Germline epigenome editing identifies H3K9me3 as a mediator of intergenerational DNA methylation recovery in mice

Domain: biology
Published: 2025-12-25
Editorial outcome: major_revision

Abstract
Inter/transgenerational epigenetic inheritance is a crucial and controversial theory that could reshape the concept of genetics. To investigate this theory directly, we invent a system for targeted reprogramming of epigenetic memory in mouse sperm. Using this system, we erase DNA methylation at the differentially methylated region of the H19 gene (H19-DMR) in sperm, which causes Silver-Russell syndrome-like phenotypes in F1 offspring. Although DNA methylation is fully lost in the sperm, it is partially restored during pre-implantation development, suggesting the existence of epigenetic memory that instructs de novo DNA methylation. Importantly, targeted removal of histone modifications in zygotes reveals that tri-methylation at lysine 9 of histone H3 (H3K9me3), which is deposited shortly after fertilization, is required for the subsequent de novo DNA methylation at the H19-DMR. Thus, our study provides a robust germline editing tool, which reveals partial intergenerational inheritance and no transgenerational inheritance at the model locus. Furthermore, we identify H3K9me3 as a mediator for DNA methylation recovery also acting at imprinted loci.

Introduction
Phenotypic inheritance is believed to be achieved via the transmission of genomic DNA. Paradoxically, ancestral environmental exposures, such as abnormal nutritional conditions, toxins, and stress, can lead to disease and phenotypic changes in offspring1–9, providing circumstantial evidence suggesting a non-Mendelian inheritance phenomenon, where epigenetic memory is passed to the next generation through the germline. While these data are still under discussion because of difficulties in showing the absence of genetic mutations, DNA methylation, histone modifications, and RNA may contribute to the primary molecular mechanisms underlying epigenetic inheritance1,2,10–14. For example, paternal prediabetes induced by feeding a high-fat diet increases the susceptibility of offspring to diabetes3, while Sun et al. reported that paternal cold exposure enhances brown adipose tissue activity in offspring4. In these cases, altered DNA methylation was detected in both sperm and offspring; however, it does not necessarily follow that germline DNA methylation changes are passed to the next generation, as it is equally possible that epimutations are first reset after fertilization and then reestablished during development by yet undetermined factors.

To provide more direct evidence for inter/transgenerational epigenetic inheritance, it is necessary to show that epimutations induced in the germline can be transmitted to the next and later generations. In this study, we define intergenerational inheritance as the transmission of epigenetic alterations originating in the gametes to the immediate offspring (F1 generation). By contrast, transgenerational inheritance refers to the sustained presence of these modifications in subsequent generations (e.g., F2 and beyond) without continued exposure to the initial trigger. Recently, Takahashi et al. showed that insertion followed by removal of a CpG-free DNA segment at CpG island promoter regions causes DNA methylation and silencing of the surrounding loci, which are maintained over multiple generations15. Curiously, the induced DNA methylation was lost during germ cell development, but then reestablished during embryonic development by unknown mechanisms15; however, the methods used in the study could not fully rule out the influence of a small genetic change required for epimutation induction16. Moreover, other concerns, including off-target mutations during insertion of CpG-free DNA by CRISPR-Cas9 genome editing and colony selection, have been raised16. Epigenome editing using a ‘dead’ Cas9 (dCas9)17,18, which does not introduce genetic changes and can rewrite the epigenetic information at a target locus, maybe a direct method that can help solve this problem. Transmission of rewritten epigenetic information in the parental germline to the next generation, according to phenotype emergence, would directly demonstrate inter/transgenerational epigenetic inheritance.

Genomic imprinting is a process wherein a gene is differentially expressed depending on whether it has been inherited from the father or mother19,20. An epimutation comprising DNA demethylation of the sperm-derived paternal allele in a differentially methylated region (DMR) of the imprinted gene, H19, (H19-DMR) is believed to cause Silver-Russell syndrome, which is characterized by intrauterine growth retardation due to Igf2 downregulation21. H19-DMR22 is located between the Igf2 and H19 imprinted genes23,24 and regulates their expression. In somatic cells of healthy individuals, H19-DMR CpG dinucleotides, including CTCF-binding sites, are normally methylated on the paternal allele and unmethylated on the maternal allele (Fig. 1a)22; this allele-specific CpG methylation upregulates paternal allele expression of Igf2 and downregulates that of H19. By contrast, in patients with Silver-Russell syndrome, demethylation of the paternal allele H19-DMR is expected to decrease the expression of Igf2 and increase that of H19 through epigenetic regulation of CTCF binding25,26, resulting in fetal intrauterine growth retardation. During establishment of genomic imprinting in the male germline, H19-DMR CpGs are progressively hypermethylated through an endogenous de novo methylation mechanism until the pro-spermatogonia stage27, while this region is unmethylated in oocytes (Fig. 1b). If the epimutation in H19-DMR occurring sperm is inherited, intrauterine growth retardation will be observed in offspring.

In this study, we developed a germline-specific DNA methylation editing system and applied it to a genomic imprinting disorder disease model28,29, to investigate inter/transgenerational inheritance of epimutations in sperm (Fig. 1b). To demonstrate this, we induced CpG demethylation in H19-DMR by germline-specific epigenome editing and investigated whether the epimutation introduced in sperm was transmitted to the next generation and led to phenotypic changes. Furthermore, we developed a targeted histone editing technique that can be used to reveal the roles of histone modifications in DNA methylation reestablishment.

Results
Germline-specific epigenome-edited mice show complete erasure of DNA methylation at H19-DMR in sperm
To induce CpG demethylation of H19-DMR, we constructed a vector that expresses epigenome editing factors driven by the Stra8 premeiotic-specific marker gene promoter (Fig. 2a)30. In this system, dCas9 with a SunTag recruits the scFv-superfolder green fluorescent protein (sfGFP)-TET1CD fusion protein31,32, which contains the catalytic domain (CD) of the ten-eleven translocation (TET)1 hydroxylase, to the H19-DMR target locus, leading to targeted demethylation during spermatogenesis. By injecting the linearized vector into fertilized eggs, three transgenic (TG) strains were established from founder mice. A previous study showed that a transgene driven by the Stra8 promoter is activated only in the postnatal testis, and not in the ovary or prenatal gonad33. RT-qPCR and immunohistochemistry analyses confirmed that epigenome editing factors were specifically expressed in adult testis, from spermatogonia to spermatocytes (Fig. 2b, c and Supplementary Fig. 1). As paternal imprinting in H19-DMR is established by the pro-spermatogonia stage27, germline-specific epigenome editing is thought to occur after paternal imprinting establishment. H19-DMR CpG methylation levels were examined in sperm and MII oocytes from TG mice by bisulfite sequencing (Fig. 2d) and combined bisulfite restriction analysis (COBRA)34 (Supplementary Fig. 2), and we found robust CpG demethylation in sperm from all TG strains. By contrast, little difference in CpG methylation levels between TG and wild-type (WT) sperm was detected on analysis of six potential off-target regions (Supplementary Fig. 3). As expected, H19-DMR was unmethylated in both TG and WT mother-derived oocytes (Fig. 2d). These data indicate that we successfully established germline-specific epigenome-edited TG mice (StrH19EpiTG), and strain 445-7 was used in subsequent experiments.

Target-specific H19-DMR demethylation is partially inherited by the next generation and causes phenotypic changes
We next compared the offspring of TG mice with those of WT control mice. Offspring derived from TG mice were obtained by crossing heterozygous TG and WT mice (Fig. 3a), and those derived from TG fathers showed intrauterine growth retardation at birth (Fig. 3b, c). To exclude the possibility that the presence of the transgene might influence phenotypes, we also explored the phenotypes in offspring with and without TG alleles (TG and non-TG, respectively). Non-TG offspring derived from TG fathers exhibited a reduced birth weight (Fig. 3c), which is thought to be associated with inherited hypomethylation of H19-DMR from sperm. As expected, CpG methylation levels at the H19-DMR (CTCF binding sites m1–m4) in offspring of TG fathers were lower than those in offspring with WT fathers (Fig. 3d). Along with lowered methylation, offspring of TG fathers showed altered gene expression patterns, with increased H19 and decreased Igf2 levels (Fig. 3e). Non-TG offspring derived from TG fathers exhibited H19-DMR demethylation and altered gene expression at comparable levels to those in TG offspring (Fig. 3d, e). In addition to intrauterine growth retardation, other Silver-Russell syndrome-like phenotypes35, including postnatal growth retardation, reduced food intake, and body asymmetry, were observed (Supplementary Fig. 4). By contrast, offspring derived from TG mothers did not differ from WT offspring in terms of birth weight or CpG methylation levels (Fig. 3b–d), consistent with the fact that H19-DMR is demethylated in oocytes (Fig. 2d). Thus, the developed system proved that induced loss of DNA methylation at the DMR in sperm was partially transmitted to the F1 offspring, which exhibited resulting phenotypes.

