G9a-mediated H3K9me2 orchestrates intestinal epithelial regeneration through epigenetic silencing of cell cycle-related genes

Domain: biology
Published: 2026-01-19
Editorial outcome: minor_revision

Abstract
Histone modifications play an important role in intestinal homeostasis and regeneration. Here, we identify histone H3 lysine 9 di-methylation (H3K9me2) as an epigenetic regulator of intestinal epithelial repair through mass spectrometry-based screening of histone modifications. We then find that H3K9me2 and its methyltransferase G9a levels are reduced during acute injury and progressively increase during regeneration in both mouse models and human clinical samples. Genetic ablation of G9a in intestinal epithelial cells or pharmacological inhibition of its enzymatic activity substantially impairs intestinal regeneration and reduces survival following irradiation. Mechanistically, integrative genomic analyses reveal that G9a-mediated H3K9me2 suppresses chromatin accessibility and transcriptional activity of cell cycle arrest genes, including Rb1cc1, Rb1, Cdkn1a, and Pten, thereby promoting intestinal stem cell proliferation. Furthermore, we elucidate that IL-4-STAT6 signaling controls G9a expression during regeneration, i.e., IL-4 upregulation leads to STAT6 phosphorylation and subsequent transcriptional activation of G9a. These findings establish the IL-4-STAT6-G9a-H3K9me2 regulatory axis as a critical epigenetic mechanism controlling intestinal regeneration with therapeutic potential for gastrointestinal disorders.

Introduction
The intestinal epithelium is a meticulously arranged system, composed of differentiated villi and actively proliferating crypts1. Continuous renewal of intestinal tissue is supported by intestinal stem cells (ISCs) positioned at the bottom of the crypts2. These ISCs, as master architects of intestinal regeneration, continually divide to generate both stem cells and a population of transit amplifying (TA) daughter cells3,4. The TA cells serve as progenitors for a suite of terminally differentiated cell types5. Balanced proliferation state of crypt cells, including ISCs and TA cells, is essential not only for the physiological integrity of the intestinal system but also for intestinal repair triggered by epithelial injury, caused by either irradiation or inflammation1,6. Hundreds of well-orchestrated genes guide crypt cell proliferation, thus ensuring the maintenance of tissue homeostasis and facilitating a timely response to injury. Although the genome is largely stable, the epigenome allows for dynamic control of gene expression and cellular identity7. Accordingly, varying levels of epigenetic regulation contribute to intestinal health through transcriptional activation or repression of specific sets of essential genes that control crypt cell proliferation and differentiation7–9.

Histone modifications, as one of the core epigenetic mechanisms, dynamically regulate chromatin accessibility and transcriptional activity in response to intracellular or extracellular signals10,11. For example, histone acetylation, primarily catalyzed by histone acetyltransferases, generally promotes chromatin opening and gene activation, while histone deacetylases remove these marks to facilitate gene silencing12. Histone methylation presents a more complex regulatory landscape, where methylation of different lysine residues (e.g., H3K4, H3K9) and varying degrees of methylation (mono-, di-, tri-methylation) can lead to distinct outcomes, such as gene activation or repression13.

The regulatory enzymes for histone methylation are particularly diverse, encompassing a large family of methyltransferases and demethylases with distinct substrate specificities14,15. For example, SET1/MLL complexes add activating H3K4 methylation marks16, G9a complex primarily deposits repressive H3K9me2 marks17,18, and SETDB1 mediates H3K9me3 deposition19. Histone demethylases such as LSD1 and JMJD2 family members provide additional regulatory control by removing specific methylation marks20. This intricate enzymatic network enables precise temporal and spatial control of gene expression through dynamic histone methylation states, allowing cells to rapidly respond to environmental stimuli.

In the intestinal epithelium, several histone modifications have been implicated in maintaining epithelial homeostasis, stem cell function, and cellular differentiation. Histone acetyltransferases such as p300 and CBP regulate intestinal stem cell proliferation and differentiation by modulating Wnt signaling target genes21. SETDB1-mediated H3K9me3 is necessary for intestinal epithelial differentiation and the prevention of intestinal inflammation by silencing endogenous viral elements in the genome22,23. Chronic stress has been observed to induce alterations in H3K9me1 at the promoters of tight junction genes in rat colon epithelial cells24. While these studies have provided valuable insights into the roles of specific histone modifications on steady-state conditions or chronic disease models, our understanding of how histone modifications dynamically regulate gene expression programs during intestinal injury and repair remains limited.

In this study, we systematically screened for differential histone modifications associated with intestinal epithelial regeneration and identified H3K9me2 as exhibiting significant changes. We then evaluated the correlation between the levels of H3K9me2 and its modifying enzyme G9a and the extent of intestinal injury repair. Subsequently, we reduced the H3K9me2 levels by either knocking out the G9a gene in intestinal epithelial cells (IECs) and ISCs or inhibiting its enzymatic activity to analyze its roles in intestinal homeostasis and injury repair in mice. Next, we explored the underlying mechanism of H3K9me2 in this process. Finally, we investigated the upstream regulatory pathways controlling this modification. Our findings reveal a significant epigenetic modification that controls ISCs and TA cells during intestinal injury and the repair process, and highlight H3K9me2 as a potential therapeutic target for interventions in gastrointestinal epithelium-related diseases.

Results
H3K9me2 and G9a levels correlate with intestinal epithelium injury and regeneration
To investigate the relationship between histone modifications and intestinal injury regeneration, we employed an irradiation-induced injury model and performed histone mass spectrometry analysis on chromatin fractions isolated from intestinal crypt cells (Fig. 1a). Among detected 14 histone H3 tail and 5 histone H4 tail modifications, H3K9me2 was significantly upregulated at 96h of post-irradiation, while H3K27me3 and H3K14ac were significantly downregulated (Fig. 1b, c). To validate these findings, we performed immunoblotting to examine the temporal dynamics of these three modifications along with other H3K9 methylation states (H3K9me1 and H3K9me3) throughout the injury-repair process. H3K9me2 exhibited a biphasic pattern, with initial reduction followed by subsequent upregulation during regeneration (Fig. 1d). G9a, the methyltransferase responsible for H3K9me2 modification, showed temporal changes that closely paralleled H3K9me2 levels (Fig. 1e). IHC analysis of H3K9me2 and G9a levels confirmed these observations (Supplementary Fig. 1a, b). To corroborate these findings, we examined a DSS-induced injury and regeneration model and observed similar dynamics (Supplementary Fig. 2a–d). These data suggested that H3K9me2 and its methyltransferase G9a are closely associated with intestinal injury and repair processes.

To further validate these findings in clinical settings, we collected large intestinal tissue samples from 20 patients with rectal cancer who had undergone radiotherapy and 15 patients who had not received radiotherapy. Within the same patients, the staining intensity for H3K9me2 and the corresponding G9a in the nuclei of IECs at the repair region was significantly higher than that in the injured region, and also exceeded that of IECs in the normal region of non-radiotherapy patients (Fig. 1f–h). Moreover, there was a positive correlation between the levels of H3K9me2 and G9a (Fig. 1i). We also analyzed these in human intestinal tissue samples from patients with CD at various stages, including moderate, severe, and remission phases. H3K9me2 level in IECs decreased during disease progression and negatively correlated with the Crohn’s Disease Activity Index (Supplementary Fig. 3a–c). Interestingly, H3K9me2 level was restored and increased during the remission phase (Supplementary Fig. 3b). A similar correlation was observed between G9a expression and CD severity, i.e., the more severe the disease, the lower the level of G9a in the nuclei of IECs (Supplementary Fig. 3a, d, e). A positive correlation was found between the levels of H3K9me2 and G9a within these tissues (Supplementary Fig. 3f). Immunoblotting confirmed the expression patterns of H3K9me2 and G9a, whereas no significant changes were observed for H3K9me1 and H3K9me3 (Supplementary Fig. 3g).

Collectively, these findings demonstrate a positive association between the nuclear levels of H3K9me2 and G9a and the degree of intestinal regeneration, with the concordance between mouse and human data suggesting an evolutionarily conserved epigenetic mechanism underlying intestinal repair.

H3K9me2 is required for intestinal regeneration
We next employed IEC-specific G9a knockout (G9aΔIEC, Supplementary Fig. 4a–d) and a G9a methyltransferase competitive inhibitor25, UNC0642, to reduce H3K9me2 levels in the intestinal epithelium, and combined these approaches with the two aforementioned models to corroborate the role of H3K9me2 in intestinal regeneration. The results showed that G9a deletion in the intestinal epithelium or treatment with UNC0642 significantly reduced H3K9me2 level without affecting H3K9me1 and H3K9me3 expression, confirming that both methods effectively decrease H3K9me2 modification (Supplementary Fig. 4e, f).

In the 8Gy irradiation-induced model, we found that G9aΔIEC mice exhibited significantly lower body weight and small intestine length than the controls (Fig. 2a–c). Histological staining of the small intestine revealed that the number of regenerating crypts, as well as the corresponding ISCs (OLFM4 positive) and proliferative cells (Ki67 positive), were significantly reduced in G9aΔIEC mice compared to controls (Fig. 2d–f), indicating that the IEC-specific knockout of G9a exacerbates post-irradiation symptoms. Similarly, treatment with UNC0642 significantly slowed down the regeneration process after irradiation, as evidenced by a more rapid decline in body weight, a greater reduction in small intestine length, and a lower number of regenerating crypts (Fig. 2g–l). When the irradiation was increased to 10Gy, both G9aΔIEC mice and UNC0642-treated mice exhibited significantly reduced survival time compared to controls (Fig. 2m).

In the DSS-induced model, G9aΔIEC mice also displayed increased susceptibility compared to G9afl/fl mice, marked by more significant weight loss, a higher disease activity index, and enhanced intestinal permeability (Supplementary Fig. 5a–d). Furthermore, analyses of small intestinal tissues collected on day 14 showed that G9a knockout resulted in a significant decrease in the number of intestinal crypts, stem cells, and proliferating cells (Supplementary Fig. 5e–g).

Taken together, these results indicate that G9a-mediated H3K9me2 plays a crucial role in the repair of intestinal injury.

H3K9me2 depletion causes villi-crypt structural abnormalities under homeostatic conditions
As components of the most vigorously self-renewing tissue, crypt ISCs and TA cells play key roles in maintaining intestinal homeostasis and regeneration26. Therefore, we evaluated the influence of H3K9me2 on the morphology and cell composition of the villi-crypt structure after G9a knockout or treatment with the UNC0642 inhibitor under homeostatic conditions without any injury treatment. In G9aΔIEC mice, we observed significantly reduced crypt depth and villus height compared to G9afl/fl mice (Supplementary Fig. 6a, b). IHC staining revealed a reduction of OLFM4-positive cells (indicating ISCs) and Ki67-positive cells (mainly representing TA cells) in G9aΔIEC mice, with corresponding downregulated expression of both genes (Fig. 3a–c). ISCs differentiate into progenitors and eventually form various cell types in the villi, including Paneth, goblet, and enteroendocrine cells (Supplementary Fig. 6c)27. Therefore, we compared these cell types in both mice and found significant reductions in their numbers and associated marker gene expressions in the G9a knockout mice (Supplementary Fig. 6d–f). Consistent with the results from G9a knockout mice, UNC0642 treatment also altered villi-crypt structure, including reduced crypt depth and villus height, and decreased numbers and marker gene expressions of ISCs and TA cells and their differentiated downstream cells (Supplementary Fig. 7a–h). Collectively, these results suggest G9a-mediated H3K9me2 in crypt cells governs ISC and TA cell numbers, thus maintaining crypt and villus structure at steady state.

