reduced levels at promoters in 2i  and accumulation at transposons after conversion to 2i + vitamin C . Together, these studies identified a global reprogramming in DNA and histone methylation patterns during the primed to naïve state. However, the specific impact of vitamin C on histone methylation patterns and the potential interplay with DNA methylation and gene expression in ES cells maintained in the naïve (2i) state has not been investigated.
We report here that vitamin C leads to a remarkably specific and global reduction of histone H3 lysine 9 di-methylation (H3K9me2) in naïve mouse ES cells. The histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced H3K9me2 demethylation, in a manner that is independent of Tet-mediated DNA demethylation. DNA methylation and H3K9me2 cooperated to repress germline gene expression in ES cells. These results highlight that histone methylation patterns are not indiscriminately sensitive to vitamin C. Moreover, our findings uncover a specific role for vitamin C in the epigenetic program of pluripotent cells, with implications for the regulation of germline development.
Vitamin C induces a specific loss of H3K9me2 in ES cells
The histone methyltransferase G9a and its binding partner G9a-like protein (GLP) can generate H3K9me2, but not H3K9me3 . We therefore compared H3K9me2 levels in vitamin C-treated ES cells to G9a −/− and GLP −/− ES cells. These mutant ES cells have almost undetectable levels of H3K9me2, a much more profound loss of this mark than that induced by vitamin C treatment (Additional file 2: Figure S2A). Consistent with a previous study , H3K9me2 in G9a −/− and GLP −/− ES cells is present in a punctate pattern similar to vitamin C treatment (Additional file 2: Figure S2B), and this minimal residual H3K9me2 in G9a −/− and GLP −/− ES cells is further reduced by vitamin C (Additional file 2: Figure S2A). Overall, these data suggest that vitamin C induces partial loss of H3K9me2 generated by G9a/GLP.
H3K9me2 is reduced at large chromosomal domains and gene promoters in ES cells treated with vitamin C
To validate the ChIP-seq data, ChIP-qPCR for H3K9me2 was performed at promoters of germline genes that are de-repressed in ES cells following vitamin C treatment . There is a two to threefold decrease in H3K9me2 in vitamin C-treated ES cells compared to untreated ES cells across all genes analyzed (Fig. 2d). A similar pattern is observed at repeat elements known to be marked by H3K9me2 (Additional file 4: Figure S4). Specificity of the ChIP was confirmed by immunoprecipitation with anti-IgG antibody as well as a primer set for the promoter of Gapdh, which is devoid of H3K9me2 (Fig. 2d). Taken together, these results indicate that vitamin C induces a widespread loss of H3K9me2 in ES cells at large chromosomal regions, repeat elements and promoters of repressed germline genes.
Kdm3a and Kdm3b mediate vitamin C-induced loss of H3K9me2
Interaction between vitamin C-induced changes in 5-methylcytosine/5-hydroxymethylcytosine and vitamin C-induced reduction in H3K9me2
Kdm3a/3b are partially required for vitamin C-induced upregulation of germline genes in ES cells
We report here that vitamin C leads to the specific and widespread loss of H3K9me2 in ES cells, via a mechanism that requires the Kdm3a/3b histone demethylases and is independent of Tet-mediated DNA demethylation. Vitamin C has also been shown to increase the efficiency of reprogramming of somatic cells to the induced pluripotent stem cell state, and this effect is likely mediated by both Tet enzymes and histone demethylases [18, 19].
The remarkable stability of other histone marks in this study raises the possibility that not all histone demethylases are equally dependent on vitamin C. Although our analysis of global histone marks by Western and mass spectrometry showed a specific loss of H3K9me2, it does not exclude the possibility that vitamin C enhances the activity of other histone demethylases at a local level. Interestingly, in contrast to the loss of H3K9me2 we observed in ES cells, vitamin C was shown to promote loss of H3K36me2/3 via KDM2A/2B in mouse embryonic fibroblasts. These results suggest that different cell types, varying in many factors including expression levels of different histone demethylases, may affect the extent to which histone methylation patterns are sensitive by vitamin C . It will be important to study the effect of vitamin C on histone methylation patterns in other cell types and investigate a potential structural basis for the differential sensitivity of histone demethylases to vitamin C.