We next investigated whether DNA methylation erasure in sperm was inherited transgenerationally (Supplementary Fig. 5a), and found that H19-DMR DNA hypomethylation was not inherited in sperm from F1 non-TG (Supplementary Fig. 5b, c) and their derivative F2 offspring (Supplementary Fig. 5d). These results indicate DMR DNA hypomethylation in F0 sperm is normalized during F1 germline cell development and not transgenerationally inherited by F2 offspring.

H19-DMR methylation is partially recovered from the 5’ end of the DMR during embryogenesis
Based on our experimental data, we surmised that DNA methylation was partially recovered in newborn mice, although it was completely erased in the sperm (Figs. 2d, 3d); we termed this phenomenon “intergenerational DNA methylation recovery”. The degree of DNA methylation recovery varied among individuals, and there was a strong positive correlation between birth weight and DNA methylation levels at the H19-DMR m3 and m4 sites and promoter regions (Supplementary Fig. 6). By contrast, DNA methylation levels at the 5’ end of the DMR (sites m1 and m2) were restored to almost WT levels (Supplementary Fig. 6), and there was no correlation between birth weight and DNA methylation levels at sites m1 and m2 (Supplementary Fig. 6). Further, time-course DNA methylation analysis across F1 development demonstrated that most DNA methylation at the 5’ end of the DMR (sites m1 and m2) was recovered before implantation, while that at 3’ end of the DMR (sites m3 and m4) was recovered after E7.5 (Fig. 4a). Furthermore, bisulfite sequencing analysis in sperm, early 1-cell zygotes (5h post-fertilization), and morula stage embryos derived from TG fathers revealed that DNA methylation recovery at m1 and m2 sites had not yet occurred in zygotes, but had occurred by the morula stage (Fig. 4b). Allele-specific bisulfite sequencing analysis confirmed that the paternal allele underwent DNA methylation recovery (Fig. 4c). These data suggest the existence of an undetermined factor that mediates DNA methylation recovery in early preimplantation embryos.

H3K9me3 is required for DNA methylation recovery
H3K9me3 is a repressive histone mark associated with heterochromatin and gene repression36, and known to co-occur with DNA methylation at the DMRs of imprinted genes37. Further, genome-wide H3K9me3 loss leads to a reduction of DNA methylation at DMRs in preimplantation embryos38. Nevertheless, although these studies suggest that H3K9me3 is required for DNA methylation maintenance, it is unknown whether H3K9me3 guides de novo DNA methylation in early embryos. According to public ChIP-seq data from gametes39,40, H3K9me3 is rarely enriched around the H19-DMR in either sperm or oocytes (Fig. 5a). To determine when H3K9me3 is deposited at the H19-DMR after fertilization, we applied an ultra-sensitive chromatin immunoprecipitation sequencing (ChIP-Seq) method, termed carrier DNA-assisted ChIP-Seq (CATCH-Seq)41,42, Interestingly, H3K9me3 CATCH-seq analysis in early 1-cell zygotes (5h post-fertilization) and morula embryos revealed that H3K9me3 could already be detected at the paternal allele of H19-DMR in early zygotes, particularly at the 5’ end of the DMR (sites m1 and m2) (Fig. 5a). H3K9me3 was further accumulated by the morula stage (Fig. 5a).

To examine the role of H3K9me3 in DNA methylation recovery, we attempted to erase H3K9me3 at the H19-DMR in early embryos. To achieve this, we conducted target-specific histone editing using an H3K9me3 demethylase, KDM4D43. Mixtures of dCas9-SunTag mRNA, scFv-GFP-Kdm4d mRNA, and two guide RNAs (gRNAs) complementary to sequence upstream of H19-DMR were injected into zygotes derived from TG fathers (Fig. 5b). Since the GFP-labeled epigenome editing factor (scFv-GFP-Kdm4d) was strongly expressed for at least 3 days until the morula stage (Supplementary Fig. 7), the H3K9me3 status of epigenome-edited embryos was examined at this stage. H3K9me3 CATCH-seq analysis of TG sperm-derived, non-injected morula embryos showed that H3K9me3 was detected around H19-DMR and upstream regions (Fig. 5c). We noted that levels of H3K9me3 at this locus in TG sperm-derived embryos were lower than those in WT embryos (see Discussion) (Fig. 5c). Nevertheless, when KDM4D was recruited to the upstream of H19-DMR by target-specific histone editing, H3K9me3 around the DMR was almost completely lost over an extensive region (approximately 10 kb). This extensive effect beyond the target sites is consistent with a recent report showing that an edited region per gRNA is comparatively broad in histone modification-edited embryonic stem cells44. By contrast, H3K9me3 was retained in DMRs of other imprinted genes (Igf2r, Rasgrf1) (Fig. 5d). Genome-wide comparative analysis demonstrated that H3K9me3 was lost at only 18 regions, including H19-DMR, in epigenome-targeted morula embryos (Fig. 5e and Supplementary Data 1). Thus, H3K9me3 demethylation by dCas9-SunTag histone editing did not affect H3K9me3 globally.

Next, these manipulated embryos were developed to embryonic stage E11.5, and their H19-DMR DNA methylation levels were examined (Fig. 6a). Importantly, target-specific H3K9me3 removal using dCas9-SunTag-Kdm4d revealed that DNA methylation recovery at H19-DMR is inhibited (Fig. 6b). Conversely, DNA methylation recovery was observed when a KDM4D mutant (H189A, lacking enzymatic activity)45 or another histone demethylase (KDM6B, H3K27 demethylase)46 was recruited (Fig. 6b). The extent of recovery in these negative controls was less than that in the non-treated control group (Fig. 6b), which could be due to steric hindrance of the epigenome editor complexes against DNA methyltransferases. In partial support of this hypothesis, a greater degree of recovery was observed when KDM4D was recruited to a different target locus, Rosa2647 (Fig. 6b). Finally, dissection of E15.5 and E18.5 embryos derived from TG sperm-derived zygotes injected or not with target-specific KDM4D revealed that H3K9me3 removal results in more severe intrauterine growth retardation than in the non-injected controls (Fig. 6c). These data indicate that H3K9me3 around H19-DMR is involved in DNA methylation recovery during embryogenesis and is physiologically relevant to the offspring’s phenotype. To further confirm the role of H3K9me3 in DNA methylation recovery, we also removed H3K9me3 globally in zygotes by injecting Kdm4d mRNA45. Similar to target-specific removal, global loss of H3K9me3 during early preimplantation development inhibited DNA methylation recovery, whereas control embryos injected with catalytic point-mutant Kdm4d mRNA, Egfp mRNA, or buffer alone led to DNA methylation recovery (Supplementary Fig. 8). Regardless of global or target-specific editing, the inhibitory effect of H3K9me3 removal on DNA methylation recovery was more prominent at the m1 and m2 sites than that at m3/m4, possibly because DNA methylation recovery occurs more robustly at the 5’ end of the DMR. Thus, both target-specific and global removal experiments confirmed that H3K9me3 is required for DNA methylation recovery at H19-DMR.

Methods
Animals
B6D2F1 mice were purchased from CLEA Japan (Kawasaki, Japan). ICR, C57BL/6NCrl (B6N) mice were purchased from Charles River Japan (Yokohama, Japan). PWK/PhJ (PWK) mice (RBRC00213, RIKEN BioResource Research Center) originated from 003715 (The Jackson Laboratory). The mouse facility was kept at 21–25°C and 40–60% humidity with a 12 h light–dark cycle. Animal had ad libitum access to food and water. All animal procedures were approved by the Animal Care and Experimentation Committee at Gunma University (No. 15-045) and carried out in accordance with approved guidelines.