H3K9me2 controls the proliferation state of ISCs under both homeostatic and injury conditions
The maintenance of crypt cell numbers relies on their proliferation capacity28. Intriguingly, we also observed a predominant localization of G9a in the nuclei of ISCs and TA cells in both human and mouse small intestine tissues (Supplementary Fig. 8a–f), indicating that G9a and its catalyzed H3K9me2 may play a role in these cells. To investigate whether G9a-mediated H3K9me2 regulates ISCs and TA cells proliferation, we employed the EdU pulse-chase assay to trace the proliferative cells under homeostatic conditions. The results showed that the labeled cells were primarily located within crypts, and their numbers were significantly reduced in G9aΔIEC mice after 1.5h of EdU incorporation (Supplementary Fig. 9a, b). At later time points, these EdU-labeled cells were positioned lower in the crypts of G9aΔIEC mice compared to G9afl/fl mice (Supplementary Fig. 9a, b), suggesting a slower proliferation rate in the presence of diminished H3K9me2 level. Immunofluorescence analysis further confirmed the notable decrease in the ISCs' proliferation after G9a deletion (Fig. 3d, e). Correspondingly, the expression levels of cell cycle-related proteins in crypt cells of G9aΔIEC mice, including Cyclin A2, Cyclin E2, Cyclin D1, and Cyclin H, were significantly reduced compared to G9afl/fl mice (Fig. 3f). When we treated these mice with UNC0642, we observed the same decreased phenomenon as in G9aΔIEC mice, further confirming the role of H3K9me2 in crypt proliferation (Supplementary Fig. 10a–e).

To further evaluate the impact of H3K9me2 on ISCs' function, we generated mice with ISC-specific G9a deletion (Supplementary Fig. 11a) and assessed its influence on crypt homeostasis and intestinal injury repair separately. Under homeostatic conditions, loss of G9a in ISCs resulted in a substantial reduction of H3K9me2 levels, leading to decreased stem cell proliferation, reduced cell numbers, and abnormal crypt architecture (Supplementary Fig. 11b–i). Furthermore, under radiation-induced injury conditions, ISC-specific G9a deficiency impaired regenerative capacity (Supplementary Fig. S12a–e), corroborating the critical role of H3K9me2 in maintaining ISC function during both homeostasis and regeneration.

To complement our in vivo findings, we established small intestinal organoids from isolated crypts and observed that G9a deficiency significantly reduced organoid area, indicating an impact on their growth (Fig. 3g, h). Individual organoid tracking revealed that those derived from G9aΔIEC crypts had significantly fewer buds compared to the ones from G9afl/fl, illustrating that H3K9me2 depletion leads to a reduction in the number of differentiated cells downstream of ISCs (Fig. 3i, j). We also treated organoids with UNC0642, which phenocopied the G9a deletion results (Supplementary Fig. 13a–d).

Overall, these results indicate that H3K9me2, regulated by G9a, controls the proliferation state of intestinal stem cells in crypts, thereby influencing their numbers; its dysfunction would damage intestinal homeostasis and regeneration capacity.

H3K9me2 suppressed cell cycle arrest genes
To elucidate the molecular events following H3K9me2 modification, we performed comprehensive genomic analyses, including transposase-accessible chromatin using sequencing (ATAC-seq), H3K9me2 and G9a chromatin immunoprecipitation with sequencing (ChIP-seq), and RNA sequencing (RNA-seq), to identify its responsive genes in isolated intestinal crypts under homeostatic conditions. ATAC-seq analysis revealed a significant increase in global chromatin accessibility in the crypt cells from G9aΔIEC mice, especially at transcription start sites (Fig. 4a). We identified 26,271 chromatin accessibility sites in G9aΔIEC crypt cells compared to 20,815 sites in G9afl/fl cells. Gene ontology enrichment analysis of these differential accessibility sites indicated primary association with regulation of cyclin-dependent protein serine/threonine kinase activity and cyclin-dependent protein kinase activity (Fig. 4b).

H3K9me2 ChIP-seq analysis showed that chromatin occupancy of H3K9me2 was significantly reduced in G9aΔIEC mice (30,861 peaks) compared to G9afl/fl mice (59,355 peaks), suggesting a global reduction in H3K9me2 levels following G9a deletion (Fig. 4c). Functional enrichment analysis of genes corresponding to significantly downregulated H3K9me2 peaks revealed enrichment in pathways related to negative regulation of cell proliferation (Fig. 4d). Similarly, G9a ChIP-seq analysis identified 20,851 G9a binding sites and their corresponding target genes, with functional annotation showing enrichment in negative regulation of cell proliferation and cell cycle pathways (Fig. 4e, f).

RNA-seq analysis revealed significant downregulation of cell cycle-promoting genes and upregulation of cell cycle-inhibiting genes in G9aΔIEC mice; differential gene enrichment analysis also pointed to cell cycle processes (Fig. 4g, h). Given that G9a-mediated H3K9me2 inhibits chromatin accessibility and thereby gene expression, we integrated the data from all three analyses to identify common differentially expressed genes, resulting in a list of 691 genes (Fig. 4i). Enrichment analysis of these differential genes primarily centered on pathways negatively regulation of cell proliferation, negatively regulation of cell cycle G1/S phase transition and regulation of cell cycle (Fig. 4j). These high-throughput data analysis confirms that in crypt cells, G9a-catalyzed H3K9me2 reduces chromatin accessibility at cell cycle-related loci and suppresses expression of genes that negatively regulate cell proliferation.

We then selected 4 genes closely related to the cell cycle from the aforementioned 691 genes, including Rb1cc1, Rb1, Cdkn1a, and Pten, for further validation. Genomic browser tracks showed that G9a knockout led to a significant reduction in H3K9me2 and G9a levels at these gene loci, resulting in increased chromatin accessibility and upregulation of gene expression (Fig. 5a). These phenomena were confirmed by ChIP-qPCR. Our data showed significant binding of H3K9me2 and G9a to Rb1cc1, Rb1, Cdkn1α, and Pten in the crypt cells of G9afl/fl mice, whereas these bindings were markedly reduced in the crypt cells of G9aΔIEC mice (Fig. 5b). Correspondingly, these genes were significantly upregulated following G9a knockout (Fig. 5c). Similarly, treatment with UNC0642 under homeostatic conditions also resulted in decreased H3K9me2 levels at these gene regions and increases in their expression levels (Supplementary Fig. 14a, b).

To establish a direct relationship between H3K9me2 and cell cycle arrest genes during intestinal regeneration, we detected H3K9me2 levels and these gene expressions in the crypts after irradiation (Fig. 5d). Indeed, the levels of H3K9me2 at these gene loci were significantly increased during the repair process. However, following G9a deletion, the H3K9me2 levels in these regions were almost vanished (Fig. 5e). Concurrently, G9a deficiency led to a slower decrease in the expression of these genes, resulting in delayed repair (Fig. 5f). These data further clarify that H3K9me2, modified by G9a, suppresses the expression of cell cycle-inhibiting genes, thereby participating in both intestinal homeostasis maintenance and injured tissue regeneration.

IL-4-STAT6 signaling regulates G9a expression during intestinal regeneration
To investigate the upstream regulatory pathway of G9a expression during intestinal epithelial regeneration initiation, we first employed a bioinformatic approach to identify potential transcription factors that could bind to the G9a promoter region. Using the JASPAR database, a collection of transcription factor DNA-binding profiles, we identified potential transcription factor binding sites in the G9a promoter (Supplementary Data 1). PANTHER signaling pathway enrichment analysis revealed that these transcription factors were associated with the JAK/STAT signaling pathway (Fig. 6a). Single-cell transcriptomic analysis of murine and human intestinal epithelium29 demonstrated high expression of Stat6 and its receptor-encoding gene Jak1 in ISCs and TA cells, which aligned with the expression pattern of G9a in the intestinal epithelium (Fig. 6b). Meanwhile, we identified specific STAT6-binding motifs in the G9a promoter region (Fig. 6c), and ChIP-qPCR analysis confirmed that phosphorylated STAT6 directly binds to the G9a promoter, with binding significantly increased at 48 and 96h post-irradiation (Fig. 6d). To determine which interleukin mediates this response, we analyzed the expression of various IL family members in isolated intestinal crypts during intestinal repair. Notably, only IL-4 showed significant upregulation during the repair process at both mRNA and protein levels, with peak expression occurring at 48–96h post-irradiation (Fig. 6e, f). Correspondingly, STAT6 phosphorylation levels increased during repair, consistent with G9a expression patterns (Fig. 6g). These findings demonstrate that G9a expression is regulated by the IL-4-STAT6 signaling pathway during intestinal injury repair (Fig. 6h).

Methods
Ethics declarations
This study complied with all relevant ethical regulations regarding the use of human study participants and was conducted in accordance with the Declaration of Helsinki. Ethical approval for the use of human subjects was obtained from the ethics committees of Second Affiliated Hospital of Zhejiang University School of Medicine (#2017-072) and Sir Run Run Shaw Hospital of Zhejiang University School of Medicine (#20210622-31). All animal studies were performed in compliance with the Guide for the Care and Use of Laboratory Animals, and adopted the protocol approved by the Medical Experimental Animal Care Commission of ZJU (#ZJU20220219updated).

Human subjects
Twenty rectal cancer patients who had received long-course neoadjuvant radiotherapy were enrolled in this study. The radiotherapy regimen consisted of 1.8 Gy per fraction administered 5 times per week for a total dose of 50.4 Gy. Tissue sections from both radiotherapy-induced injury regions and adjacent repair regions were obtained during radical resection at the Pathology Department of the Second Affiliated Hospital of Zhejiang University School of Medicine (Supplementary Table 1). Meanwhile, 15 non-radiotherapy patients were enrolled to serve as controls. Crohn’s disease (CD) patient samples used in this study were collected from the Inflammatory Bowel Disease Center at the Sir Run Run Shaw Hospital, affiliated with the Zhejiang University School of Medicine (Supplementary Table 2)41,42. The diagnosis of CD was performed according to a standard combination of clinical, endoscopic, histological, and radiological criteria. Patient characteristics including age at diagnosis (A1, <16 years; A2, 16–40 years; A3, >40 years), disease localization (L1, ileal; L2, colonic; L3, ileocolonic; L4, upper gastrointestinal tract), and disease behavior (B1, non-stricturing and non-penetrating; B2, stricturing; B3, penetrating) were documented according to the Montreal classification. Disease activity was assessed using the Crohn’s Disease Activity Index (CDAI), a standardized scoring system that incorporates abdominal pain, general well-being, stool frequency, and other clinical parameters to provide a numerical score ranging from 0 to 600, with higher scores indicating more active disease43. Sex-based analysis was not performed in this study for the following reasons: (1) the primary objective was to investigate the epigenetic mechanisms of intestinal epithelial regeneration, which are fundamental cellular processes not expected to differ substantially between sexes; (2) the sample size was not powered to detect potential sex differences; and (3) patients were enrolled based on clinical diagnosis and tissue availability rather than sex-stratified recruitment. Both sexes were included to enhance the generalizability of findings. Future studies with larger, sex-balanced cohorts will be needed to explore potential sex-specific differences in intestinal regeneration.