Given the genome-wide loss of H3K9me2 (this study) and DNA methylation  induced by vitamin C, it is interesting that the transcriptome of ES cells is largely unchanged, with the exception of a subset of germline-associated genes . It is possible that other genetic programs remain silenced by other repressive mechanisms, such as H3K27me3 , which we show here is largely insensitive to vitamin C, and/or the lack of transcriptional activators. We found that vitamin C-induced loss of H3K9me2 is independent of Tet-mediated DNA demethylation at specific germline genes and that both H3K9me2 and DNA methylation contribute to their repression in ES cells. Our results indicate that pluripotent cells have the ability to express germline genes, but that these are repressed by H3K9me2 and DNA methylation. Interestingly, both H3K9me2 and DNA methylation are detected at high levels in pluripotent epiblast cells in vivo and are extensively erased during germline (but not soma) development [22–25]. Moreover, methods to generate germline-like cells from pluripotent stem cells in vitro involve culture in the presence of knockout serum replacement (KSR), which contains vitamin C [26, 27]. It will be of interest to assess whether vitamin C regulates H3K9me2, DNA methylation or gene expression during germline development, in vivo or in vitro. In addition, our results raise the possibility that vitamin C availability may regulate other processes where H3K9me2 has been shown to play a role, such as in hematopoietic lineage commitment , brain development  or adult metabolism .
In this study, we investigated the role of vitamin C in regulating histone methylation in naïve ES cells. We performed comprehensive profiling of histone methylation by mass spectrometry following vitamin C treatment and found a specific reduction in H3K9me2. The distribution pattern of H3K9me2 was not changed by vitamin C, instead levels were reduced throughout the genome, including at gene promoters. Mechanistic studies showed that vitamin C-induced loss of H3K9me2 is via Kdm3a/b enzymes. Vitamin C reduces both H3K9me2 and DNA methylation independently at germline gene promoters, allowing expression of these genes in ES cells.
ES cell lines and cell culture
Oct4-GiP, TT2, G9a −/−, GLP −/−, V6.5, and Tet1 −/− Tet2 −/− ES cells were used in this study. ES cells were cultured on tissue culture plates coated with 0.1% gelatin in 2i medium, which consists of N2B27 base medium  supplemented with the MEK inhibitor, PD0325901 (1 μM, Stemgent), the GSK3β inhibitor, CHIR99021 (3 μM, Selleck Chemicals), and with ESGRO leukemia inhibitory factor (LIF) at 1000 U/ml (Millipore). Vitamin C (L-ascorbic acid 2-phosphate, Sigma, A8960) was added on day 1 after seeding at 100 μg/ml. Medium was replaced daily.
Cultured cells were fixed in 4% paraformaldehyde for 15 min, washed 3× with PBS, and blocked with blocking solution (PBS + 0.5% Tween20 + 5% FBS) for 1 h at room temperature (RT). Primary antibodies were diluted in blocking solution and incubated with cells overnight at 4 °C. Primary antibodies included H3K9ac (1:1000, Active Motif, 39917), H3K9me1 (1:2000, Abcam, ab9045), H3K9me2 (1:1000, Abcam, ab1220), H3K9me3 (1:1000, Diagenode, pAb-056-050). Cells were then washed for 5 min 3× in PBS and incubated with secondary antibodies in blocking solution for 2 h at RT. Secondary antibodies included 594-conjugated donkey anti-mouse (1:1000, Life Technologies, A21203) and 594-conjugated donkey anti-rabbit (1:1000, Life Technologies, A21207). DAPI (1 μg/ml) was added with secondary antibodies. Cells were washed for 5 min 3× in PBS prior to imaging.
Histone extraction for quantitative mass spectrometry and Western blotting
Core histones were extracted from cultured ES cells using the Histone Purification Mini Kit (Active Motif, 40026). Following extraction, core histones were precipitated in 4% perchloric acid overnight at 4 °C, washed, and resuspended in water.
Isolated histones were resolved on a 4–20% gradient TGX gel and transferred to PVDF membranes. Membranes were blocked in blocking solution (Li-Cor Odyssey Buffer diluted 1:1 with PBS) for 1 h at RT. Primary antibodies were diluted in blocking solution and incubated for 3 h at RT. Membranes were washed for 10 min 3× in TBST (TBS + 0.5% Tween) and incubated with secondary antibodies diluted in blocking solution for 2 h at RT. Membranes were washed for 10 min 3× in TBST and visualized with Pierce ECL Plus.