Vector construction for germline-specific demethylation
The all-in-one epigenome editing vector, including dCas9 fused with five copies of GCN4 and an anti-GCN4 peptide antibody (scFv)-sfGFP-TET1CD fusion protein (pPlatTET-gRNA2, Addgene plasmid 82559), was previously reported by this laboratory31. An all-in-one epigenome editing vector, including nine gRNAs (Supplementary Data 2) targeting H19-DMR (pPlatTET-gRNA2-H19DMRx9), was also constructed as previously reported32. In this study, the CAG promoter of the pPlatTET-gRNA2-H19DMRx9 vector was replaced with the promoter of the Stra8 gene, which is specifically expressed during gametogenesis (pStrPlat-TET-gRNA2-H19DMRx9). The vector was linearized by digestion with ApaLI before microinjection.

Generation of StrH19EpiTG mice
B6D2F1 female mice aged 8–10 weeks were induced to superovulate by injecting 7.5 units of pregnant mare’s serum gonadotropin (SEROTROPIN; ASKA Pharmaceutical, Tokyo, Japan), followed 48 h later by 7.5 units of human chorionic gonadotropin (hCG; GONATROPIN, ASKA Pharmaceutical). After administration of hCG, females were mated with B6D2F1 males and zygotes isolated from the oviduct 21 h later. After treatment with M2 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.1% hyaluronidase (Sigma-Aldrich) for 3 minutes, zygotes were washed with M2 medium and then transferred to drops of M16 medium (Sigma-Aldrich) supplemented with penicillin and streptomycin at 37°C. Pronuclear injection was performed at 24–27 h post-hCG injection32. Linearized pStrPlat-TET-gRNA2-H19DMRx9 vector (35 ng/μl) was injected into the pronuclei of zygotes. Injected embryos were cultured in M16 medium at 37°C under 5% CO2 in air. The next day, embryos that had developed to the 2-cell stage were transferred into the ampulla of the oviduct (20–25 embryos per oviduct) of pseudopregnant ICR females. For vector integration analysis, genomic DNA was extracted from tail tips of obtained mice using a DNA extraction kit (DirectPCR Lysis Reagent, Mouse Tail; Viagenbiotech, CA, USA). PCR analysis using the primer set for dCas9 (Supplementary Data 2) demonstrated that 9 of 41 founder mice possessed the transgene. Three reproducible TG lines (441-2, 445-7, and 445-14) and their sub-lines were finally established; the 445-7 line was used in the present study and maintained by backcrossing WT B6D2F1 females with TG males. A TG male mouse that had been backcrossed twice with a PWK strain was used for allele-specific analysis.

Collection of sperm, oocytes, embryos, and newborn mice
Sperm (Fig. 2) were isolated from the cauda epididymis of TG or WT mice aged 10–21 weeks by dissecting tissue in 500 μl modified Human Tubal Fluid (mHTF) medium (Kyudo, Saga, Japan). Sperm were allowed to swim up for 1 h at 37°C under an atmosphere of 5% CO2 in air. The upper 200 μl fraction was collected, and the collected sperm were then resuspended and incubated with hypotonic buffer to eliminate red blood cells for 3 min at room temperature (RT), followed by washing with phosphate-buffered saline (PBS). Newborn (P0) mice (Fig. 3) were generated by natural mating of TG or WT (B6D2F1) parents. Blastocyst, E7.5, and E11.5 embryos (Fig. 4) were generated by in vitro fertilization followed by embryo transfer. Briefly, sperm were obtained from the caudal epididymis of adult TG or WT male mice, and capacitated by 1 h incubation in mHTF medium. Cumulus–oocyte complexes were harvested from B6D2F1 female mice aged 8–10 weeks 15–17 h after hCG administration, and then inseminated with activated sperm in mHTF medium. At 4 h post-insemination, zygotes with two pronuclei were subjected to in vitro culture in KSOM medium (ARC Resource, Kumamoto, Japan) at 37°C under 5% CO2 in air. The next day, a proportion of embryos that had developed to the 2-cell stage were transferred into the ampulla of the oviduct of pseudopregnant ICR females and harvested at E7.5 and E11.5. Wild-type preimplantation embryos (Fig. 5a) were generated by in vitro fertilization using PWK sperm and B6N oocytes65,66. Preparation of histone-edited embryos (Figs. 5b–e, 6) is described below.

DNA methylation analysis
Sperm pellets were re-suspended in RSB (10 mM NaCl, 10 mM Tris pH 7.5, and 25 mM EDTA) containing 2% sodium dodecyl sulfate (SDS), 2% 2-mercaptoethanol, and 1 mg/ml Proteinase K. Embryos were re-suspended in TNES (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 25 mM EDTA, and 1% SDS) containing 0.2 mg/ml Proteinase K. After incubation overnight at 55°C, DNA was isolated by phenol/chloroform extraction followed by ethanol precipitation. Genomic DNA from whole bodies of newborn mice was extracted using an AllPrep DNA/RNA Mini Kit (QIAGEN, Venlo, The Netherlands). Purified DNA samples (500 ng) were processed with an Epitect Plus DNA Bisulfite Kit (QIAGEN) according to the manufacturer’s instructions. Modified DNA was amplified using TaKaRa Taq (TaKaRa, Kusatsu, Japan) and the PCR primers described in Supplementary Data 2. Percentages of demethylated CpG sites were determined by COBRA34. Amplified fragments were cleaved with restriction enzymes (Supplementary Data 2) with recognition sequences located in CpG sites. PCR products were separated and quantified using capillary and microchip electrophoresis (MCE-202 MultiNA, Shimadzu, Kyoto, Japan). Methylation level was calculated as the percentage of cleaved PCR products (mV･μm) among total PCR products (mV･μm). Alternatively, PCR products were cloned using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA). Around 24 positive clones were sequenced for each sample using the BigDye terminator method (ABI PRISM 3500xL, Applied Biosystems, Foster City, CA, USA). Alignment, visualization, and quantification of bisulfite sequenced data were performed using QUantification tool for Methylation Analysis (QUMA; http://quma.cdb.riken.jp/top/index.html). Single-nucleotide polymorphisms (SNPs) of PWK and B6N strains were utilized for allele-specific DNA methylation analysis.

Bisulfite amplicon sequencing analysis
For comprehensive CpG methylation analysis of H19-DMR, genomic DNA samples treated with the Epitect Plus DNA Bisulfite Kit (QIAGEN) were amplified using specific primer pairs (Supplementary Data 2). A fragment library was prepared from pooled PCR products using the NEBNext Ultra Ⅱ DNA Library Prep Kit for Illumina (NEB). Briefly, 100 ng of PCR product was sheared using the Covaris® S220 System (Covaris Inc, MA) at 5°C for 2 cycles, treatment time 150 sec, with duty factor 10%, and 200 cycles/burst. To generate a fragment library, NEBNext Adapters (NEB) were ligated to both ends of the DNA. Barcoded libraries were amplified by PCR for 5 cycles using NEBNext Multiplex Oligos for Illumina (NEB). Prepared libraries were then sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA). Raw sequenced data generated in FASTQ format were imported into CLC Genomics Workbench 24.0.1 (QIAGEN), trimmed using the Trim reads 3.0 tool, and mapped to 15 kb reference sequence regions (NC_000073, Mus musculus strain C57BL/6J chromosome 7, GRCm38.p6 C57BL/6J) using the Map Bisulfite Reads to Reference tool. For CpG methylation analysis, trimmed libraries were mapped to the reference sequence described above using Map Bisulfite Reads to Reference, and 5-mC percentages calculated using Call Methylation levels 1.4.

RT-qPCR
Total RNA was isolated from the whole bodies of newborn mice using an AllPrep DNA/RNA Mini Kit (QIAGEN). Isolated RNA (8.5 μl, 2 μg) was treated with DNase I (50 μml−1) in a total volume of 10 μl at 37°C for 20 min. DNase I was inactivated by adding 0.8 μl 25 mM EDTA and incubating samples at 75°C for 10 min. cDNA was produced from each RNA sample using random primers (Invitrogen, Carlsbad, CA) and SuperScript II (Invitrogen), according to the manufacturer’s instructions. Total RNA extraction and cDNA synthesis from zygotes and preimplantation embryos were performed using the superPrep Cell Lysis & RT Kit for qPCR (TOYOBO, Osaka, Japan). Reverse transcription reactions were diluted 10-fold with water before qPCR. Gene expression levels of Igf2, H19, and dCas9 were measured using a LightCycler 96 (Roche) with TB Green Premix Ex Taq II (TakaRa), according to the manufacturer’s instructions. Expression levels were normalized against those of Gapdh. Primer sequences are listed in Supplementary Data 2.

