Histone and DNA methylation control by H3 serine 10/threonine 11 phosphorylation in the mouse zygote
© The Author(s) 2017
Received: 31 August 2016
Accepted: 23 January 2017
Published: 14 February 2017
In the mammalian zygote, epigenetic reprogramming is a tightly controlled process of coordinated alterations of histone and DNA modifications. The parental genomes of the zygote show distinct patterns of histone H3 variants and distinct patterns of DNA and histone modifications. The molecular mechanisms linking histone variant-specific modifications and DNA methylation reprogramming during the first cell cycle remain to be clarified.
Here, we show that the degree and distribution of H3K9me2 and of DNA modifications (5mC/5hmC) are influenced by the phosphorylation status of H3S10 and H3T11. The overexpression of the mutated histone variants H3.1 and 3.2 at either serine 10 or threonine 11 causes a decrease in H3K9me2 and 5mC and a concomitant increase in 5hmC in the maternal genome. Bisulphite sequencing results indicate an increase in hemimethylated CpG positions following H3.1T10A overexpression suggesting an impact of H3S10 and H3T11 phosphorylation on DNA methylation maintenance.
Our data suggest a crosstalk between the cell-cycle-dependent control of S10 and T11 phosphorylation of histone variants H3.1 and H3.2 and the maintenance of the heterochromatic mark H3K9me2. This histone H3 “phospho-methylation switch” also influences the oxidative control of DNA methylation in the mouse zygote.
The epigenetic reprogramming in mouse zygote involves an extensive rearrangement of the epigenetic landscape, including chromatin reorganization and comprehensive changes in DNA modifications. These changes require a coordinated control of epigenetic “writers”, “readers”, “erasers” and “remodelers” on the level of histones and DNA after fertilization. The interplay between histone variants, chromatin modifications and DNA modifications has been studied to a great detail. Here, we analyse the synergetic dynamics of different post-translational modifications in histone H3 variants H3.1, H3.2 and H3.3 which are found in different epigenetic compartments of chromatin . In mouse zygotes, these histone variants show an asymmetrical deposition into parental pronuclei: H3.3 is a predominant histone variant in the newly formed paternal pronucleus, while H3.1 and H3.2 only appear during the first replication of the paternal chromatin. In contrast, the maternal chromatin is initially enriched for H3.1/H3.2 and accumulates H3.3 at later zygotic stages [2–4]. This asymmetry in histone variant composition is accompanied by an asymmetric allocation of histone modifications in both pronuclei [2, 5, 6]. While the paternal chromatin is mainly marked by open chromatin modifications such as H3K4me3, the maternal chromatin shows a high abundance of H3K9me2 heterochromatic mark, which is slightly reduced during the first cell division [5, 7]. In pre-replicative paternal chromatin, H3K9me2 is almost absent and only becomes detectable at late replication stages. In both pronuclei, the abundance of H3K9me2 is linked to differences in DNA modifications. The H3K9me2 containing maternal pronucleus maintains 5mC as the predominant modification, and the level of DNA methylation decreases only slightly during the first DNA replication . It has been shown that the presence of H3K9me2 in the maternal pronuclei protects against Tet3-mediated oxidation of 5mC to 5hmC . As a consequence of this, the maternal chromosomes appear to maintain 5mC levels in contrast to the more oxidized paternal chromosomes, which are practically devoid of H3K9me2 at early stages of DNA replication and where 5mC is extensively converted to 5hmC by Tet3, reducing DNA methylations by about 50% at the end of the first cell cycle .