Mice
The G9a conditional knockout mice (G9afl/fl) and intestinal epithelial-specific Cre recombinase-expressing mice (Villin-cre) were obtained from the Shanghai Model Organisms Center. The C57BL/6 mice and Lgr5-EGFP-creERT2 knock-in mice were obtained from Shanghai Slack Company and Jackson Laboratory, respectively. Intestinal epithelial cell-specific G9a knockout mice (G9aΔIEC) and their littermate controls (G9afl/fl) were generated by crossing Villin-cre mice with G9afl/fl mice. Intestinal stem cell-specific G9a knockout mice were generated by crossing Lgr5-EGFP-creERT2 mice with G9afl/fl mice, followed by tamoxifen induction (75mg/kg body weight, intraperitoneally, once daily for 5 consecutive days). Genotypes were confirmed by PCR amplification using primers listed in Supplementary Table 3. Littermates were randomly assigned to experimental groups. All mice were maintained under pathogen-free conditions at the Laboratory Animal Centre of Zhejiang University with a controlled 12-h light/dark cycle, ambient temperature of 22 ± 1°C, and relative humidity of 50 ± 10%. Mice were provided with water and a standard laboratory diet ad libitum unless otherwise noted. Male mice aged 8–10 weeks were used in all experiments unless otherwise specified. Sex was considered in the study design; male mice were selected to minimize potential confounding effects of estrous cycle-related hormonal fluctuations on intestinal epithelial dynamics and to ensure experimental consistency. Data are reported for male mice, and findings may not be generalizable to female mice without further investigation.

Intestinal injury and regeneration mouse model
For the irradiation-induced model, eight-week-old C57BL/6 male mice were subjected to 8Gy or 10Gy whole-body IR with an RS2000 X-source irradiator at a dose rate of 60 or 85 cGy/min. Intestinal tissue samples were collected at 0, 1, 6, 12, 24, 48, and 96h post-irradiation for subsequent analyses. In this model, IECs typically undergo an apoptotic phase within hours post-irradiation, followed by a proliferation and repair phase starting around 24h later, during which surviving cells proliferate and restore the intestinal lining44.

For the dextran sulfate sodium (DSS) induced model, eight-week-old C57BL/6 male mice were treated with DSS (2.5% w/vol, molecular weight: 36–50kDa, #160110; MP Biomedicals) in drinking water for 7 days, followed by regular drinking water. Intestinal tissue samples were collected at 0, 7, 10, and 14 days after DSS treatment initiation for subsequent analyses. In this model, mice reached peak injury severity on day 10 of DSS treatment before gradually returning to baseline levels45.

G9a pharmacological inhibition
The G9a inhibitor UNC0642 (CAS: 1481677-78-4; #A15487; AdooQ Bioscience) was dissolved in a vehicle solution containing 10% DMSO, 20% β-cyclodextrin, and normal saline. The stock solution was prepared at a 5mg/mL concentration and stored at −20°C until use. For treatment, the stock solution was diluted 10-fold with sterile saline to achieve a working concentration of 0.5mg/mL. Eight-week-old male C57BL/6J mice were administered with UNC0642 via intraperitoneal injection at a dose of 5mg/kg body weight every other day for four consecutive weeks. Control mice received an equivalent volume of vehicle solution following the same injection schedule. Body weight was monitored throughout the treatment period to assess potential toxicity. Following the treatment period, mice were used for subsequent experimental analyses, including tissue collection, organoid culture, and phenotypic assessments.

Histone modifications screening and identification
For comprehensive histone modification analysis in intestinal regeneration, we performed liquid chromatography-mass spectrometry (LC-MS) analysis46. Intestinal crypts were isolated from mice before and after irradiation, then lysed in buffer containing 2mM EDTA, 3μM trichostatin A (TSA; #HY-15144, MedChemExpress), 50mM nicotinamide (NAM; #72340, Sigma-Aldrich), 1% Triton X-100, and protease inhibitor cocktail (#04693132001, Roche) at 4°C for 30 min. Following centrifugation at 20,000 × g for 10 min, the pellet was resuspended in 0.2M H₂SO₄, sonicated on ice, and centrifuged again to obtain the histone-enriched supernatant. Protein concentrations were determined using a BCA assay kit.

Extracted histones were propionylated in 1M triethylammonium bicarbonate (TEAB; #T7408, Sigma-Aldrich) buffer at room temperature, followed by overnight trypsin digestion at 37°C at an enzyme-to-histone ratio of 1:20 (w/w). Samples were subsequently treated with hydroxylamine (final concentration 0.25%) and ammonia water (final concentration 4%) to remove nonspecific chemical modifications, then quenched with formic acid (final concentration 1%). The resulting peptides were analyzed by LC-MS/MS using an EASY-nLC 1200 UPLC system coupled to an Orbitrap Exploris 480 mass spectrometer (both Thermo Fisher Scientific) operating with FAIMS compensation voltage set at −45V. LC-MS/MS analysis was performed by PTM BIO (Hangzhou, China). Data were processed using Spectronaut software v16 (Biognosys) with a comprehensive variable modification search including acetylation, mono-, di-, and tri-methylation of lysine residues, enabling identification and quantification of histone post-translational modifications across experimental conditions.

Hematoxylin and eosin (H&E), immunohistochemical (IHC), and Periodic Acid-Schiff (PAS) staining
The intestinal tissue samples were washed with 70% ethanol and then fixed in 10% formalin solution overnight. The fixed tissues were embedded in paraffin and sectioned. Sections were deparaffinized in xylene for 30 min and rehydrated through a graded ethanol series (100%, 95%, and 80%) for 5 min each, followed by a 5-min incubation in distilled water. For H&E staining, the sections were immersed in hematoxylin for 5 min and then in phosphate-buffered saline (PBS) for 3 min to prevent background staining, followed by staining in eosin for 2 min and distilled water for 5 min. For IHC staining, the sections were subjected to high-temperature antigen retrieval in 0.01M citrate buffer (pH 6.0) for 5 min, blocked in 1.5% normal goat serum (#ZLI-9056, ZSGB-BIO) for 30 min at room temperature, and then incubated with mouse anti-Ki67 antibody (#ab15580, Abcam), rabbit anti-OLFM4 antibody (#39141, Cell Signaling Technology), rabbit anti-LYZ antibody (#ab108508, Abcam) and rabbit anti-Chromogranin A antibody (#ab237979, Abcam) overnight at 4°C. The primary antibody was detected using an avidin/biotin-based peroxidase system (#ab64261 or #ab64264, Abcam) according to the manufacturer’s instructions. Briefly, the sections were incubated with a biotinylated goat anti-rabbit IgG or anti-mouse IgG secondary antibody for 60 min. After three washes in PBS, the sections were treated with a solution of avidin-biotin-peroxidase complex for 60 min and then incubated with 3,3′-diaminobenzadine (DAB) and H2O2 in PBS for 45–90 s to develop the insoluble brown chromogen. Hematoxylin was used as a counterstain for 5 min to visualize the nuclei. For PAS staining, the deparaffinized and rehydrated sections were immersed in a 0.5% periodic acid solution (#DG0005, LEAGENE) for 5 min, followed by a wash in distilled water. The sections were then submerged in Schiff’s reagent (#DG0005, LEAGENE) for 15 min and subsequently washed in distilled water. Counterstaining was performed using Hematoxylin solution for 1 min. After H&E, IHC, or PAS staining, the sections were dehydrated through a graded ethanol series (80%, 90%, and 100%) for 5 min each, followed by a 15-min incubation in xylene. The stained sections were mounted using neutral resin and a coverslip, and observed and digitally photographed using a Leica DM2000 LED.

Histological analysis
Histological assessment of stained sections was performed by a board-certified pathologist who was blinded to the experimental groups. The scoring criteria for staining intensity were as follows: 0 = no staining; 1 = weak staining (light yellow); 2 = moderate staining (yellow-brown); and 3 = strong staining (brown). For each sample, five randomly selected high-power fields were examined. Any discrepancies in scoring between the pathologist and a second observer were resolved by consensus. For the evaluation of colitis severity in mouse samples, the extent and severity of inflammation and ulceration of the mucosa were assessed using the following criteria. The severity score for inflammation was as follows: 0, normal (within the normal limit); 1, mild (small, focal, or widely separated, limited to the lamina propria); 2, moderate (multifocal or locally extensive, extending to the submucosa); and 3, severe (transmural inflammation with ulcers covering >20 crypts). The score for ulceration was as follows: 0, normal (no ulcers); 1, mild (1–2 ulcers involving up to a total of 20 crypts); 2, moderate (3–4 ulcers involving a total of 20-40 crypts); and 3, severe (>4 ulcers or >40 crypts). The overall colitis score was calculated as the sum of the inflammation severity score and the ulceration score, resulting in a total score ranging from 0 to 6. Higher scores indicate more severe colitis.

Immunofluorescence staining
Intestinal tissues were fixed with 4% paraformaldehyde (PFA) at 4°C for 2 h. After washing with cold PBS, the tissues were dehydrated in 30% sucrose at 4°C for 12 h and then embedded in optimal cutting temperature (OCT) compound for cryosectioning. The frozen sections were hydrated in PBS for 5 min and blocked with 1.5% normal goat serum for 1 h at room temperature. The sections were then incubated with rabbit anti-EHMT2/G9a antibody (#ab185050, Abcam) overnight at 4°C. The primary antibody was detected using a fluorescent-conjugated species-specific secondary antibody (Goat anti-Rabbit IgG antibody/Alexa Fluor 555, #A31572, Invitrogen). Nuclei were counterstained with Hoechst 33258 dye (#H3570, Life Technologies) for 15 min at room temperature. After three washes in PBS, the sections were mounted using neutral resin and coverslips. The sections were then examined and digitally photographed using a Nikon A1R Confocal Microscope.

EdU pulse-chase assay
To assess cell proliferation and track the fate of EdU-labeled cells, mice were administered a single injection of 8mg/kg EdU (#A10044, Thermo Fisher Scientific) and analyzed at the indicated timepoints (1.5h, 24h, and 48h post-injection). EdU staining was performed using the BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 594 (#C0078S, Beyotime Biotechnology) according to the manufacturer’s protocol. Briefly, intestinal tissue sections were fixed with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 in PBS for 20 min. EdU labeling was visualized by incubating the sections with fresh-prepared Click-iT reaction buffer containing azide-conjugated Alexa Fluor 594 for 30 min at room temperature. To delineate the cell boundaries, EdU-stained sections were washed with 3% bovine serum albumin (BSA, #ST025, Beyotime Biotechnology) in PBS and incubated with a primary rabbit anti-E-cadherin antibody (#3195, Cell Signaling Technology) overnight at 4°C. The sections were then incubated with a secondary goat anti-rabbit IgG antibody conjugated to Alexa Fluor 647 (#A21446, Thermo Fisher Scientific) for 1 h at room temperature. Finally, the sections were stained with Hoechst 33342 for 15 min to visualize the nuclei. Images were acquired using a Nikon A1 confocal microscope system.