Immunohistochemistry
Testes specimens were fixed in 4% paraformaldehyde diluted in PBS, embedded in paraffin, and 4 μm sections prepared. Sections were deparaffinized, autoclaved (121°C, 5 min) in 10 mM sodium citrate buffer pH 6.0 for antigen retrieval, and treated with methanol containing 0.3% H2O2 for 10 min. After blocking with 3% bovine serum albumin diluted in PBS containing Tween 20, an anti-GFP antibody (1:500 dilution, ab290, Lot No. GR3184825-1; Abcam, Cambridge, UK) was applied and incubated overnight at 4°C. Sections were reacted with Histofine Simple Stain MAX-PO(R) (Nichirei, Tokyo, Japan) for 30 min at room temperature, signals visualized using 3-3’-diaminobenzidine, and samples counterstained with hematoxylin.

Preparation of RNA
Kdm4d and its enzymatic mutant, Kdm4dMut (H189A)45, Kdm5b, EGFP, dCas9, dCas9-SunTag (L15aa), scFv-Kdm4d, scFv-Kdm4dMut, and scFv-Kdm6b were amplified from template plasmid using Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs (NEB), MA, USA) and primer sets for in vitro transcription (Supplementary Data 2). Amplified PCR products were gel-purified and subjected to in vitro transcription with an mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). In vitro transcribed RNA was eluted into RNase-free water, and the quality was checked by gel electrophoresis. Single guide RNAs (sgRNAs) for H19-DMR and Rosa26 (Supplementary Data 2) were purchased from Integrated DNA Technologies (IDT).

Histone editing by RNA injection
Zygotes derived from TG fathers were generated by in vitro fertilization. Sperm were obtained from adult TG male mouse caudal epididymis and capacitated by 1 h incubation in mHTF medium (Kyudo, Saga, Japan). Cumulus–oocyte complexes were harvested from B6D2F1 female mice 15–17 h after hCG administration, and then inseminated with activated sperm in mHTF medium. At 4 h post-insemination, zygotes with two pronuclei were subjected to cytoplasmic injection of RNA67 for histone editing. For global histone editing, in vitro transcribed Kdm4d/Kdm4dMut (1300 ng/μl) and EGFP (1300 ng/μl) mRNA samples were injected into the cytoplasm of zygotes in M2 medium. For target-specific histone editing, in vitro transcribed dCas9-SunTag mRNA (239 ng/μl), scFv-GFP-Kdm4d/Kdm4dMut mRNA (161 ng/μl), or scFv-GFP-Kdm6b mRNA (172 ng/μl), and two sgRNAs targeting sites upstream of H19-DMR (97 ng/μl each) or a sgRNA targeting Rosa26 (194 ng/μl) were injected into the cytoplasm of zygotes in M2 medium. Injected embryos were cultured in KSOM medium at 37°C under 5% CO2 in air. The next day, a proportion of the embryos that had developed to the 2-cell stage were transferred into the ampulla of the oviduct of pseudopregnant ICR females and harvested at E11.5–E18.5 for DNA methylation analyses or weight measurement.

H3K9me3 Carrier Assisted ChIP-seq (CATCH-seq)
H3K9me3 CATCH-seq libraries were prepared as previously described41,42, with some modifications. Zygotes (n=200–250) and morula embryos (n=30–45) were permeabilized using Nuclei EZ lysis buffer supplemented with 0.1% Triton X-100, 0.1% deoxycholate, complete EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonylfluoride on ice. Chromatin was fragmented in situ using 2 μ/μl MNase (M0247S, NEB) at 37°C for 7.5 min; the reaction was stopped by adding 1/10 volume of 100 mM EDTA and diluted with freshly prepared immunoprecipitation buffer. Then, 30 ng of annealed I-SceI carrier DNA was added to each sample. The forward and reverse strand sequences of the carrier DNA were as follows: /5AmMC6/Gtagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaat*c/3AmMO/ and /5AmMC6/Gattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatcccta*c/3AmMO/, respectively, where asterisks represent phosphorothioate bonds. Oligos were synthesized by IDT. For each immunoprecipitation reaction, 1.0 μl of rabbit polyclonal anti-H3K9me3 antibody (Active Motif, #39162, Lot No. 30220003) conjugated to precleared Dynabeads Protein A and G mixture was used. After immunoprecipitation at 4°C overnight, chromatin-Dynabeads were washed in low and high-salt wash buffers, and chromatin eluted in freshly prepared ChIP elution buffer at 65°C for 1 h. DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation. Adapter ligation was conducted using an NEBNext Ultra Ⅱ DNA Library Prep Kit for Illumina (E7645, NEB) at half scale relative to the manufacturer’s instructions, and libraries purified using 1.8× SPRIselect beads (B23318, Beckman Colter). DNA was amplified using KAPA Hifi 2× master PCR mix (KK2605) with indexing primers for 13–14 PCR cycles. After purification with 0.9× SPRIselect beads, samples were digested with I-SceI (5 μ/μl, NEB, R0694) at 37°C for 2 h, followed by heat inactivation at 65°C for 20 min, then purified using 0.9× SPRIselect beads. A second amplification was not performed. Libraries were sequenced on the NextSeq 500 (Illumina, CA, USA) or NovaSeq (Illumina) platforms.

CATCH-seq data analysis
Allele-specific analysis of hybrid embryos (B6N×PWK) was performed as described previously66, with some modifications. Reads were trimmed using fastp68 and mapped using bowtie269 with the options “-N 1”. The modified mm10 reference genome, in which PWK strain single-nucleotide polymorphisms were masked as N, was used for mapping. Multiply mapped reads were retained for downstream analysis in this study. PCR duplicates were removed using Picard MarkDuplicates (https://broadinstitute.github.io/picard/). SNPsplit70 was used to assign reads to their parental origin. For analysis of previous data39,40, reads were aligned to the original mm10 reference genome, and subsequent processes performed as described above. For analysis of TG embryo CATCH-seq data, reads were extended to 250 bp and processed as single-end reads to minimize the difference in sequencing conditions among samples, and subsequent processes were performed as described above. To visualize H3K9me3 enrichment, reads from biological replicates were pooled, and bigwig files generated using bamCoverage from deepTools71, with the options “--normalizeUsing RPKM --binSize 50”. Scatter plots showed a strong correlation between biological replicates of H3K9me3 CATCH-seq (Supplementary Fig. 9).

To confirm the specificity of epigenome editing by KDM4D, biological replicates of WT_control were pooled for peak calling using bdgpeakcall from macs272 with options “-l 3000 -c 4”. H3K9me3 enrichment peaks were compared using DiffBind73; differences of p<0.05 and |log2 fold change|>2 were considered significant changes induced by KDM4D (Fig. 5e and Supplementary Data 1).

Micro-CT and 3D reconstruction
Eight-week-old mice were scanned using a computed tomography instrument (DELPet μCT100, DEL Bio, Taoyuan County, Taiwan (R.O.C.)). Three-dimensional cross sections were generated with a resolution of one cross-section per 22.5 μm. Images were transformed into DICOM format and landmarks placed within the 3D representation at the endpoints of limb bones in the forelimb (humerus and ulna) and hindlimb (femur and tibia) using VivoQuant 2020 software (Invicro, MA, USA). Linear measurements were obtained for each pair of landmarks.