The current knowledge suggests that H3K9me2 has an important protective role for the maintenance of 5mC. Work by Nakamura et al. showed that Stella protein while present in both pronuclei only protects the maternal DNA against Tet3 oxidation due to the presence of H3K9me2 [9, 11]. However, previous data also suggest that this epigenetic control could be linked to the asymmetric distribution of the major histone variants H3.1, H3.2 and H3.3 . H3S10 phosphorylation has been shown to negatively control H3K9 methylation in fruit fly . In vitro biochemical assays demonstrated a protective role of H3T11 phosphorylation against H3K9me3 demethylation [13, 14]. However, interactions of H3S10phos and H3T11phos with H3K9me2 in mammalian cells have not yet been described. The vicinity of the K9, S10 and T11 residues in the N-terminus of H3 suggests a possible influence or crosstalk of modifications at these residues. This crosstalk might influence “writers” or “erasers” of individual modifications or alternatively affect the interaction with modification “readers”. Phosphorylation of histone H3 fulfils multiple roles: it participates in mitotic chromosomes condensation and segregation, but also modulates gene expression in the context-dependent manner (reviewed in ). Our intention was to examine the potential links between the asymmetric distribution of histone variants and the various layers of epigenetic control in both pronuclei before and after replication. In particular, we analysed the dynamics of H3S10 and H3T11 phosphorylation in the three histone H3 variants during the first cell cycle and their impact on the replication-dependent control of H3K9me2 and DNA modifications in both zygotic pronuclei. Our data reveal a direct or indirect crosstalk between H3S10 and H3T11 phosphorylation, histone variant-dependent H3K9me2 methylation and DNA methylation.
H3S10phos and H3T11phos have different dynamics and associations with histone H3 variants in the mouse zygote
We next investigated the dynamic of H3S10 and H3T11 phosphorylation on all three histone variants. We therefore microinjected mRNAs encoding either histone wild-type H3 variants (WT) or S10A or T11A mutated forms, respectively, into early pre-replicative (2–3 h post-fertilization) mouse zygotes. The ectopically expressed wild-type or mutated forms of H3 variants were fused to GFP reporter (C-terminal fusion). This allowed us to follow their import in the pronuclei. We observe that all WT and mutant forms were readily expressed and efficiently imported into the pronuclei (Additional file 1). We assumed that the mutated non-phosphorylatable H3 variants will be incorporated into nucleosomes generating a “dominant-negative” phosphorylation effect in nucleosomes after replication at PN4/5.
Note that the overexpression of H3WT-GFP variants in the majority of cases did not change the H3S10 and H3T11 phosphorylation pattern. However, in each experiment we observe a few examples in which overexpression leads to a reduction in the respective phosphorylation signals (Additional file 2). This effect may be caused by the time of injection or a variable amount of injected material, leading to a higher abundance of H3.1WT-GFP and H3.2WT-GFP overexpressed proteins competing with endogenous H3 for the kinase activity. Zygotes injected with H3.3WT-GFP mRNA did not show variation in H3T11phos signals (Additional file 2), supporting the notion that H3.3 is not a substrate for H3T11 phosphorylation-specific reaction.
H3S10 and H3T11 phosphorylation is coupled to H3K9me2 histone methylation
From all these experiments, we conclude that H3S10 and H3T11 phosphorylation particularly of variants H3.1 and H3.2 strongly influences the post-replicative levels of H3K9me2.
A simple explanation for a reduction in H3K9me2 signals following mutant overexpression is that the recognition and binding of anti-H3K9me2 antibody to its epitope are affected by the mutation. To examine this possibility, we co-expressed either H3.1-GFPT11A or H3.1-GFPWT together with the catalytical domain of G9a histone methyltransferase (G9aCat) in E. coli cells. The wild-type or mutated histones were partially purified and probed by Western blot using anti-H3K9me2 antibody. Indeed, the antibody clearly detects the H3K9me2 modification after co-expression on both WT and T11A mutated form (Additional file 4).
H3S10 and H3T11 phosphorylation influences DNA modifications
Additional H3K9me2 methylation causes only subtle changes in DNA modifications
Having shown that H3.1-GFPT11A mutant can be methylated by G9aCat-NLS-GFP in E. coli (see above), we next asked whether the decrease in H3K9me2, caused by the expression of H3T11A mutants, may be compensated by ectopic G9a overexpression in the mouse zygote. We co-injected mRNA encoding G9aCat-NLS-GFP with either H3.1-GFPT11A or H3.2-GFPT11A or H3.3-GFPT11A mRNAs, respectively. Indeed, the co-injection partially compensates for the H3K9me2 loss from maternal chromatin observed for H3.1-GFPT11A or H3.2-GFPT11A expressing groups alone, while the co-expression with H3.3T11A did not cause this effect and the G9A mediated H3K9me2 methylation was as high, as in zygotes, injected only with G9aCat-NLS-GFP alone (Additional file 9). The paternal chromatin, which is naturally depleted with H3K9me2, had an increased amount of this modification in all three mutated H3 variants (Additional file 9b). These results support the notion that the maintenance of H3K9me2 on H3.1 and H3.2 (and partially on H3.3) during replication is controlled by the H3T11 phosphorylation status.