Crypt isolation
To isolate intestinal crypts, the mouse small intestine was dissected and opened longitudinally. The tissue was briefly washed with ice-cold Dulbecco’s phosphate-buffered saline (DPBS, #14190144, Thermo Fisher Scientific) and further dissected into small pieces. Using a glass coverslip, the villi were gently scraped off, and the tissue fragments were washed with cold DPBS to remove unattached epithelial fragments. The intestinal fragments were then incubated in DPBS containing 3mM EDTA for 10 min on ice. After removing the EDTA solution, the fragments were washed once with DPBS and vigorously suspended in DPBS using a 10 mL pipette. The supernatant containing the villous fraction was discarded, and the sediment was resuspended in PBS. To enrich for crypts, the resuspended sediment was vigorously suspended once more, and the resulting supernatant was collected. This crypt-enriched fraction was passed through a 70 μm cell strainer to remove residual villous material and then centrifuged at 70 g for 3 min to separate the crypts from single cells. The purified crypts were counted and subsequently used for organoid culture or fluorescence-activated cell sorting (FACS), depending on the experimental requirements.

Organoid culture
For organoid culture, 100–300 isolated crypts were embedded in 30 μL of Matrigel (#356231, BD Biosciences) and cultured in IntestiCult Organoid Growth Medium (#06005, STEMCELL Technologies) supplemented with 100U/mL penicillin and 100μg/mL streptomycin (#15070063, Thermo Fisher Scientific). The culture medium was replaced every 2–3 days, and the organoids were maintained under standard tissue culture conditions (37°C, 5% CO2). To assess organoid growth and morphology, bright-field images of the organoids were acquired using an inverted microscope (ECLIPSE Ti2, Nikon) at various timepoints during culture. The images were analyzed using ImageJ software v1.53 (National Institutes of Health). Quantitative analysis of organoid growth was performed by determining the number of de novo buds and measuring the area of the organoids between day 3 and day 5 of culture.

Fluorescence-activated cell sorting (FACS)
To isolate specific cell populations from the intestinal crypt, a single-cell suspension was prepared by filtering the crypt cells through a 40 μm cell strainer. The cells were then simultaneously labeled with the following fluorescence-conjugated antibodies in staining buffer (2mM EDTA and 3% fetal bovine serum (FBS, #10100147, Thermo Fisher Scientific) in PBS): CD31-BV510 (#102407, BD Biosciences), CD45-BV510 (#103105, BD Biosciences), FVD450-BV450 (to distinguish live cells from dead/dying cells) (#65-0863-14, eBioscience), EpCAM-APC (#17-5791, Thermo Fisher Scientific) and CD24-PerCP-Cyanine5.5 (#562360, BD Biosciences). The cells were gently shaken for 30 min on ice to allow for antibody binding. After labeling, the cells were washed three times with staining buffer and resuspended at a concentration of 5 × 10^6 cells/mL in Advanced DMEM/F12 medium (#12634028, Gibco). The labeled cells were then analyzed and sorted using a BD FACS Aria II flow cytometer. The following markers were used to identify specific cell populations: LGR5-EGFPhi; EpCAM+; CD24med/−; CD31−; FVD450−; CD45−; TA cells were LGR5-EGFPlow; EpCAM+; CD24med/−; CD31−; FVD450−; CD45−; and Paneth cells were LGR5-EGFPneg; EpCAM+; CD24hi; CD31-; FVD450-; CD45−; Side scatter hi. The sorted cell populations were collected for further analysis or downstream applications. Cell population analysis was performed using FlowJo software v10.4.

RNA isolation and real-time quantitative PCR (RT-qPCR)
For mRNA analysis, total RNA was extracted from the samples using TRIzol reagent (#15596026, Thermo Fisher Scientific) following the manufacturer’s instructions. The quality and quantity of the isolated RNA were assessed using a Nanodrop 2000 spectrophotometer. Reverse transcription of mRNA to cDNA was performed using Moloney murine leukemia virus (MMLV) reverse transcriptase (#639524, Takara Bio). The resulting cDNA was then subjected to RT-qPCR using SYBR Green master mix (#RR420A, Takara Bio) on a LightCycler 480 real-time PCR system (Roche). The PCR primers used were designed using the NCBI Primer-BLAST tool and listed in Supplementary Table 4. The relative quantities were calculated using the ΔΔCt method. To ensure the integrity of RNA samples, all equipment used for RNA manipulations was sterilized according to standard laboratory protocols, and diethylpyrocarbonate-treated water was used throughout the process.

RNA sequencing (RNA-Seq) and data analysis
The mRNA was enriched from 500 ng of total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490L, NEB) according to the manufacturer’s instructions. The enriched samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation using the Ultra II Directional RNA Library Prep kit (#E7760L, NEB). The libraries were quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies) and equimolarly pooled and sequenced with 75bp single-end reads on a NextSeq 500 (Illumina). After sequencing, RNA-SeQC (1.1.8) was used to perform basic quality control, including the assessment of exonic, intronic, and intergenic distribution of the reads and the rRNA rate within each sample. Alignment of the reads to the mouse reference genome (mm10) was performed using GSNAP (v2018-07-04), and Ensembl gene annotation V.92 was used to detect splice sites. Uniquely aligned reads were counted with featureCounts (V.1.6.3) using the same Ensembl annotation. Normalization of the raw read counts based on library size and testing for differential expression between the two conditions was performed using the DESeq2 R package (V.1.24) and IHW (V.1.12).

Chromatin immunoprecipitation sequencing (ChIP-Seq) and data analysis
At least 2 × 10^5 intestinal crypts were lysed in 1mL single-cell dissociation buffer (TrypLE Express Enzyme (#12604-021, Gibco), 1% DNase I (260U/μL, #18047019, Invitrogen)). After incubation for 2 min at 32°C, the sample was filtered through a 40 μm cell strainer. Following centrifugation at 1300 × g for 3 min at 4°C, the sample was resuspended in 1mL ice-cold phosphate-buffered saline. To enable spike-in normalization for accurate quantification of histone modifications across experimental conditions, the ChIP DNA from HEK293T cells, equivalent to 1% of the murine crypt cell ChIP DNA amount, was added to the murine ChIP samples, following the established protocol for spike-in control of epigenomic analysis47. The cells were crosslinked in 1% formaldehyde, quenched in 0.125M glycine, and lysed in cell lysis buffer (50mM Tris pH 8.1, 10mM EDTA, 1% SDS). Lysates were sonicated with Bioruptor Pico (Diagenode) and diluted in dilution buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100) with a pre-incubated mixture of rabbit anti-H3K9me2 antibody (#ab1220, Abcam), rabbit anti-G9a/EHMT2 antibody (#68851, Cell Signaling Technology), rabbit anti-Phospho-Stat6 antibody (#56554, Cell Signaling Technology), or species-matched IgG (#2729, Cell Signaling Technology) and Dynabeads Protein A and Protein G (#80106G, Invitrogen). After overnight incubation, samples were washed five times with 1mL low salt washing buffer (75mM NaCl, 50mM Tris-HCl pH 7.5, 10mM EDTA, and 0.01% NP40), 1mL high salt washing buffer (100mM NaCl, 50mM Tris-HCl pH 7.5, 10mM EDTA, and 0.01% NP40), and 1mL LiCl washing buffer (50mM HEPES pH 7.6, 0.5M LiCl, 1mM EDTA, 0.7% sodium deoxycholate, 1% NP40), followed by two washes with Tris-EDTA Buffer (10mM Tris pH 8, 1mM EDTA). DNA was eluted overnight in decrosslinking Buffer (0.1M NaHCO3, 1% SDS) and purified using the QIAquick PCR Purification Kit (#28106, QIAGEN). The ChIP DNA was further treated with RNase A (#EN0531, Invitrogen) and Protease K (#25530049, Invitrogen), followed by DNA purification using a PCR purification kit (#28104, QIAGEN). The DNA samples were quantified using Qubit fluorometric quantification (Thermo Fisher Scientific). The libraries were constructed through end repair, A-tailing, adapter ligation, size selection, and PCR amplification, followed by fragment size analysis using the Agilent 5400 system and quantification to 1.5nM via qPCR. and sequenced on the Illumina platform, generating paired-end reads of 150bp to achieve an average of 20M reads per sample at the Novogene Genomics Center (China).

For analysis, reads were trimmed using fastp (v 0.20.0) and aligned to the mouse reference genome (mm10) using BWA (v0.7.12). Spike-in reads were aligned to the human reference genome (hg38) to calculate spike-in normalization factors for each sample. Normalization was performed by scaling ChIP-seq signal intensities based on the ratio of spike-in reads between samples, following established guidelines to correct for technical variations in chromatin preparation and immunoprecipitation efficiency. After mapping reads to the reference genome, the MACS2 (v2.2.7.1) peak calling software was used to identify regions of IP enrichment over background. A q-value threshold of 0.05 was used for all data sets. DiffBind was used to predict and annotate differential peaks with the following parameters. Peak calling and differential analysis incorporated spike-in normalization factors to ensure accurate detection of biologically relevant changes in histone modifications. Differential peaks with a false discovery rate (FDR) P < 0.05 were considered statistically significant.

For qPCR validation of ChIP-seq data, the DNA samples were diluted appropriately and analyzed using SYBR Green-based real-time PCR on a LightCycler 480 real-time PCR system. The PCR primers used were designed using the NCBI Primer-BLAST tool and listed in Supplementary Table 5. The relative enrichment of each target region was calculated using the percent input method, where the amount of immunoprecipitated DNA is normalized to the input DNA for each sample.

Assay for transposase-accessible chromatin using sequencing (ATAC-Seq) and data analysis
The single cells from intestinal crypts were lysed in lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, and 0.15% NP40) for 10 min on ice to prepare the nuclei. Immediately after lysis, nuclei were centrifuged at 500 × g for 5 min to remove the supernatant, and incubated with Tn5 transposome (#53150, Active Motif) and tagmentation buffer (#53150, Active Motif) at 37°C for 30 min. Following tagmentation, the stop buffer (#53150, Active Motif) was directly added to the reaction. PCR amplification of the library was performed for 15 cycles using the following conditions: 72°C for 3 min; 98°C for 30 s; and thermocycling at 98°C for 15 s, 60°C for 30 s, and 72°C for 3 min; followed by a final extension at 72°C for 5 min. After PCR, libraries were purified with 1.2× AMPure beads (A63880, Beckman) before proceeding to mitochondrial DNA depletion. The libraries were quantified using the Qubit dsDNA HS Assay Kit (#Q32854, Thermo Fisher Scientific), and the size distribution was assessed using the Agilent 2100 Bioanalyzer. The libraries were then diluted to a final concentration of 2 nM and pooled in equimolar ratios. 2 × 150bp paired-end (PE150) sequencing was performed on an Illumina NovaSeq 6000 platform at a depth of approximately 50 million reads per sample. Raw reads were trimmed using Trim Galore (v0.4.4) and aligned to the mouse reference genome (mm10) using Bowtie 2 (v2.2.6). Peak detection was performed using MACS2 software to identify open chromatin regions across the genome for each sample. HOMER (v4.9) was used to predict and annotate differential peaks. Differential peaks with a fold change ≥1.5 and an FDR < 0.001 were considered statistically significant. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) was performed using the ShinyGO (http://bioinformatics.sdstate.edu/go/).