Statistical analysis
Box plots show the median (centerline), the 25th and 75th percentiles (box edges), and the minimum and maximum values (whiskers). Data shown in bar and line graphs are presented as mean and standard deviation (s.d.) values. DNA methylation, mRNA expression, body weight, and food intake were analyzed by Welch’s t test (two-tailed) for pairwise comparisons or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc HSD test for multiple comparisons. To analyze correlations between these variables, Pearson’s correlation coefficient (r) values were calculated. Fisher’s exact probability test was used to compare the frequency of body asymmetry. A p-value <0.05 was considered significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "As the authors correctly state transgenerational epigenetic inheritance is a controversial topic. In order to avoid just adding another “controversial paper on this topic to the literature, it would be needed that the results presented in this study are supported by strong experimental data and conceptual power. Moreover, writing should be as clear as possible to avoid misunderstanding and inaccuracies. Unfortunately, both these conditions are not full-filled.\n1) One of the critical claims made by the authors is that „DNA methylation was fully lost in the sperm“… but „it was partially restored during pre-implantation development, suggesting the existence of epigenetic memory that instructs de novo DNA methylation.“ This finding must be seen in the well-established context of massive global de novo DNA methylation ongoing in the early development. While it is shown that DNA methylation is restored in the early embryo (lines 172-190), the author do not provide convincing evidence that this remethylation is instructed by methylation information present in the sperm and later the paternal pronucleus and paternal genome.\n2) Of note, intergenerational inheritance in this paper is studied in the context of an imprinted locus. It will be established that several methylation marks at DMRs act together to mediate biological responses. Hence it is well-conceivable that other differentially methylated genomic regions that were not affected by epigenome editing were responsible for the recovery of methylation at the H19 locus.\n3) „Tri-methylation at lysine 9 of histone H3 (H3K9me3) was required for de novo DNA methylation.“ This is correct and convincingly shown in lines 231-242, but this is expected in the context of „normal“ de novo DNA methylation. There are no data providing evidence for a role of H3K9me3 in transgenerational inheritance.\n4) “..our study … identifies H3K9me3 as a mediator of intergenerational DNA methylation recovery.” This claim is not supported by data. There is no experimental evidence showing that H3K9me3 transmits epigenetic information through generations.\n5) Investigation of transgenerational inheritance would require the presence of the epimutation in the germline of the F1 generation and in F2. The authors demonstrate that this is not happening (Line 165-170). So claims on transgenerational inheritance are unfounded.\n6) Line 130: Fig. 2C also shows some expression of the epigenome editor in oocytes, different than stated by the authors. Oocyte DNA methylation data should be provided as well. Moreover, expression should be tested in the zygote and early embryo.\nMy conclusion is that the authors have presented an epigenome editing system in the germline, though some doubts remain on the germline-only expression of the factors. The application of the system to study intergenerational epigenetic inheritance is not convincing and the conclusions are not justified by the provided data.\nMinor comments:\nThere are language inaccuracies suggesting that concepts are not clear to the authors. For example:\nLine 55: Mendel did not know about DNA based inheritance.\nLine 58: This is only true if the absence of genetic changes is convincingly documented, which is not the case in many of these reports.", "reviewer_2": "The manuscript \" Germline epigenome editing identifies H3K9me3 as a mediator of intergenerational DNA methylation recovery\" by Horii et al. addresses several longstanding and critical questions regarding epigenetic inheritance, the regulation of imprints, and DNA methylation stability. Importantly, the authors tackle these issues using state-of-the-art methodologies, including in vivo epigenetic engineering, which represents a significant technical and conceptual advance in the field. The combination of cutting-edge approaches and well-designed, convincing strategies makes this study a highly valuable contribution to the field of epigenetics.\nThis is an exciting and timely manuscript that deserves publication. The results presented are not only robust but also, in some cases, unexpected, particularly regarding the recovery of DNA methylation. While the authors do not yet present a fully conclusive mechanism underlying these observations, this should not detract from the significance of their findings. In fact, the partially surprising behaviors of DNA methylation, particularly on differentially methylated regions (DMRs) following perturbations, are somewhat consistent with previous unresolved phenomena observed in the imprinting field. These findings further underscore the complexity of DNA methylation dynamics and open new avenues for investigation.\nThe authors' use of epigenome editing techniques, specifically for targeted DNA de-methylation and H3K9 modification, is noteworthy. These approaches are not only difficult to establish but also significantly more specific and precise compared to previous methodologies. The surprising nature of some of the findings only adds to the intrigue and importance of the work.\nOverall, this manuscript provides an impressive array of insights into the mechanisms governing DNA methylation stability and epigenetic regulation. I do not have any major concerns, as the study is well-conceived and executed. However, there are a few minor points that the authors could address to further enhance the clarity and comprehensiveness of their presentation:\n- “DNA methylation recovery occurred specifically at the paternal, but not the maternal, DMR after fertilisation”\nIt stays unclear on which basis this statement has been made. The data I found concerning DNA methylation, Fig.2, Fig.3,Fig.4, EXF1, EXF2, EXF4 do not distinguish the parental alleles. If there is information about the allelic dynamics of recovery this should be clearly and prominently analysed and depicted.\n- There are some typos in the figure legends", "reviewer_3": "The stable epigenetic inheritance across cell division of 5-cytosine DNA methylation (5meC) makes it a compelling vector for the transmission of non-genetic information across generations. However, DNA methylation reprogramming in the mammalian embryo and germline makes transmission of the mark inter/transgenerationally difficult in theory. Recently, a study from the Belmonte lab showed that artificially induced 5meC indeed is lost during germline reprogramming but, in some contexts, returns during the DNA methylation establishment phase (PMID: 36754048). This is a very compelling result, as it implies there is another epigenetic signature in addition to 5meC that allows for a memory of gene silencing. Horii, Hatada and colleagues have been leaders in the epigenome editing field since their breakthrough paper describing the dCas9-SunTag/TET in 2016 (PMID: 27571369). Here, they adapt their system to make its expression male germline-specific (Stra8 promoter), and target demethylation to the H19-Igf2 paternal imprint. In this way, they can demonstrate if sperm-specific demethylation is sufficient to eliminate transmission of the imprint, which results in a Silver-Russell Syndrome-like phenotype. As they previously showed (PMID: 32234052), their system is very efficient at in vivo removal of the methyl-mark. Now, they show that this signature can be maintained intergenerationally, and lead to a phenotype. While this is a satisfying result, it was also somewhat expected. More surprisingly, they went on to show that regions of the H19-Igf2 DMR reacquire DNA methylation in the preimplantation embryo. Furthermore, by targeting an H3K9me3-demethylase (KDM4D) using dCas9-SunTag, they elminate his DNA methylation gain. Hence, H3K9me3 is a second epigenetic signature that contributes to imprint regulation.\nI really appreciated this paper. The experiments are very straightforward, the data are clear, and the authors do a good job of explicating their study. Also, they make a breakthrough by establishing a link with H3K9me3 methylation and imprint transmission. This finding demonstrates the power of the dCas9-SunTag tool, and also is a strong hint at the mechanism underlying the observations made in the Belmonte paper mentioned above. This paper should be added to the list of important advances made in epigenome editing by Horii and Hatada. A major question, and one for future studies, is what is the signal for de novo deposition of H3K9me3 in the early embryo? I do have a list of ideas/concerns that I would like to see addressed:\n• The authors mentioned that CTCF protection could be an explanation for why the maternal imprint was not H3K9me3-methylated. However, they failed to mentioned the study from Burton et al. (PMID: 32601371) that showed SUV39H2 is required for H3K9me3 deposition on the paternal genome. This may be akin to the patenral-specific DNMT3A activity reported by Richard Albert et al, and already cited. In other words, protection of the maternal allele is one potential mechanism, but active targeting of the paternal allele is a non-exclusive second mechanism.