We observe that dynamics and the variant-specific distribution of S10 and T11 phosphorylation are distinct and only partially overlapping. H3T11phos is preferentially linked to S-phase and the histone variants H3.1 and H3.2, while H3S10phos is present at G1 and G2 and found on all histone variants (Figs. 2, 3, respectively). Despite this differential dynamics, mutations in S10 and T11 phosphorylation sites cause very similar global effects on both H3K9me2 histone methylation and 5mC/5hmC DNA modifications. It remains to be clarified which regulatory proteins (histone demethylases or DNA modification controller) are influenced by S10 and T11 phosphorylation and how and where in the genome this occurs for the different histone H3 variants.
Some of our data suggest that the conversion of 5mC into 5hmC is directly linked to the histone phospho-methylation switch model discussed above. The overexpression of both H3.1/2-GFPS10A and H3.1/2-GFPT11A mutated histones indeed induces significant accumulations of 5hmC in the maternal pronucleus (Figs. 6a, 7a and Additional file 5). It is tempting to assume that the altered S10 and T11 phosphorylation influences changes in maternal H3K9me2 levels, which in turn influence the Tet3-mediated conversion of 5mC to 5hmC. However, one experiment argues against such a simple scenario. When overexpressing G9A in the zygote, we could massively increase the levels of H3K9 without comparably dramatic changes in the presence or the ratio of 5mC and 5hmC in maternal and paternal pronuclei (Fig. 10a, b). Hence, while the decrease in maternal H3K9me2 causes a strong change on maternal 5hmC amounts, the G9aCat-NLS-GFP-induced increase in H3K9me2 mainly in the paternal chromosome had no or little influence on 5hmC (Fig. 10a). This finding indicates that H3K9me2 (in combination with Stella) might not constitute the exclusive chromatin modification protecting chromatin against Tet-mediated oxidation.
In all our IF experiments, we observed a strong decrease in 5mC and an increase in 5hmC in zygotes overexpressing S10 and T11 mutants. When examining the DNA methylation patterns by hairpin bisulphite sequencing after the first DNA replication, we observe a massive increase in hemimethylated sites in zygotes overexpressing H3.1-GFPT11A mutants (Fig. 8). A lack of T11 (and probably also S10) phosphorylation apparently increases the presence of 5hmC, which in turn affects the maintenance of full methylation after DNA replication. This observation is in line with our previous findings, showing that 5hmC may interfere with DNA methylation maintenance [20, 21].
Our findings provide novel insights into the crosstalk between DNA and histone modifications in the mammalian zygote. We find that serine 10 and threonine 11 phosphorylation of histone H3 shows a different dynamic and pronuclear appearance during the first cell cycle. Overexpression of variant-specific serine 10 and threonine 11 mutants reveals that the phosphorylation at both positions is important for the maintenance of the maternally inherited H3K9me2 as well as for the maintenance of symmetric CpG DNA methylation (5mC/5hmC). Mechanistically our data suggest that H3S10phos and H3T11phos help to stabilize H3K9me2 during the first cell cycle most likely by protecting maternal chromatin against a K9-specific demethylation activity.