Immunoblotting
Intestinal tissues, organoids, or cells were lysed in radio-immunoprecipitation assay buffer (RIPA, #P0013C, Beyotime Biotechnology) supplemented with protease inhibitor cocktail (#04693132001, Roche). Protein concentrations were quantified using a BCA protein assay kit (#P0012, Beyotime Biotechnology). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a 0.45µm polyvinylidene fluoride (PVDF) membrane (#FFP39, Beyotime Biotechnology). The membrane was blocked with 5% BSA prepared in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 1 h, washed with TBST for 5 min, and incubated with primary antibodies diluted in 5% BSA overnight at 4°C on a rocker. After washing with TBST for 5 min, the membrane was incubated with HRP-conjugated secondary antibodies (anti-mouse IgG, #SA00001-1; anti-rabbit IgG, #SA00001-2; Proteintech) diluted in 5% BSA for 1 h at room temperature. Chemiluminescence signals were detected using SuperSignal West Pico Chemiluminescent Substrate (#34580, Thermo Fisher Scientific), and images were acquired using a chemiluminescence imager, Amersham Imager 600 (GE Healthcare Life Sciences). The following primary antibodies were used in this study: rabbit anti-EHMT2/G9a (#ab185050, Abcam), rabbit anti-Mono-Methyl-Histone H3K9 (#A2358, ABclonal), rabbit anti-Di-Methyl-Histone H3K9 (#4658, Cell Signaling Technology), rabbit anti-Tri-Methyl-Histone H3K9 (#A2360, ABclonal), rabbit anti-Acetyl-Histone H3K14 (#7627, Cell Signaling Technology), rabbit anti-Tri-Methyl-Histone H3K27 (#9733, Cell Signaling Technology), rabbit anti-Phospho-Stat6 antibody (#56554, Cell Signaling Technology), rabbit anti-STAT6 antibody (#ab32520, Abcam), rabbit anti-Cyclin A2 (#A19036, ABclonal), rabbit anti-Cyclin H (#A4076, ABclonal), rabbit anti-Cyclin E2 (#A9305, ABclonal), rabbit anti-Cyclin D1 (#A19038, ABclonal), mouse anti-Histone H3 (#sc-517576, Santa Cruz), and mouse anti-ACTB (#60004-1-Ig, Proteintech).

Sample size and sample collection
No statistical methods were used to predetermine sample size for in vivo and in vitro experiments, but at least three samples were used per experimental group and condition. The number of samples used in each experiment is indicated in the Source Data. Samples and experimental animals were randomly assigned to experimental groups. Animal procedures (that is, genotyping and treatments) were performed by investigators unaware of the experimental design.