\n• On this note, do the authors propose the signal for this deposition? Genetic? Epigenetic? If it’s a genetic signal, the 5’ of the DMR could be injected into paternal pronuclei, and observed if H3K9me3 accumulates. If it’s epigenetic, what could that signal be? Another histone mark or chromatin complex?\n• One experiment I felt was lacking was phenotypic characterization of the TG+KDM4D pups. Was the Silver-Russell phenotype exacerbated? This would be important to show the physiological relevance of the embryonic H3K9me3 deposition."}, "author_response": "Ref.: NCOMMS-24-84758\nTitle: Germline epigenome editing identifies H3K9me3 as a mediator of intergenerational\nDNA methylation recovery\nResponses to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nWe are grateful to reviewer #1 for the critical comments and useful suggestions that have\nhelped us to improve our paper. As indicated in the responses that follow, we have taken\nall these comments and suggestions into account in the revised version of our paper.\nAs the authors correctly state transgenerational epigenetic inheritance is a controversial\ntopic. In order to avoid just adding another “controversial paper on this topic to the\nliterature, it would be needed that the results presented in this study are supported by\nstrong experimental data and conceptual power. Moreover, writing should be as clear as\npossible to avoid misunderstanding and inaccuracies. Unfortunately, both these\nconditions are not full-filled.\n1) One of the critical claims made by the authors is that „DNA methylation was fully lost\nin the sperm“… but „it was partially restored during pre-implantation development,\nsuggesting the existence of epigenetic memory that instructs de novo DNA\nmethylation.“ This finding must be seen in the well-established context of massive global\nde novo DNA methylation ongoing in the early development. While it is shown that DNA\nmethylation is restored in the early embryo (lines 172-190), the author do not provide\nconvincing evidence that this remethylation is instructed by methylation information\npresent in the sperm and later the paternal pronucleus and paternal genome.\nResponse: We sincerely apologize for any lack of clarity in our previous response. The\ncentral objective of this study is to validate the applicability of our gamete-specific\nepigenome editing system in assessing whether inter/transgenerational epigenetic\ninheritance occurs at specific genomic loci. Using this system, we directly revealed that\nepigenetic information of H19-DMR is inherited intergenerationally but not\ntransgenerationally. Through targeted and global H3K9me3 removal experiments, we\nobserved that H3K9me3 plays a pivotal role in mediating the restoration of DNA\nmethylation at the H19-DMR. We believe this is a major discovery that shows how two\nkey epigenetic marks—DNA methylation and H3K9me3—work together to control\ngenomic imprinting in early embryos.\nAs the reviewer rightly pointed out, our data suggest that H3K9me3 itself may not serve\nas the intergenerational epigenetic mark. Instead, it is plausible that an upstream\nregulatory factor—yet to be identified—initiates this process. We acknowledge this as an\nimportant avenue for future investigation. Accordingly, we have incorporated the\nfollowing clarification into the Discussion section to better reflect our interpretation and\nthe future direction of our research:\n“Target-specific and global H3K9me3 removal experiments in zygotes revealed that\nH3K9me3 mediates DNA methylation recovery in the H19-DMR. By contrast, H3K9me3\nis nearly absent at the H19-DMR in sperm, suggesting that H3K9me3 is not the primary\nintergenerational memory directly transmitted across generations, but rather that some\nother factor(s) upstream of H3K9me3 is involved. Identification of the upstream factors,\nwhich could be genetic or epigenetic, awaits further investigations. Future studies will be\ndirected toward uncovering the mechanism of these upstream signals to further advance\nour understanding of epigenetic inheritance (L291-L298).”\n2) Of note, intergenerational inheritance in this paper is studied in the context of an\nimprinted locus. It will be established that several methylation marks at DMRs act\ntogether to mediate biological responses. Hence it is well-conceivable that other\ndifferentially methylated genomic regions that were not affected by epigenome editing\nwere responsible for the recovery of methylation at the H19 locus.\nResponse: We are grateful to the reviewer for highlighting this possibility. In this study,\nwe found that removal of H3K9me3 from the H19-DMR prevented DNA methylation\nrecovery after fertilization. Therefore, we believe that it is not necessary to consider the\ninvolvement of other potential DMRs in methylation recovery. We apologize if our\nresponse does not appropriately address or misses the point of the reviewer’s comment.\n3) „Tri-methylation at lysine 9 of histone H3 (H3K9me3) was required for de novo DNA\nmethylation.“ This is correct and convincingly shown in lines 231-242, but this is expected\nin the context of „normal“ de novo DNA methylation. There are no data providing\nevidence for a role of H3K9me3 in transgenerational inheritance.\nResponse: We sincerely apologize for any lack of clarity in our previous response. As\nthe reviewers commented, it is assumed that H3K9me3 itself may not be the primary\nepigenetic mark transmitted across generations, but rather that it is an early-deposited\nmediator required for subsequent DNA methylation recovery. There is likely to be another\nfactor(s) further upstream of H3K9me3 that intergenerationally transmits the information\nacross generations. It could be epigenetic marks or genetic sequence, and determining\nwhich one it is will be an important avenue for future investigations. The text in the\nAbstract pointed out by the reviewer has been corrected as follows: “Tri-methylation at\nlysine 9 of histone H3 (H3K9me3), which is deposited shortly after fertilization, is required\nfor subsequent de novo DNA methylation at the H19-DMR (L47-L49).” In addition, due\nto technical challenges and low throughput of germline targeted DNA demethylation, we\nadmit that it currently remains unknown how much H3K9me3-mediated recovery\noperates generally at other loci. We have mentioned this limitation in the Discussion as\nfollows: “Our study suggests that de novo H3K9me3 in zygotes is a prerequisite for\nsubsequent de novo DNA methylation, although the degree to which this mechanism is\nshared at loci other than H19-DMR is currently unknown (L321-L324).”\n4) “..our study … identifies H3K9me3 as a mediator of intergenerational DNA methylation\nrecovery.” This claim is not supported by data. There is no experimental evidence\nshowing that H3K9me3 transmits epigenetic information through generations.\nResponse: We apologize for any confusion caused by the lack of clarity in our previous\nresponse. In this context, the term ‘mediator’ refers to a factor that receives epigenetic\nmemory from upstream signals and transmits it downstream to facilitate functional\noutcomes. H3K9me3 is a “mediator,” but is not necessarily the primary memory in sperm.\nThere may be other factor(s) operating upstream of H3K9me3. At present, it remains\nunclear whether this upstream signal is genetic or epigenetic in nature. We acknowledge\nthat this as an important avenue for future investigations.\n5) Investigation of transgenerational inheritance would require the presence of the\nep of the\nepimutation in the germline of the F1 generation and in F2. The authors demonstrate\nthat this is not happening (Line 165-170). So claims on transgenerational inheritance are\nunfounded.\nResponse: We apologize for not being able to explain clearly what we meant previously.\nWe would like to argue that our gamete-specific epigenome editing system can be\napplied to verify whether inter/transgenerational inheritance occurs at a target region.\nIn this study, the germline-specific epigenome editing system revealed that the epigenetic\ninformation of H19-DMR is inherited intergenerationally but not transgenerationally. To\navoid any confusion, we have modified the manuscript as follows: In this study, we\ndeveloped a germline-specific DNA methylation editing system and applied it to a\ngenomic imprinting disorder disease model, to investigate inter/transgenerational\ninheritance of epimutations in sperm (L104-L106). We hope that revised text in the\nmanuscript is now clearer.\n6) Line 130: Fig. 2C also shows some expression of the epigenome editor in oocytes,\ndifferent than stated by the authors. Oocyte DNA methylation data should be provided\nas well.\nResponse: We agree that DNA methylation data of oocytes was missing from the\nmanuscript and should be added to bolster the findings of the study. Accordingly, we\nperformed bisulfite sequencing analysis of both TG and WT mother-derived MII oocytes,\nand confirmed that H19-DMR is originally hypomethylated in WT oocytes and that this\nhypomethylation is not altered in TG-derived oocytes (Fig. 2d).\nMoreover, expression should be tested in the zygote and early embryo.\nResponse: We also performed fluorescent microscopy analysis and RT-qPCR to detect\nexpression of the epigenome editor in zygotes and early embryos. Fluorescence\nmicroscopy analysis to detect sfGFP revealed no significant differences in fluorescence\nintensity between TG and WT samples (Supplementary Fig. 1a).\nFurthermore, RT-qPCR analysis did not detect the expression of the epigenome editor\ndCas9 at any developmental stage. Therefore, we concluded that the epigenome editors\nwere hardly expressed in zygotes or early embryos.