Quantitative mass spectrometry (qMS)
Approximately 20 μg of extracted histones was resuspended in 30 μl of 100 mM ammonium bicarbonate, at pH 8.0. Chemical propionylation derivatization, digestion and desalting of histones followed by analysis by LC–MS and MS/MS were performed as described previously [32, 33]. In brief, purified peptides were loaded onto 75-μm-ID fused-silica capillary columns packed with 12 cm of C18 reversed-phase resin (Reprosil-pur 120 C18, aq-3 μm particles, Fisher Scientific). Peptides were separated using EASY-nLC nano-HPLC (Thermo Scientific, Odense, Denmark) and introduced into a hybrid linear quadrupole ion trap–Orbitrap mass spectrometer (ThermoElectron) and resolved with a gradient from 0 to 35% solvent B (A = 0.1% formic acid; B = 95% MeCN, 0.1% formic acid) over 30 min and from 34 to 100% solvent B in 20 min at a flow-rate of 250 nL/min. The Orbitrap was operated in data-dependent mode essentially as previously described . Relative abundances of peptide species were calculated by chromatographic peak integration of full MS scans using EpiProfile . Where necessary, peptide and PTM identity were verified by manual inspection of MS/MS spectra.
Isolated ES cells were resuspended in PBS and crosslinked with 1% formaldehyde for 5 min at RT. Crosslinking was quenched with 125 mM glycine. Crosslinked material was sonicated on a Covaris sonicator for 12 min at duty 5%, intensity 3, and bursts 200. ChIP was performed using the Diagenode LowCell# Kit with a mouse anti-H3K9me2 antibody (Abcam, ab1220). A mouse anti-IgG antibody (Abcam, ab18413) was used as control. Isolated DNA was used for qPCR analysis with the KAPA SYBR Fast ABI Prism qPCR kit on an Applied BioSystems 7900HT Sequence Detection System. Primer sequences are listed in Additional file 7: Table S1. For ChIP-seq isolated DNA was resuspended in an elution buffer (10 mM Tris–HCl pH 7.6, 200 mM NaCl, 5 mM EDTA, 0.5% SDS) with 10 μl RNAse and reverse-cross-linked at 65 °C for 4 h. Proteinase K was added and samples were incubated at 55 °C for 1 h. DNA was purified with QIAquick and quantified with a Qubit Fluorometer (Invitrogen). ChIP-seq libraries were constructed from immunoprecipitated DNA as described . Libraries were sequenced using paired-end 100nt sequencing V3 chemistry on an HiSeq 2000 sequencer following the manufacturer’s protocols (Illumina, Hayward, CA.) using multiplex custom index adapters added during library construction to distinguish pooled samples.
Dot blot analysis
Dot blot analysis was performed as previously described . Briefly, isolated DNA was denatured and then serially diluted twofold. DNA samples were spotted on a nitrocellulose membrane using a Bio-Dot apparatus (Bio-Rad) and immunoblotted with rabbit anti-5-hydroxymethylcytosine polyclonal antibody (Active Motif, 1:5000) in Odyssey:PBS (Li-Cor). The membrane was washed and then incubated with HRP-conjugated goat anti-rabbit IgG (Abcam, 1:10,000) secondary antibody in Odyssey:PBS. The membrane was visualized by chemiluminescence with GE ECL Plus.
5mC DNA immunoprecipitation
DNA immunoprecipitation was performed using the Diagenode MagMeDIP as previously described . Briefly, DNA was sonicated into short fragments with a Diagenode Bioruptor. Sonicated DNA (1 μg) was immunoprecipitated with 1 μg of mouse anti-5-methylcytosine monoclonal antibody (Active Motif, 1 μg/μl). Isolation of immunoprecipitated DNA was performed according to the kit instructions, and qPCR was performed in combination with the KAPA SYBR Fast ABI Prism qPCR kit on an Applied BioSystems 7900HT Sequence Detection System. Primer sequences are listed in Additional file 7: Table S1.
Total RNA was isolated from cultured cells using Qiagen RNeasy with on-column DNase I treatment. cDNA was generated from 1 μg of RNA using random hexamers to prime the reaction. Quantitative RT-PCR was performed with the KAPA SYBR Fast ABI Prism qPCR kit on an Applied BioSystems 7900HT sequence detection system. Primer sequences are listed in Additional file 7: Table S1. The relative amount of each gene was normalized using two housekeeping genes (L7 and Ubb), unless otherwise indicated.
Oct4-GiP ES cells were transfected in suspension with Dharmacon siGENOME SMARTpool siRNAs (4 siRNAs per gene) against Kdm3a, Kdm3b, or Kdm3c at 50 nM and seeded into tissue culture plates (Day 0). Cells were re-transfected on day 2 followed by treatment with vitamin C for 24 or 72 h. For double knockdown of Kdm3a and Kdm3b, cells were transfected with 50 nM of both Kdm3a and Kdm3b siRNAs for a total final concentration of 100 nM. Dharmacon siGENOME non-targeting siRNA (NT2) was used as a control. Dharmafect reagent was used for transfections according to manufacturer’s instructions.