Mouse superovulation and in vitro fertilization
For superovulation, females were injected with 6 IU pregnant mare serum gonadotropin (PMSG) and 6 IU human chorionic gonadotropin (hCG) with a time interval of around 48 h. Spermatozoa isolation, oocytes collection and IVF procedures were carried out as previously published protocol with small modifications . Briefly, sperm was isolated from the cauda epididymis of male mice and capacitated in pre-gassed modified KSOM medium supplemented with 30 mg/ml BSA for 2 h. Mature oocytes were collected 15 h post-hCG injection of female mice. Cumulus–oocyte complexes were placed into a 400-μl drop of KSOM medium, mixed with capacitated sperm and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
The cDNAs of H3.1, H3.2 (gifts from Prof. Dr. Fugaku Aoki), H3.3 (purchased from ImaGenes GmbH) were cloned to pEGUP1 (a modified pET28b vector containing GFP coding sequence) by using NcoI and XhoI sites. The cDNA of G9a full length (gift from Prof. Dr. Y. Shinkai, Prof. Dr. M. Tachibana, Prof. Dr. M. Brand and Prof. Dr. M. R. Stallcup) was subcloned to pEGUP1. The same strategy was applied to G9aCat-GFP, HDAC1 (obtained from Addgene) and HDAC2 (gift from Prof. Dr. Richard M. Schultz). To create G9aCat-NLS-GFP expressing construct, G9aCat encoding DNA fragment was inserted to pEGUP1-NLS (pEGUP1 version with nuclear localization signal). For co-expression experiments in E. coli, G9aCat encoding fragment was cloned into pET15Am (a modified pET28b containing ampicillin resistance marker, p15 replication origin and no His-tag sequence). All positive constructs were verified by Sanger sequencing. All the primers used are listed in Additional file 12, also including the ones for mutagenesis, in vitro transcription as well as hairpin bisulphite sequencing.
The generation of all histone H3 mutants (K9R, S10A, T11A) was done by overlapping PCR-based mutagenesis. Briefly, two PCRs, namely PCR1 and PCR2, were applied with two sets of primers in which reverse primer for PCR1 and forward primer for PCR2 are complementary and contain the desired mutation. After gel purification, the cleaned-up fragments from PCR1 and PCR2 were mixed as templates in overlapping PCR. The expected PCR products were then double-digested by NcoI and XhoI, followed by insertion into pEGUP1. The positive clones containing the corresponding mutations were selected and confirmed by Sanger sequencing.
In vitro transcription of mRNAs
To prepare the DNA templates, highly purified DNA templates were extracted with phenol:chloroform, and capped mRNAs were generated by in vitro transcription using AmpliCap-Max T7 High Yield Message Maker Kit (CellScript, Inc.), followed by purification using RNA Clean & Concentrator™-25 Kit (Zymo research) according to the manufacturers’ instructions. Finally, transcribed mRNAs were eluted with nuclease-free water (Life technologies) and stored at −80 °C for later use.
Expression of G9aCat and histone H3 variants in E. coli
For co-expression of G9aCat with histone H3.1-GFPWT, or H3.1-GFPT11A or H3.3-GFPWT, both constructs were transformed into Rosetta™ 2 Competent Cells. The transformed cells were grown in SOC medium with ampicillin (100 mg/mL) and kanamycin (25 mg/mL) until OD value reached 0.5–0.6 at 37 °C, followed by 0.5 mM isopropyl-b-d-thiogalactopyranoside (IPTG) induction for 24 h at 16 °C. Modified proteins of H3.1-GFPWT, H3.1-GFPT11A, H3.3-GFPWT were purified through Ni–NTA resin by taking advantage of the 6xHis tag. The protein expression was confirmed by Coomassie blue staining of SDS-PAGE gel. The presence of H3K9me2 or H3K9me3 on recombinant histones was verified by Western blotting using specific antibodies, which were also used for immunofluorescence.
The detailed information regarding the antibodies used is listed in Additional file 13.
BIX 01294 treatment of zygotes
At 2 h post-fertilization, zygotes were collected and transferred into the 100 μl drop of KSOM medium containing BIX 01294 (5.4 μM). The zygotes were cultured to post-replication stages (PN4/5), followed by fixation for immunostaining.