\nMinor comments:\nThere are language inaccuracies suggesting that concepts are not clear to the authors.\nFor example:\nLine 55: Mendel did not know about DNA based inheritance.\nResponse: We agree with the reviewer’s suggestion. This sentence has been changed\nto “Phenotypic inheritance is believed to be achieved via the transmission of genomic\nDNA (L53).”\nLine 58: This is only true if the absence of genetic changes is convincingly documented,\nwhich is not the case in many of these reports.\nResponse: We thank the reviewer and fully agree with the comments. Indeed, genetic\nmutations might have occurred, but this has not been documented in many of these\nreports. Therefore, we are considering adding the following sentence to the Introduction\n“This is only true if the absence of genetic changes is convincingly documented, which\nis not the case in many of these reports”. However, we think that adding this sentence\ncould disrupt the flow of the surrounding text, so we decided not to add it to the revised\nmanuscript. However, if the reviewer could provide us with advice about how to\nincorporate it, we’d be happy to add it to the manuscript. .\nReviewer #2 (Remarks to the Author):\nWe are grateful to reviewer #2 for the critical comments and useful suggestions that have\nhelped us to improve our paper. As indicated in the responses that follow, we have taken\nall these comments and suggestions into account in the revised version of our paper.\nThe manuscript \" Germline epigenome editing identifies H3K9me3 as a mediator of\nintergenerational DNA methylation recovery\" by Horii et al. addresses several\nlongstanding and critical questions regarding epigenetic inheritance, the regulation of\nimprints, and DNA methylation stability. Importantly, the authors tackle these issues using\nstate-of-the-art methodologies, including in vivo epigenetic engineering, which\nrepresents a significant technical and conceptual advance in the field. The combination\nof cutting-edge approaches and well-designed, convincing strategies makes this study a\nhighly valuable contribution to the field of epigenetics.\nThis is an exciting and timely manuscript that deserves publication. The results\npresented are not only robust but also, in some cases, unexpected, particularly regarding\nthe recovery of DNA methylation. While the authors do not yet present a fully conclusive\nmechanism underlying these observations, this should not detract from the significance\nof their findings. In fact, the partially surprising behaviors of DNA methylation, particularly\non differentially methylated regions (DMRs) following perturbations, are somewhat\nconsistent with previous unresolved phenomena observed in the imprinting field. These\nfindings further underscore the complexity of DNA methylation dynamics and open new\navenues for investigation.\nThe authors' use of epigenome editing techniques, specifically for targeted DNA de-\nmethylation and H3K9 modification, is noteworthy. These approaches are not only\ndifficult to establish but also significantly more specific and precise compared to previous\nmethodologies. The surprising nature of some of the findings only adds to the intrigue\nand importance of the work.\nOverall, this manuscript provides an impressive array of insights into the mechanisms\ngoverning DNA methylation stability and epigenetic regulation. I do not have any major\nconcerns, as the study is well-conceived and executed. However, there are a few minor\npoints that the authors could address to further enhance the clarity and\ncomprehensiveness of their presentation:\n- “DNA methylation recovery occurred specifically at the paternal, but not the maternal,\nDMR after fertilisation”\nIt stays unclear on which basis this statement has been made. The data I found\nconcerning DNA methylation, Fig.2, Fig.3,Fig.4, EXF1, EXF2, EXF4 do not distinguish\nthe parental alleles. If there is information about the allelic dynamics of recovery this\nshould be clearly and prominently analysed and depicted.\nResponse: We thank the reviewer for this helpful suggestion. As the reviewers pointed\nout, it is important to investigate allele-specific methylation in this study. In order to\ndistinguish parental alleles, it is necessary to create TG mice with sub-species\nbackground (e.g., PWK strain). To this end, we attempted to backcross TG mice with\nsub-strain PWK mice, but the reproductive ability of hybrid TG mice was extremely low,\nand thus, we were unable to prepare them in time for the initial submission. Subsequently,\nwe obtained a male TG mouse that had been backcrossed twice with the PWK strain,\nwhich allowed us to perform bisulfite sequencing analysis using this mouse. Finally, we\nconfirmed that DNA methylation recovery occurs in the paternal allele but not in the\nmaternal allele (Fig. 4c).\n- There are some typos in the figure legends\nResponse: We thank the reviewer for this suggestion. We have proofread the\nmanuscript again to remove the typos.\nReviewer #3 (Remarks to the Author):\nWe are grateful to reviewer #3 for the critical comments and useful suggestions that have\nhelped us to improve our paper. As indicated in the responses that follow, we have taken\nall these comments and suggestions into account in the revised version of our paper.\nThe stable epigenetic inheritance across cell division of 5-cytosine DNA methylation\n(5meC) makes it a compelling vector for the transmission of non-genetic information\nacross generations. However, DNA methylation reprogramming in the mammalian\nembryo and germline makes transmission of the mark inter/transgenerationally difficult\nin theory. Recently, a study from the Belmonte lab showed that artificially induced 5meC\nindeed is lost during germline reprogramming but, in some contexts, returns during the\nDNA methylation establishment phase (PMID: 36754048). This is a very compelling\nresult, as it implies there is another epigenetic signature in addition to 5meC that allows\nfor a memory of gene silencing. Horii, Hatada and colleagues have been leaders in the\nepigenome editing field since their breakthrough paper describing the dCas9-\nSunTag/TET in 2016 (PMID: 27571369). Here, they adapt their system to make its\nexpression male germline-specific (Stra8 promoter), and target demethylation to the\nH19-Igf2 paternal imprint. In this way, they can demonstrate if sperm-specific\ndemethylation is sufficient to eliminate transmission of the imprint, which results in a\nSilver-Russell Syndrome-like phenotype. As they previously showed (PMID: 32234052),\ntheir system is very efficient at in vivo removal of the methyl-mark. Now, they show that\nthis signature can be maintained intergenerationally, and lead to a phenotype. While this\nis a satisfying result, it was also somewhat expected. More surprisingly, they went on to\nshow that regions of the H19-Igf2 DMR reacquire DNA methylation in the preimplantation\nembryo. Furthermore, by targeting an H3K9me3-demethylase (KDM4D) using dCas9-\nSunTag, they elminate his DNA methylation gain. Hence, H3K9me3 is a second\nepigenetic signature that contributes to imprint regulation.\nI really appreciated this paper. The experiments are very straightforward, the data are\nclear, and the authors do a good job of explicating their study. Also, they make a\nbreakthrough by establishing a link with H3K9me3 methylation and imprint transmission.\nThis finding demonstrates the power of the dCas9-SunTag tool, and also is a strong hint\nat the mechanism underlying the observations made in the Belmonte paper mentioned\nabove. This paper should be added to the list of important advances made in epigenome\nediting by Horii and Hatada. A major question, and one for future studies, is what is the\nsignal for de novo deposition of H3K9me3 in the early embryo? I do have a list of\nideas/concerns that I would like to see addressed:\n• The authors mentioned that CTCF protection could be an explanation for why the\nmaternal imprint was not H3K9me3-methylated. However, they failed to mentioned the\nstudy from Burton et al. (PMID: 32601371) that showed SUV39H2 is required for\nH3K9me3 deposition on the paternal genome. This may be akin to the paternal-specific\nDNMT3A activity reported by Richard Albert et al, and already cited. In other words,\nprotection of the maternal allele is one potential mechanism, but active targeting of the\npaternal allele is a non-exclusive second mechanism.\nResponse: We are grateful for the reviewer’s request to include the study by Burton et\nal., and we apologize for our oversight in not referencing this important work earlier. We\nagree that the DNA methylation recovery of H19-DMR via H3K9me3 may occur through\nthe mechanism outlined by the reviewer. Therefore, we have now cited this paper, and\nadded the following sentence to the Discussion:\n“Burton et al. reported that de novo H3K9me3 catalyzed by SUV39H2 (H3K9\nmethyltransferase) occurs in the paternal genome immediately after fertilization, which\nis consistent with our observation that H3K9me3 emerges on the paternal allele of H19-\nDMR in early zygotes (L315-L318).”\n• On this note, do the authors propose the signal for this deposition? Genetic?\nEpigenetic? If it’s a genetic signal, the 5’ of the DMR could be injected into paternal\npronuclei, and observed if H3K9me3 accumulates. If it’s epigenetic, what could that\nsignal be? Another histone mark or chromatin complex?