Statistical analysis
All data in this paper are presented as mean ± standard error of the mean (SEM). Normality of the data was assessed using the Shapiro–Wilk test. For comparisons between two groups, a two-tailed unpaired t-test (Student’s t-test) was used for normally distributed data, and the Mann-Whitney U-test was used for non-normally distributed data. Differences among variables were evaluated using one-way analysis of variance (ANOVA) and two-way ANOVA for normally distributed data, and the Kruskal–Wallis test for non-normally distributed data. All statistical analyses were performed using GraphPad Prism 10. Statistical significance was defined as P < 0.05 unless otherwise specified, and exact P values are reported where applicable.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The RNA-seq, ATAC-seq, and ChIP-seq data generated in this study have been deposited in the NCBI’s Sequence Read Archive (SRA) database under accession code PRJNA1135937, PRJNA1136160, and PRJNA1136210, respectively. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study revealed the role of H3K9 di-methylation (H3K9me2) in the intestinal epithelial homeostasis and\nregeneration.The authors examinined that the levels of H3K9me2 and its modifying enzyme G9a in intestinal epithelial cells\n(IECs) in patients with Crohn's disease CD and concluded that the H3K9m2 level and G9a expression in the IECs\ndecreased and negatively correlted with the disease activity. Simultaneously, they observed that the level of H3K9me2 and\nthe G9a expression was in a possitive correction in both CD patients and in cnacer patients with radiotherapy. Consistently,\nwe used animal models to confirm the correlation of H3K9me2 and the G9a expression. Using IEC-sepcific G9a knockout\nmodel, they provided evidence that G9a-mediated H3K9me2 was critical for the repair of intestinal injury by regulating the\nISC and TA cell proliferation. By ATAC-seq, ChIP-seq, and RNA-seq analyses, the authors identified critical genes\nregulated by H3K9me2 in the IECs.They validated Rb1cc1, Rb1, Cdkn1a, and Pten, which were directly controled by G9a\ninduced H3K9me2. Overall, this is an interesting study and provides new insight in the mechanism of IEC regeneration. One\nof my concern is that the authors did not reveal why and G9a is regulated duing the initiation of IEC regeneration. Is it\nboosted by inflammation or the intrisic factors during this process? Another question is why G9a is specifically regulating\nthese 91 gene expression.", "reviewer_2": "In this manuscript Chen and colleagues report a link between the G9a histone lysine methyltransferase enzyme and\nintestinal epithelial maintenance and regeneration. G9a catalyses the histone modification H3K9 di-methylation.\nThe mechanism of how this histone modification contributes to gene regulation and chromatin structure is poorly understood,\nunlike many other histone modifications.\nThe intestinal epithelium is a heavily studied tissue to model stem cell compartments and differentiation pathways due to the\nwell-established spatial organisation of intestinal crypts. Other chromatin regulators, including histone modifying enzymes,\nhave been shown to be essential for maintenance of stem cell compartments, or indeed for differentiation into specific cell\ntypes from intestinal stem cells (goblet, tuft, Paneth etc).\nHere the authors report a correlative increase in G9a and its associated histone modification H3K9me2 in regenerating\nintestinal tissue. Constitutive genetic loss and chemical inhibition of G9a both have a phenotype that results in a shallower\nintestinal crypt. The authors show that loss/ inhibition of G9a causes changes in cell proliferation that explain this phenotype.\nThey attempt to mechanistically link this proliferative phenotype to changes in H3K9me2 levels at specific genes. Overall,\nthe phenotypic data and data linking changes in cell cycle to this phenotype is robust. However, the mechanistic link to\nepigenetic processes governed by G9a is not entirely convincing.\nThe overall findings of the paper therefore lack a cohesive mechanism, and while robust the biological phenotype of G9a\nloss/ inhibition is unsurprising, and in fact quite mild, compared to other studies examining histone modifying enzymes in the\nsame compartments. Given that G9a has already been linked to controlling cell cycle my opinion is that the authors should\naim for a more specialised journal.\nMajor comments\n• The introduction is very short and something that is missing is the logic, or hypothesis behind examining G9a and\nH3K9me2 in the intestinal crypt in the first place. Many histone modifications likely change levels in different cell types in the\ncrypt- as their levels are tightly linked to cell cycle changes. Why was H3K9me2 picked over the others? Histone mass\nspectrometry to detect changes in methylation, acetylation etc in the different conditions examined in figure 1 would likely\nreveal many changes in different histone modifications. Only H3K9 methylations are examined. What happens to H3K4 and\nH3K27 methylation/acetylation? These modifications have much better-defined roles in transcriptional control than\nH3K9me2.\n• A major comment throughout the manuscript is that results, figure legends and methods provide very little detail. They are\noften lacking information on which cell type is being examined, how many mouse/ organoids are used, how long G9a\ninhibition has been administered for. These need to be updated to provide sufficient information for interpreting the\nexperiment correctly. This is also a major problem in figure 5 where it is unclear whether all peaks, common peaks, de novo\npeaks, upregulated genes etc are being examined at different points.\n• The above point is particularly important for Figure 2C and 2J where the results are reported as significant, but the\nphenotype is extremely mild (5% change in body weight- or possibly less?) and is very surprising that it reached\nsignificance. The legend does not state how many mice were used here or included error bars.\n• The phenotype shown of disrupted intestinal epithelium in figure 3 is very mild compared to genetic ablation of other\nhistone modifiers in skin or intestinal stem cell niches (EZH2, RING1B for example). Here constitutive loss of G9a has a mild\nphenotype while inducible loss of EZH2/ RING1B has rapid and gross intestinal problems for mice. The authors should\nconsider this in their results/discussion as they need to put this mild phenotype in the context of other work that has been\nshown in the field previously.\n• Some of the plots seem to use average per mouse/organoid. In figure 4G for example 60 organoids were tested per mouse\nbut only the average organoid size per mouse is plotted. The same is true for figure 3B, D, H, I. These data points should all\nbe plotted together to account for variation in organoid size/ crypt depth/ cell number generally. Violin/cello plots should be\nused instead.\n• The methods for the ChIP-seq do not mention any spike-in control. This is now a well established standard for such\nepigenomic analyses (https://doi.org/10.1038/s41587-024-02377-y). Without this it is unclear how reliable the changes in\nH3K9me2 detected in ChIP-seq are. While there seems to be a complete loss of H3K9me2 in figure 5C these changes look\nminimal in figure 6A. This is a really critical point as these figures attempt to link G9a catalytic activity to the phenotypic\nchanges observed in mice. The H3K9me2 signal shown in Figure 5C does not have the typical ‘peak’ pattern at TSS that\nmany histone modifications do. This is a further reason it is so important to include internal ChIP-seq spike in normalisation\ncontrols. Also, the legend does not say what genes are being examined in Fig 5C, is this all genes, all H3K9me2 positive\ngenes, or all genes that lose H3K9me2 signal in G9a knockout?\n• In Fig 5G it is unclear if these are differentially expressed/regulated sites for each dataset or if its up/down specifically.\nATAC-up, RNA-up, H3K9me2 down sites are those that should examined to make the mechanistic link the authors are\nproposing. But that does not seem to be the case. This analysis presented shows there is poor correlation between\nH3K9me2 loss and increases in accessibility (only 450 of 5474 accessible sites overlap with H3K9me2). Therefore, the\nstatement saying H3K9me2 compacts chromatin in the text is entirely incorrect. These results are very descriptive and do not\nseem to be mechanistically linked to the phenotype of the mouse without a more comprehensive analysis of the ATAC and\nChIP-seq data.\n• Due to the aforementioned limitations in quality of the H3K9me2 ChIP-seq the authors could perform G9a CUT&RUN/\nChIP-seq to assess which targets it binds to. This would be a more useful mechanistic link than mild changes in H3K9me2.\nMinor comments\n• In Figure 1, define the “Disease Activity Index” and how it was calculated in the present study.\n• Western blots to detect minor changes in histone modifications levels are notoriously unreliable. The authors should\nquantify the changes observed across experimental replicates in histone modifications- particularly in figure 1, 2 and S2B.\nBlots in Figure 2A are quite poor with several being overexposed and band signal bleeding into neighbouring lanes. This is\nparticularly important in figure 2A and 2B to establish how well the model systems targeting G9a are working. The G9a\ninhibitor seems to have a very mild effect on H3K9me2 levels based on these blots.\n• Fig S4C western blot has some important technical issues. The image shown as a ‘blot’ is missing a membrane in both\nG9a blots. Lines above the G9a band are clearly visible showing where the membrane has been cut. The box shapes\naround these should be readjusted accordingly to avoid showing empty space where it seems like a blot should be.\n• Figure 3 and 4 make the same broad conceptual point as figure 2 but without additional interesting details that aid\nmechanistic interpretations of the data. G9a is clearly important for proliferation and maintenance of the stem cell\ncompartment- and therefore intestinal epithelia. However, this same point is made repetitively using different assays over\nthere three figures. There I no apparent specificity of this phenotype in any particular differentiated cells, it seems to arise\nfrom issues in the stem cells. Much of this data could be moved to a supplementary figure supporting figure 2.\n• The discussion states that H3K27me3 is present only in the villi and not the crypt. However, this is not true (doi:\n10.15252/embj.201694550). The citations the authors provide for this section are also incorrect and cite two studies (#30\nand #31) that do not examine the intestinal epithelium at all.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point Response to Reviewers’ Comments\nWe thank the reviewers for their constructive remarks on our manuscript, and have taken the comments\non board to improve and clarify the manuscript. Please find below a detailed point-by-point response\nto all comments (please note that we have labeled the reviewers’ comments in black and our replies in\nblue, and organized the supporting data as Figure 1-x for the Reviewer #1, Figure 2-x for the Reviewer\n#2, etc.).\nReviewer #1:\nThis study revealed the role of H3K9 di-methylation (H3K9me2) in the intestinal epithelial\nhomeostasis and regeneration. The authors examined that the levels of H3K9me2 and its modifying\nenzyme G9a in intestinal epithelial cells (IECs) in patients with Crohn's disease (CD) and concluded\nthat the H3K9m2 level and G9a expression in the IECs decreased and negatively correlated with the\ndisease activity. Simultaneously, they observed that the level of H3K9me2 and the G9a expression was\nin a positive correction in both CD patients and in cancer patients with radiotherapy. Consistently, they\nused animal models to confirm the correlation of H3K9me2 and the G9a expression. Using IEC-\nspecific G9a knockout model, they provided evidence that G9a-mediated H3K9me2 was critical for\nthe repair of intestinal injury by regulating the ISC and TA cell proliferation. By ATAC-seq, ChIP-seq,\nand RNA-seq analyses, the authors identified critical genes regulated by H3K9me2 in the IECs. They\nvalidated Rb1cc1, Rb1, Cdkn1a, and Pten, which were directly controlled by G9a induced H3K9me2.\nOverall, this is an interesting study and provides new insight in the mechanism of IEC regeneration.\nResponse: I sincerely thank you for your positive assessment on our manuscript and for recognizing\nthe significance of our findings regarding the role of H3K9me2 in intestinal epithelial homeostasis and\nregeneration, especially you pointed out that our study provides new insight into the mechanism of\nIEC regeneration. Thanks to your and the other reviewer’s valuable comments and suggestions, we\nhave conducted additional experiments and integrated the new data into the RESULTS to strengthen\nour conclusion. After substantially rewriting the manuscript, I believe now our work is suitable for\npublication in Nature Communications.\n1. One of my concern is that the authors did not reveal why and G9a is regulated during the initiation\nof IEC regeneration. Is it boosted by inflammation or the intrinsic factors during this process?\nResponse: Thank you for raising this critical point to uncover the regulatory mechanism of G9a\nexpression during the initiation of IEC regeneration. To address this issue, we have redesigned our\nresearch rationale, performed additional experiments, and found that it was the inflammatory signaling\npathway which activated G9a expression during the regeneration process (Figure 1-1).\nFigure 1-1. IL-4－STAT6 signaling regulates G9a expression during intestinal regeneration\na PANTHER signaling pathway enrichment analysis of transcription factors predicted to bind to G9a promoter\nregion. b Expression levels of STAT and JAK family members in ISCs and TA cells from mouse and human\nsingle-cell RNA sequencing data. Circle size represents expression level and color indicates cell type. c STAT6\nconsensus binding motif identified in G9a promoter region. d Phosphorylated STAT6 (pSTAT6) binding to G9a\npromoter at different time points post-irradiation. n=3 mice/group. e Heatmap showing expression of IL family\nmembers at different time points post-irradiation. f Quantitative analysis of IL-4 expression at different time\npoints post-irradiation. n=6 mice/group. g Immunoblotting of phosphorylated STAT6 and total STAT6 levels at\ndifferent time points post-irradiation. Numbers below each blot represent relative band intensities normalized\nto 0 h. h Schematic model illustrating IL-4-STAT6-G9a signaling pathway during intestinal regeneration. Data\nare presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA in (d and f).\nTo investigate the upstream regulation of G9a, we employed a comprehensive approach\ncombining bioinformatic analysis and experimental validation. Using the JASPAR database, a\ncollection of transcription factor DNA-binding profiles, we first identified the potential transcription\nfactors (TFs) which could bind to the G9a promoter region, then used PANTHER signaling pathway\nenrichment analysis and found that these TFs were primarily associated with the JAK/STAT signaling\npathway, a critical mediator of inflammatory responses and cellular homeostasis. Further single-cell\ntranscriptomic analyses of murine and human intestinal epithelium revealed high expression of Stat6\nand its receptor-encoding gene Jak1 in ISCs and TA cells, which aligned with the expression pattern\nof G9a in the intestinal epithelium. Meanwhile, we identified specific STAT6-binding motifs in the\nG9a promoter region, and ChIP-qPCR analysis confirmed that phosphorylated STAT6 directly bound\nto the G9a promoter, with significantly increased binding at 48 and 96 hours post-irradiation.