Microinjection of mRNAs into zygotes
At around 1hpf, just before microinjection, zygotes were collected, washed in M2 medium for several times and transferred into 100 μl drops of pre-gassed KSOM medium. Microinjection was performed under a Zeiss Axiovert 200 M inverted microscope (Zeiss) equipped with a FemtoJet microinjector and piezo-driven micromanipulators (Eppendorf). After a quick set-up, zygotes were placed into a 20-μl drop of M2 medium and cytoplasm injection was done with mRNAs encoding for H3.1/2/3-GFPWT/K9R/S10A/T11A, G9aFL-GFP, G9aCat-GFP, G9aCat-NLS-GFP or in combinations. Each microinjection experiment was replicated at least once, and groups of 10–30 zygotes were used for each experiment.
Live monitoring chromatin incorporation of H3.1/2/3-GFPK9R and H3.1/2/3-GFPWT
After being microinjected with corresponding mRNAs, the zygotes were transferred into a KSOM drop of 10 µl on a glass bottom culture dish and placed into the chamber of Nikon BioStation live cell imaging System under the condition of 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The pictures were captured every 15 min for 97 cycles with the exposure time 1/250 s.
The detailed protocol was described earlier elsewhere [10, 25]. After brief treatment in acidic Tyrode’s solution, zona-free zygotes and cleavage stage embryos were fixed by incubating in 3.7% paraformaldehyde solution in PBS for 30 min at RT, followed by permeabilization in 0.2% TritonX100/PBS at RT for 15 min. Then, the embryos were blocked at RT for 4 h or overnight at 4 °C in PBS, containing 0.1% TritonX100 and 3 mg/ml BSA. Later, the embryos were incubated with primary antibody overnight at 4 °C in blocking solution. Note that for 5mC, 5hmC, 5caC and ssDNA staining DNA was denatured: the fixed embryos were incubated in 4 M HCl for 15 min and then neutralized in 100 mM TrisCl pH 8.0 for 10 min at RT. For simultaneous 5hmC detection and PI DNA staining, the denaturation time was reduced to 6 min 30 s. After incubation with primary antibody, the embryos were washed 3–4 times in blocking solution and then incubated at RT for 2 h with fluorescently labelled secondary antibody. Finally, the embryos were washed and mounted on a glass slide in Vectashield mounting medium (Vector Laboratories). All antibodies used in this study are listed in Additional file 13.
IF microscopy, quantification and statistical analysis
The mounted embryos were analysed on Zeiss Axiovert 200 M inverted microscope equipped with the fluorescence module and B/W digital camera for imaging. The IF images were captured, pseudocoloured and merged using AxioVision software (Zeiss). GIMP and Image J software were used together to complete quantification of the signals of z-stack computed images. For each group, at least 10 zygotes were analysed from at least two repeated experiments. Student’s t test was employed for statistical analysis.
Hairpin bisulphite sequencing
Hairpin bisulphite high-throughput sequencing was performed as previously described with no modifications to the protocol .
histone H3 serine 10
histone H3 threonine 11
histone H3 serine 10 phosphorylation
histone H3 threonine 11 phosphorylation
histone H3 lysine 9 dimethylation
histone H3 lysine 9 trimethylation
histone H3 lysine 4 trimethylation
catalytical domain of G9a histone methyltransferase
full-length G9a histone methyltransferase
intracisternal A-particle element
nuclear localization signal
pregnant mare serum gonadotropin
human chorionic gonadotropin
JW, KL and JL conceived the project. JW and KL wrote the manuscript with contributions and approval by all authors. JL and KL designed the experiments, performed the experiments and evaluated the data. PG performed the hairpin bisulphite sequencing and evaluated the sequencing data. All authors read and approved the final manuscript.
We thank F. Aoki for providing cDNAs of histone H3.1 and H3.2, Y. Shinkai, M. Tachibana, M. Brand and M. R. Stallcup for providing cDNA of G9a.
The authors declare that they have no competing interests.
Availability of supporting data
All plasmid-based constructs used in this study are available upon the request.
Ethical approval and consent to participate
All animal experiments were performed according to the German Animal Welfare law in agreement with the authorizing committee.
This work was supported by a Grant from Deutsche Forschungsgemeinschaft (DFG) WA 1029. J.L. was supported by fellowship offered by Chinese Government Scholarship Council. KL was partially supported through the BLUEPRINT project (European Union’s Seventh Framework Programme grant agreement 282510).
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