\nResponse: We thank the reviewer for the insightful suggestion. At this point, it is unclear\nwhether this is a genetic or an epigenetic signal, but we have obtained preliminary data\nthat we think potentially addresses this point. We generated mice lacking the 5’ region of\nthe H19-DMR, and examined phenotypes of the offspring of the TG father lacking the 5’\nregion of the DMR (Figures a, b). The results showed no DNA methylation recovery in\nthese offspring (Figure c) and the offspring had a more severe Silver-Russell syndrome-\nlike phenotype, including intrauterine growth retardation at E18.5 (Figure d). This\nindicates that the genetic sequence of the 5’ DMR is necessary for DNA methylation\nrecovery. On the other hand, it cannot be ruled out that histone marks other than\nH3K9me3 and chromatin complexes are transmitted from sperm. We hope to clarify this\nin a future study.\nIn addition, we added the following description to the Discussion:\n“Target-specific and global H3K9me3 removal experiments in zygotes revealed that\nH3K9me3 mediates DNA methylation recovery at the H19-DMR. By contrast, H3K9me3\nis nearly absent at the H19-DMR in sperm, suggesting that H3K9me3 is not the primary\nintergenerational memory directly transmitted across generations, but rather that some\nother factor(s) upstream of H3K9me3 is involved. Identification of the upstream factors,\nwhich could be genetic or epigenetic, awaits further investigations. Future studies will be\ndirected toward uncovering the mechanism of these upstream signals to further advance\nour understanding of epigenetic inheritance (L291-L298).”\nRegarding the reviewer’s proposal that we perform DNA fragment injection followed by\nH3K9me3 ChIP-seq, we think that this would be pose many problems, not least because\nsuch an experiment would be completely new and to the best of our knowledge, has not\nbeen previously performed in this field of research. There are too many concerns to\novercome, including optimization of DNA concentration, fragment length, protection\nchemistry for the fragment (otherwise it could be degraded too soon), and ChIP-seq\noptimization for the injected fragments (we don’t know if our protocol optimized for\nchromatin will work for a DNA fragment). So, we’d like to avoid performing this\nexperiment, at least until after the publication of this work. We think the question about\nwhether the signal is genetic or epigenetic can be left open for investigation in a future\nstudy.\n• One experiment I felt was lacking was phenotypic characterization of the TG+KDM4D\npups. Was the Silver-Russell phenotype exacerbated? This would be important to show\nthe physiological relevance of the embryonic H3K9me3 deposition.\nResponse: We thank the reviewer for this suggestion. The weights of TG+KDM4D\nembryos were examined at E15.5 and E18.5. We found that removal of H3K9me3 by\ntarget-specific histone editing resulted in more severe intrauterine growth retardation at\nE18.5 (Fig. 6c), demonstrating the physiological relevance of H3K9me3 deposition."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In general, the authors made a good job to clarify the paper and add additional data and they resolved most of my concerns.\nOverall, this is a very nice and convincing paper. I only see little room for improvement at few places in the manuscript. I recommend acceptance of an appropriately adjusted manuscript.\nComments\n1) The authors showed at their model locus full establishment of a DNA methylation epimutation. Still there was “incomplete” intergenerational inheritance and no transgenerational inheritance. These important conclusions should be spelled out clearly in the abstract to become visible to the community.\n2) It would be of interest, if editing of DNA methylation and H3K9me3 leads to transgenerational inheritance of the combined epimutation.\n3) H3K9me3 has been shown to support incorporation of DNA methylation in general. Please clarify if there is a specific effect at imprinted loci that goes beyond that and adjust the writing accordingly.\n4) Line 39: “validate” implies the authors aim to show the concept is correct. I recommend more neutral wording like “investigate”, “examine” or even “challenge”, which comes more from the friendly critical side.\n5) Line 50: Maybe it is better to say: “H3K9me3 as a mediator for DNA methylation recovery also acting at imprinted loci”?\n6) Line 58 and based on the rebuttal letter. One may state: “germline. While these data are still under discussion because of difficulties in showing the absence of mutations, DNA methylation…”\n7) Line 68: It will be good here to provide the definition of inter and transgenerational inheritance used in this paper.\n8) Line 298: “Candidates include those *factors* known to be …”,", "reviewer_2": "The authors clarified all my concerns.", "reviewer_3": "The authors have satisfactorily responded to my comments. I look forward to publication of these exciting results.\nMaxim Greenberg"}, "author_response": "Ref.: NCOMMS-24-84758A\nTitle: Germline epigenome editing identifies H3K9me3 as a mediator of intergenerational\nDNA methylation recovery\nResponses to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nWe are grateful to reviewer #1 for the critical comments and useful suggestions that have\nhelped us to improve our paper. As indicated in the responses that follow, we have taken\nall these comments and suggestions into account in the revised version of our paper.\nIn general, the authors made a good job to clarify the paper and add additional data and\nthey resolved most of my concerns. Overall, this is a very nice and convincing paper. I\nonly see little room for improvement at few places in the manuscript. I recommend\nacceptance of an appropriately adjusted manuscript.\nComments\n1) The authors showed at their model locus full establishment of a DNA methylation\nepimutation. Still there was “incomplete” intergenerational inheritance and no\ntransgenerational inheritance. These important conclusions should be spelled out clearly\nin the abstract to become visible to the community.\nResponse: Thank you for the helpful suggestion. We consider it essential to highlight\nthe findings from the inter/transgenerational inheritance experiments at the H19-DMR\nlocus in the abstract section. While we mostly agree, we felt that \"incomplete\" could\nsound slightly negative, so we replaced it with the more balanced term \"partial\" as shown\nbelow:\n“Our study provides a robust germline editing tool, which reveals partial intergenerational\ninheritance and no transgenerational inheritance at the model locus.” (L50-L51)\n2) It would be of interest, if editing of DNA methylation and H3K9me3 leads to\ntransgenerational inheritance of the combined epimutation.\nResponse: We appreciate the reviewer’s insightful comment. While this intriguing\npossibility has not yet been investigated in our current study, we consider it a compelling\ndirection for future research and intend to explore it further.\n3) H3K9me3 has been shown to support incorporation of DNA methylation in general.\nPlease clarify if there is a specific effect at imprinted loci that goes beyond that and adjust\nthe writing accordingly.\nResponse: We appreciate the reviewer’s thoughtful comment. While H3K9me3 is\nindeed known to generally support DNA methylation, our observations suggest that its\nrole at imprinted loci may involve additional specificity. However, further investigation is\nrequired to determine whether this effect goes beyond the general mechanism.\nTherefore, we revised manuscript as follows:\n“Although H3K9me3 is generally recognized for its role in supporting DNA methylation,\nfurther research is necessary to determine whether this H3K9me3-mediated DNA\nmethylation recovery is a general phenomenon or specific to imprinted loci.” (L335-L338)\n4) Line 39: “validate” implies the authors aim to show the concept is correct. I recommend\nmore neutral wording like “investigate”, “examine” or even “challenge”, which comes\nmore from the friendly critical side.\nResponse: Thank you for the helpful suggestion. In accordance with the reviewer's\nrecommendation, we have revised \"validate\" to \"investigate\". (L39)\n5) Line 50: Maybe it is better to say: “H3K9me3 as a mediator for DNA methylation\nrecovery also acting at imprinted loci”?\nResponse: Following the reviewer’s comment, we revised the sentence as follows:\n“Furthermore, we identify H3K9me3 as a mediator for DNA methylation recovery also\nacting at imprinted loci.” (L51-L52)\n6) Line 58 and based on the rebuttal letter. One may state: “germline. While these data\nare still under discussion because of difficulties in showing the absence of mutations,\nDNA methylation…”\nResponse: We truly appreciate the excellent phrasing the reviewer provided. We have\nrephrased it as follows. “…germline. While these data are still under discussion because\nof difficulties in showing the absence of genetic mutations, DNA methylation…” (L59-\nL60)\n7) Line 68: It will be good here to provide the definition of inter and transgenerational\ninheritance used in this paper.\nResponse: We thank the reviewer for this valuable suggestion. To improve clarity, we\nhave now included definitions of intergenerational and transgenerational inheritance in\nthe revised manuscript as follows:\n“In this study, we define intergenerational inheritance as the transmission of epigenetic\nalterations originating in the gametes to the immediate offspring (F1 generation). By\ncontrast, transgenerational inheritance refers to the sustained presence of these\nmodifications in subsequent generations (e.g., F2 and beyond) without continued\nexposure to the initial trigger.” (L73-L77)\n8) Line 298: “Candidates include those *factors* known to be …”\nResponse: Following the reviewer’s suggestion, we included the term \"factors\". (L309)"}, {"round": 3, "reviewer_comments": {"reviewer_1": "All my comments were addressed. I recommend acceptance of this paper."}, "author_response": ""}]

Decision letter