\nImportantly, our analysis of IL family expression during intestinal repair revealed that only IL-4\nwas significantly upregulated during the repair process at both mRNA and protein levels, with peak\nexpression occurring at 48-96 hours post-irradiation. Correspondingly, STAT6 phosphorylation levels\nincreased during repair, consistent with G9a expression pattern.\nThese results demonstrate that G9a is regulated by inflammation-mediated signaling through\nthe IL-4－STAT6 pathway during intestinal injury repair, representing a novel mechanism wherein\ninflammatory cytokine IL-4 promotes intestinal stem/progenitor cell regeneration through STAT6-\nmediated G9a transcriptional activation and subsequent epigenetic reprogramming. We think this IL-\n4－STAT6－G9a axis complements existing knowledge by demonstrating G9a-mediated chromatin\nremodeling as a previously unrecognized repair pathway, which bridges inflammatory signaling with\nepigenetic regulation of regeneration.\nWe have incorporated these mechanistic insights as a new section in our results (i.e., the Fig. 6 in\nour revised manuscript) to provide a comprehensive understanding of how inflammatory factors\nregulate G9a during IEC regeneration initiation.\n2. Another question is why G9a is specifically regulating these 91 gene expression.\nResponse: We appreciate this insightful question regarding the specificity of the 91 genes regulated\nby G9a. Actually, our study has provided several lines of evidence which could explain this selective\nregulatory phenomenon.\nFirst, our expanded analysis identified 691 genes that are enriched with G9a-mediated H3K9me2\nmodifications. Conserved sequence analysis showed that their promoter regions are selectively\nenriched with C2H2 zinc finger motifs (Figure 1-2). This sequence-specific binding pattern suggests\nthat G9a does not randomly target genes but rather recognizes specific chromatin contexts and DNA\nmotifs, which may explain the selective regulation of this gene subset.\nFigure 1-2 Analysis of predicted motifs in promoter regions of four cell cycle arrest genes\nHeight of the letters on the y-axis represents the frequency of occurrence of corresponding base at each position\nacross the sequences. Width of the x-axis represents the length of motifs. Data correspond to Supplementary\nFig. 15 in the revised manuscript.\nSecond, functional analysis of these G9a-regulated genes revealed they are predominantly\ninvolved in cell cycle arrest and negative regulation of cell proliferation pathways. This functional\nclustering is not coincidental but reflects G9a's specialized role in maintaining a specific regulatory\nnetwork.\nThird, during intestinal regeneration, G9a expression is dynamically regulated by the IL-4－\nSTAT6 signaling pathway. This temporal control, combined with the pre-existing chromatin landscape\nof intestinal stem cells, ensures that G9a preferentially targets genes in permissive chromatin states\nthat require epigenetic silencing for proper cell cycle progression.\nIn summary, G9a's regulatory specificity results from three coordinated mechanisms: recognition\nof specific DNA motifs, functional specialization in cell cycle pathways, and context-dependent\nexpression during regeneration, which together enable targeted epigenetic regulation of intestinal stem\ncell proliferation.\nReviewer #2 (Remarks to the Author):\nIn this manuscript Chen and colleagues report a link between the G9a histone lysine methyltransferase\nenzyme and intestinal epithelial maintenance and regeneration. G9a catalyses the histone modification\nH3K9 di-methylation. The mechanism of how this histone modification contributes to gene regulation\nand chromatin structure is poorly understood, unlike many other histone modifications.\nThe intestinal epithelium is a heavily studied tissue to model stem cell compartments and\ndifferentiation pathways due to the well-established spatial organisation of intestinal crypts. Other\nchromatin regulators, including histone modifying enzymes, have been shown to be essential for\nmaintenance of stem cell compartments, or indeed for differentiation into specific cell types from\nintestinal stem cells (goblet, tuft, Paneth etc).\nHere the authors report a correlative increase in G9a and its associated histone modification\nH3K9me2 in regenerating intestinal tissue. Constitutive genetic loss and chemical inhibition of G9a\nboth have a phenotype that results in a shallower intestinal crypt. The authors show that loss/inhibition\nof G9a causes changes in cell proliferation that explain this phenotype. They attempt to mechanistically\nlink this proliferative phenotype to changes in H3K9me2 levels at specific genes. Overall, the\nphenotypic data and data linking changes in cell cycle to this phenotype is robust. However, the\nmechanistic link to epigenetic processes governed by G9a is not entirely convincing.\nThe overall findings of the paper therefore lack a cohesive mechanism, and while robust the\nbiological phenotype of G9a loss/ inhibition is unsurprising, and in fact quite mild, compared to other\nstudies examining histone modifying enzymes in the same compartments. Given that G9a has already\nbeen linked to controlling cell cycle my opinion is that the authors should aim for a more specialised\njournal.\nResponse: I sincerely appreciate your comprehensive evaluation of our manuscript and insightful\ncomments on our work. To be frankly, I am happy that you pointed out that “The mechanism of how\nthis histone modification contributes to gene regulation and chromatin structure is poorly understood,\nunlike many other histone modifications”, and you recognized “the phenotypic data and data linking\nchanges in cell cycle to this phenotype is robust”. However, you questioned “the mechanistic link to\nepigenetic processes governed by G9a is not entirely convincing”. To address your concerns, we have\nredesigned our research rationale, performed additional experiments, and rewrited the manuscript.\nMost importantly, we have:\nStrengthened the mechanistic foundation: We repeated our ChIP-seq experiments with proper\nspike-in normalization controls and added G9a ChIP-seq analysis, as you suggested. These\nimprovements resulted in robust correlations (>70% overlap) between G9a binding, H3K9me2\ndeposition, chromatin accessibility, and gene expression changes, providing the mechanistic link that\nwas initially lacking.\nEnhanced experimental rigor: We improved our data presentation by plotting individual\nmeasurements rather than averaged values and implemented violin plots to better represent biological\nvariation. We also repeated key immunoblot experiments to eliminate technical issues and provided\ncomprehensive quantitative analyses across experimental replicates.\nClarified the rationale and context: Following your suggestion, we conducted comprehensive\nhistone modification screening with mass spectrometry, which led us to focus on H3K9me2 as it\nshowed the most pronounced changes among 19 examined histone modifications. We substantially\nrevised the Introduction part of our manuscript to provide a clear rationale and added comparative\ndiscussion of G9a's specialized modulatory role versus master epigenetic regulators such as EZH2 and\nRING1B.\nImproved manuscript organization: We streamlined redundant figures while maintaining\nmechanistic insights, and corrected factual errors in our discussion regarding histone modification\ndistributions.\nWhile I understand your perspective that the phenotype appears mild compared to other histone\nmodifiers, our survival analysis using lethal irradiation demonstrates the critical importance of this\npathway under stress conditions (Fig.2m in the revised mansucript). Thus, I believe G9a represents a\nspecialized \"fine-tuning\" mechanism that complements master regulatory systems, which may explain\nboth its manageable baseline effects and its crucial role during tissue regeneration.\nI hope that these comprehensive revisions have addressed your concerns and sufficiently\nstrengthened the mechanistic foundation of our work, warranting its publication in Nature\nCommunications.\nMajor comments\n1. The introduction is very short and something that is missing is the logic, or hypothesis behind\nexamining G9a and H3K9me2 in the intestinal crypt in the first place. Many histone modifications\nlikely change levels in different cell types in the crypt as their levels are tightly linked to cell cycle\nchanges. Why was H3K9me2 picked over the others? Histone mass spectrometry to detect changes\nin methylation, acetylation etc in the different conditions examined in figure 1 would likely reveal\nmany changes in different histone modifications. Only H3K9 methylations are examined. What\nhappens to H3K4 and H3K27 methylation/acetylation? These modifications have much better-\ndefined roles in transcriptional control than H3K9me2.\nResponse: I highly appreciate this instructive comment regarding the rationale for focusing on\nH3K9me2 and G9a. Indeed, our initial introduction was too brief, and there was lack of\nexplanation/evidence for why H3K9me2 was picked for further research. Thanks to your suggestion,\nwe have performed additional experiments to address these issues.\nFirst, we conducted liquid chromatography-mass spectrometry (LC-MS) analysis on chromatin\nfractions isolated from intestinal crypts before and after irradiation-induced injury (Figure 2-1a). In\ntotal, we detected and quantified 14 histone H3 tail modifications and 5 histone H4 tail modifications\n(Figure 2-1b, c). Among these 19 modifications, H3K9me2 exhibited the most pronounced changes.\nWhile H3K27me3 and H3K14ac also showed significant alterations, we did not detect changes in\nH3K4 methylation in our system. To validate these findings, we then performed immunoblotting to\nexamine the temporal dynamics of H3K9me2 and related modifications, including H3K9me1,\nH3K9me3, H3K27me3, and H3K14ac, across multiple time points during the injury-repair timeline\n(Figure 2-1d). Notably, H3K9me2 exhibited a distinct biphasic pattern, with initial reduction during\ninjury followed by subsequent upregulation during regeneration (Figure 2-1d), suggesting its potential\ninvolvement in the injury-repair process.\nFigure 2-1. H3K9me2 levels correlate with intestinal epithelium injury and regeneration\na Schematic illustration of the irradiation-induced intestinal injury model and experimental workflow. b-c LC-\nMS quantification of histone modifications in intestinal crypt cells. Relative normalized intensity of 14 histone\nH3 tail modifications (b) and 5 histone H4 tail modifications (c) at baseline (0 h, blue) and 96 hours post-\nirradiation (96 h, red). d Immunoblotting of H3K9me1, H3K9me2, H3K9me3, H3K27me3, and H3K14ac at\nindicated time points during the injury-regeneration process. Total H3 was used as loading control. Numbers\nbelow each blot represent relative band intensities normalized to 0 h. Data are presented as mean ± SEM.\nStatistical analysis was determined by two-tailed unpaired t-test in (b and c).\nBased on these results, I have made two major revisions to strengthen the research rationale and\nlogical foundation of our study. First, I have substantially rewritten the INTRODUCTION to provide\na more comprehensive overview of histone modifications in intestinal epithelial homeostasis. The\nrevised INTRODUCTION (Line 52-76) now discusses the established roles of various histone\nmodifications in maintaining intestinal homeostasis while emphasizing that their dynamic regulation\nduring injury and regeneration remains poorly understood. Second, I have reorganized the RESULTS\nsection (Line 412-425) to incorporate the screening process described above as the first part of our\nfindings (Fig.1a-d in the revised manuscript), thereby establishing a clear logical foundation for our\nfocus on H3K9me2 and G9a.\n2. A major comment throughout the manuscript is that results, figure legends and methods provide\nvery little detail. They are often lacking information on which cell type is being examined, how\nmany mouse/organoids are used, how long G9a inhibition has been administered for. These need to\nbe updated to provide sufficient information for interpreting the experiment correctly. This is also\na major problem in figure 5 where it is unclear whether all peaks, common peaks, de novo\npeaks, upregulated genes etc are being examined at different points.\nResponse: I feel sorry for not providing this basic information throughout the previous manuscript,\nand acknowledge that this is a significant issue which affects the interpretability of our experiments.\nFollowing your instruction, I have now comprehensively addressed these details in this revised\nmanuscript.\nFirst, I have clarified cell type specifications throughout the manuscript. In this study, the intestinal\ncrypt cell population was used for all major analyses including LC-MS analysis, immunoblot, ATAC-\nseq, ChIP-seq, and RNA-seq experiments. This population mainly contains TA cells and ISCs. For\norganoid analyses, these cultures contain various intestinal epithelial cell types, as organoids are\nderived from intestinal crypts and undergo differentiation to recapitulate the cellular diversity of the\nintestinal epithelium.\nSecond, we have provided detailed sample sizes and replication information in the figure legends:\nmouse experiments typically included n=4-6 mice/group for molecular analyses and n=5-8 mice/group\nfor phenotypic studies. For cell counting, at least 20-60 crypts/fields were counted per mouse\ndepending on the specific assay. Organoid studies assessed at least 50-90 organoids per mouse with\nn=4 mice/group.\nThird, for G9a inhibition experiments, eight-week-old male C57BL/6J mice were administered\nUNC0642 via intraperitoneal injection at a dose of 5 mg/kg body weight every other day for four\nconsecutive weeks, then used for subsequent experimental analyses including tissue collection,\norganoid culture, and phenotypic assessments.\nFourth, regarding the original Figure 5 (now Fig.4 in the revised manuscript) peak analysis\nclarification, we have added detailed descriptions in the results section specifying the exact analytical\napproach: we examined differential H3K9me2 peaks (sites with significantly decreased H3K9me2\noccupancy in G9aΔIEC vs G9afl/fl mice), differential G9a binding sites (regions with reduced G9a\noccupancy), differentially accessible chromatin regions (sites with increased accessibility in ATAC-\nseq), and upregulated genes from RNA-seq analysis. The 691 overlapping genes represent the\nintersection of genes with all four characteristics: decreased H3K9me2, decreased G9a binding,\nincreased chromatin accessibility, and upregulated expression in G9aΔIEC mice.\nI hope you are now satisfied with the detailed experimental information described in the methods,\nresults, and figure legends sections of our revised manuscript.\n3. The above point is particularly important for Figure 2C and 2J where the results are reported as\nsignificant, but the phenotype is extremely mild (5% change in body weight- or possibly less?) and\nis very surprising that it reached significance. The legend does not state how many mice were used\nhere or included error bars.