KDM3a biochemical assay
The KDM3A assay was performed in 384-well black flat-bottom plates (Corning, Cat# 3654). Enzyme and substrate solutions were prepared in assay buffer consisting of 5 mM HEPES pH 7.0, 15 mM HEPES pH 7.5, 25 mM NaCl, 1 µM (or 1 mM) Alpha-ketoglutarate, 3.75 µM FeSO4, 0.014% Tween 20, 0.03% BSA. Dose–response concentrations of vitamin C (MP Biochemicals, 100769), DTT (Sigma, D0632) or glutathione (Affymetrix, 16315; Chem-Impex, 00158) were as indicated in the graphs. The KDM3A enzyme was produced in house . H3K9me1 substrate was purchased from Perkin Elmer. Plates were covered and incubated for 30 min at room temperature. The reaction is stopped with detection buffer consisting of 100 mM HEPES pH 7.0, 800 mM KF and 0.2% BSA containing the detection reagents Eu-antiH3K9me0 (purchased from Perkin Elmer) and SA:APC (i.e., XL-665, purchased from Cisbio). The final concentrations of the various reagents were: KDM3A 2 nM, H3K9me1 500 nM, Eu-antiH3K9me0 0.75 nM, SA:APC 1.67 µg/ml. Plates were read in a Perkin Elmer Envision Plate Reader (ex/em 340/615 and 340/665).
ChIP-seq raw sequences were examined for quality, sample swap and reagent contamination using custom in house scripts. Sequence reads were aligned to mm10 using BWA 0.5.7 and default parameters and assessed for overall quality using Findpeaks 3.1 . For rescaling, we calculated average ChIP-seq coverage in 500 bp genomic bins. We considered ~150 bins with the highest signal in untreated data samples and calculated distributions of the fold change between vitamin C-treated and untreated data. These bins typically correspond to alignment artifacts and have extreme coverage not due to enrichment. They appear in both IP and control data sets with a very similar coverage profile, as validated by visual inspection of the majority of those locations in the UCSC genome browser. The alignment artifacts have a universal nature (mostly due to incomplete knowledge of genome) with the expectation that for similar cell types and the same read length and DNA fragment length distributions, the difference in the coverage for these ‘outliers’ will be a reflection of the differences in the sequencing depth. We observed that, as expected, for the input sample, the fold change for the total genomic coverage between vitamin C-treated and untreated data is in agreement with median fold change measured from ‘outliers’ bins. For the H3K9me2 signal, these twofold change values are very different, and for the ‘outliers’ the fold change is larger than a value of 1, suggesting that vitamin C-treated data had to be rescaled with a factor ~0.57 (Additional file 3: Figure S3). We verified that the value for the scaling factor depends only weakly on the exact threshold for choice of the outliers within a reasonable range. Using the rescaled data, the coverage profiles for DNA fragments corresponding to properly paired aligned reads were calculated and used to determine the average coverage in 500 bp bins genome wide (5,459,336 bins) and in the promoter regions (TSS ± 2 Kb) of the coding genes (21,958 genes, Ensembl v81).
M.R.-S. directed the study. K.T.E. and K.M. designed and performed experiments. K.T.E., K.M. and M.R.-S. analyzed and interpreted the data. M.B. and M.H. developed and performed ChIP sequencing data processing. S.L. and B.A.G. performed histone methylation quantitative mass spectrometry and analysis. A.F. and M.A. performed in vitro histone demethylation assays. K.T.E., K.M., and M.R.-S. wrote the manuscript with input from all the authors. All authors read and approved the final manuscript.
We thank Matthew C. Lorincz and Yoichi Shinkai for TT2, G9a −/−, GLP −/− and M.M. Dawlaty and R. Jaenisch for the V6.5, Tet1 −/− Tet2 −/− ES cells.
The authors declare that they have no competing interests.
Availability of data and materials
The ChiP-seq datasets used in this study have been deposited in the Gene Expression Omnibus under the accession number GSE84009. Correspondence and requests for materials should be addressed to M.R.-S. (firstname.lastname@example.org).
Ethics approval and consent to participate
This work was supported by NIH Grant R01GM110174 to B.A.G., Grants CCSRI 22R22583 and TFRI NI 22R22545 to M.H., and by NIH Grants R01OD012204 and R01GM113014 to M.R.-S.
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