\nResponse: Again, I thank you for reminding us to provide these basic experimental details related to\nthe original Figures 2C and 2J. I also acknowledge that the observed differences, while statistically\nsignificant, appear modest in absolute terms and appreciate the opportunity to clarify this issue.\nFirst, I would like to point out that in radiation-induced intestinal injury models, crypt architectural\ndamage and cellular dysfunction occur substantially earlier than systemic manifestations such as body\nweight loss. Histological changes in the intestinal epithelium represent more sensitive and direct\nindicators of tissue injury than body weight alterations, as established in previous studies (Dose\nResponse. 2020;18(3):1559325820956783; Cell Death Discov. 2025;11(1):235). The modest body\nweight changes observed in our system are consistent with the early timepoints we set for examination,\nand reflect the initial stages of intestinal injury response. To more definitively establish the biological\nsignificance of G9a-mediated H3K9me2 in intestinal injury and regeneration, we performed additional\nexperiments using a lethal dose of 10 Gy irradiation. Both G9a knockout mice and UNC0642-treated\nmice showed significantly earlier mortality compared to controls (Figure 2-2), demonstrating the\ncritical importance of G9a-mediated H3K9me2 in mouse survival following severe intestinal injury.\nThis new survival data has been integrated into Fig. 2m in the revised manuscript.\nFigure 2-2. Survival curve of mice following 10 Gy irradiation\nKaplan-Meier survival analysis of G9afl/fl mice (cyan line), G9aΔIEC mice (red line), vehicle-treated mice (blue\nline), and UNC0642-treated mice (purple line) subjected to 10 Gy whole-body irradiation. n=8 mice/group.\nStatistical analysis was performed using log-rank (Mantel-Cox) test.\nActually, in the original Figures 2C and 2J (now Fig. 2a and 2g in the revised manuscript), 5 mice\nwere included per group with data presented as mean ± SEM. I have thoroughly revised all figure\nlegends in this revised manuscript, ensuring clearly stated sample information and other experimental\ndetails.\n4. The phenotype shown of disrupted intestinal epithelium in figure 3 is very mild compared to genetic\nablation of other histone modifiers in skin or intestinal stem cell niches (EZH2, RING1B for\nexample). Here constitutive loss of G9a has a mild phenotype while inducible loss of EZH2/\nRING1B has rapid and gross intestinal problems for mice. The authors should consider this in their\nresults/discussion as they need to put this mild phenotype in the context of other work that has been\nshown in the field previously.\nResponse: Thank you for this important reminding and instructive suggestion. Indeed, the observed\nphenotype of constitutive G9a loss was mild, compared to the severe defects observed with inducible\nEZH2 or RING1B ablation.\nIn our opinion, the phenotypic differences reflect distinct functional roles of these epigenetic\nmodifiers. For example, EZH2 and RING1B, as components of Polycomb repressive complexes,\nfunction as master regulators controlling fundamental developmental programs and core stem cell\nidentity maintenance. Their acute loss could cause immediate disruption of essential transcriptional\nnetworks, resulting in rapid and severe intestinal pathology. In contrast, our data indicate that G9a\noperates as a specialized modulatory regulator, rather than a master controller. As a matter of fact, our\nresults revealed that G9a functions through a unique \"fine-tuning\" mechanism that provides regulatory\nprecision without disrupting core transcriptional networks, which could explain why constitutive G9a\nloss produces manageable baseline effects while becoming critically important under stress conditions.\nI think the selective suppression of cell cycle arrest genes by G9a represents a specialized regulatory\nlayer that complements, rather than replaces, the fundamental circuits controlled by Polycomb\ncomplexes.\nFollowing your instruction, I have incorporated this comparative analysis into our discussion (Line\n623-636) to properly contextualize G9a's specialized role within the hierarchy of epigenetic regulation\nin intestinal stem cell biology, emphasizing how this modulatory function complements master\nregulatory system.\n5. Some of the plots seem to use average per mouse/organoid. In figure 4G for example 60 organoids\nwere tested per mouse but only the average organoid size per mouse is plotted. The same is true\nfor figure 3B, D, H, I. These data points should all be plotted together to account for variation in\norganoid size/ crypt depth/ cell number generally. Violin/cello plots should be used instead.\nResponse: I highly appreciate this friendly reminding. Following your instruction, we have\nsystematically revised our data presentation manner throughout the manuscript.\nFor the original Figure 4G (now Fig. 3h in the revised manuscript), we now plot all 60 individual\norganoid measurements rather than averaging them per mouse, thereby fully representing the\nbiological variation in organoid size. Similarly, for the original Figure 3B, D, H, and I (now\nSupplementary Fig. 6b, 6e, and Fig. 3b in the revised manuscript), all individual measurements of\ncrypt depth, cell number, and related parameters are now displayed as individual data points rather\nthan averaged values per mouse.\nFurthermore, we have replaced bar graphs with violin plots throughout the manuscript to better\nvisualize the complete data distribution and present natural biological variation. These changes have\nbeen implemented in multiple figures including Fig. 3e, 3h, 3j, and Supplementary Fig. 5f, 5g, 7e, 7g,\n9b, 10b, 10d, 11d, 11f, 11h, 11i, 12d, 13b, and 13d in the revised manuscript.\nTo appropriately handle the hierarchical nature of our data (multiple measurements per\nmouse/organoid), we have updated our statistical analyses accordingly. Where appropriate, we have\nemployed nested statistical models or clearly indicated the biological replicates (number of mice)\nversus technical replicates (measurements per mouse) in the figure legends.\nI believe these revisions substantially improve the transparency and rigor of our data presentation,\nand hope now you will find the updated figures satisfactory.\n6. The methods for the ChIP-seq do not mention any spike-in control. This is now a well established\nstandard for epigenomic analyses (https://doi.org/10.1038/s41587-024-02377-y). Without this it is\nunclear how reliable the changes in H3K9me2 detected in ChIP-seq are. While there seems to be\na complete loss of H3K9me2 in figure 5C these changes look minimal in figure 6A. This is a really\ncritical point as these figures attempt to link G9a catalytic activity to the phenotypic changes\nobserved in mice. The H3K9me2 signal shown in Figure 5C does not have the typical ‘peak’\npattern at TSS that many histone modifications do. This is a further reason it is so important to\ninclude internal ChIP-seq spike in normalisation controls. Also, the legend does not say what genes\nare being examined in Fig 5C, is this all genes, all H3K9me2 positive genes, or all genes that lose\nH3K9me2 signal in G9a knockout?\nResponse: Thank you very much for your instruction to improve our experiment design and data\nquality. I acknowledge that our initial ChIP-seq experiments did not include spike-in controls, which\nis indeed a well-established standard for reliable epigenomic analyses as highlighted in the Nature\nBiotechnology paper you mentioned.\nFollowing your instruction, we have repeated our ChIP-seq experiments with the following\nimprovements: (1) Implementation of spike-in normalization control: We incorporated external\nspike-in chromatin (from HEK293T cells) to allow for proper normalization and accurate\nquantification of H3K9me2 changes between different conditions. (2) Use of more specific antibodies:\nWe employed anti-H3K9me2 antibodies with higher specificity (#ab1220, Abcam) to reduce potential\ncross-reactivity and improve signal-to-noise ratio.\nThe results from our improved ChIP-seq experiments now demonstrate (Figure 2-3):\n1. Clear H3K9me2 enrichment at TSS regions in G9afl/fl control mice, showing the expected peak\npattern characteristic of this histone modification at TSS.\n2. Complete loss of H3K9me2 enrichment in G9a-deficient mice, confirming that the observed\nchanges are indeed due to G9a catalytic activity rather than technical artifacts.\nFigure 2-3. H3K9me2 ChIP-seq analysis\nHeatmap and average intensity plots (top row) illustrating H3K9me2 occupancy within ± 3kb of the TSS for all\nH3K9me2-positive genes. Analysis includes spike-in normalization control for accurate quantification.\nI think these improved results provide robust evidence to link G9a catalytic activity to H3K9me2\ndeposition and the subsequent phenotypic changes observed in our mouse model. Accordingly, I have\nupdated our MATERIALS AND METHODS section (Line 310-347) to include detailed spike-in\nnormalization protocol and have revised the figure legends to clearly specify that the analysis includes\nall H3K9me2-positive genes with appropriate spike-in normalization control.\nI hope now you will find that our epigenomic analysis methodology is feasible and the produced\nresults are sound.\n7. In Fig 5G it is unclear if these are differentially expressed/regulated sites for each dataset or if its\nup/down specifically. ATAC-up, RNA-up, H3K9me2 down sites are those that should examined to\nmake the mechanistic link the authors are proposing. But that does not seem to be the case. This\nanalysis presented shows there is poor correlation between H3K9me2 loss and increases in\naccessibility (only 450 of 5474 accessible sites overlap with H3K9me2). Therefore, the statement\nsaying H3K9me2 compacts chromatin in the text is entirely incorrect. These results are very\ndescriptive and do not seem to be mechanistically linked to the phenotype of the mouse without a\nmore comprehensive analysis of the ATAC and ChIP-seq data.\nResponse: Thank you for raising this critical mechanistic question. I understand your concern about\nthe need to establish a clear mechanistic link between H3K9me2 loss, chromatin accessibility, and\ngene expression changes.\nIn response to your comment, we have optimized our H3K9me2 ChIP-seq experimental conditions\nand additionally performed G9a ChIP-seq analysis to provide a more comprehensive mechanistic\nunderstanding. Our revised intersection analysis now reveals a much stronger correlation between the\ndatasets (Figure 2-4):\nIntersection analysis results:\n Triple overlap (H3K9me2 loss + G9a loss + ATAC-up): 3,913 genes\n H3K9me2 loss + G9a loss: 3,913 genes\n H3K9me2 loss + ATAC-up: 4,718 genes\nProportion Analysis:\n Of total H3K9me2-decreased sites (5,463): 3,913 sites (72%) show the expected correlation\nwith G9a loss and/or chromatin opening\n Of total G9a-decreased sites (5,086): 3,913 sites (77%) correlate with H3K9me2 loss and/or\nchromatin opening\n Of total ATAC-accessible sites (5,456): 3,913 sites (72%) correlate with H3K9me2/G9a loss\nI think these results strongly support our proposed mechanism: G9a-mediated H3K9me2\ndeposition → chromatin compaction → gene repression. Upon G9a loss, the removal of H3K9me2\nleads to chromatin decompaction (increased accessibility) and subsequent gene expression changes.\nI hope now you could agree that the substantially improved correlation (>70% overlap across\ndatasets), compared to our initial analysis, supports that G9a-catalyzed H3K9me2 modification\ndirectly regulates chromatin accessibility and gene expression in intestinal epithelial cells, thus\nproviding the mechanistic link between epigenetic regulation and the observed mouse phenotypes.\nFigure 2-4. Integration of H3K9me2 ChIP-seq, ATAC-seq, and RNA-seq analyses\nVenn diagram showing overlap of differentially regulated genes identified in ATAC-seq, ChIP-seq, and RNA-\nseq analysis.\n8. Due to the aforementioned limitations in quality of the H3K9me2 ChIP-seq the authors could\nperform G9a CUT&RUN/ ChIP-seq to assess which targets it binds to. This would be a more useful\nmechanistic link than mild changes in H3K9me2.\nResponse: We highly appreciate this constructive suggestion to improve our work, and completely\nagree that direct assessment of G9a binding targets would provide a more robust mechanistic\nunderstanding than relying solely on H3K9me2 changes. Following your instruction, we have\nperformed G9a ChIP-seq analysis (we employed ChIP-seq rather than CUT&RUN for technical\nconsistency with our other experiments) with the following improvements: (1) Direct G9a binding\nassessment: We conducted G9a ChIP-seq using high-specificity G9a antibodies to directly map G9a\noccupancy across the genome in both control (G9afl/fl) and knockout conditions. (2) Improved\nexperimental design: The G9a ChIP-seq experiments were performed with proper spike-in\nnormalization control, following the same rigorous standard we applied to our revised H3K9me2\nanalysis. (3) Comprehensive binding site analysis: We identified G9a binding sites genome-wide and\nperformed intersection analysis with our ATAC-seq and RNA-seq datasets to establish direct\nmechanistic links. From the results, we could find:\n1. G9a shows specific binding pattern at promoter regions of target genes.\n2. G9a occupancy correlates with H3K9me2 deposition at the same loci.\n3. Loss of G9a binding directly corresponds to increased chromatin accessibility and gene expression\nchanges.\n4. The intersection analysis (3,913 genes with G9a binding loss + H3K9me2 decrease + chromatin\nopening) provides direct evidence for the mechanistic pathway.\nI think these data have established a clear mechanistic explanation: G9a directly binds to target\ngene promoters, catalyzes H3K9me2 deposition, and regulates chromatin accessibility, and hope to get\nyour understanding.\nMinor comments\n1. In Figure 1, define the “Disease Activity Index” and how it was calculated in the present study.\nResponse: Thank you for your suggestion to improve the quality of our manuscript. “Disease Activity\nIndex” refers to the Crohn's Disease Activity Index (CDAI) which is a standardized scoring system\nused to assess disease severity in Crohn's disease patients (please refer to Gastroenterology.\n1976;70(3):439-44.).\nThe CDAI is calculated as a composite measure that incorporates multiple clinical parameters\nincluding abdominal pain, general well-being, stool frequency, and other relevant symptoms to provide\na numerical score typically ranging from 0 to 600, with higher scores indicating more active disease.\nThe specific CDAI values for each patient included in our study are provided in Source data file.\nWe have revised the y-axis labels in the original Figure 1C and 1E (now in Supplementary Fig.3c\nand 3e in the revised manuscript) as “Crohn's Disease Activity Index scores” and added a brief\nexplanation in the figure legend for clarity. I believe this revision will make the term understandable\nfor the potential readers.\n2. Western blots to detect minor changes in histone modifications levels are notoriously unreliable.\nThe authors should quantify the changes observed across experimental replicates in histone\nmodifications- particularly in figure 1, 2 and S2B. Blots in Figure 2A are quite poor with several\nbeing overexposed and band signal bleeding into neighbouring lanes. This is particularly important\nin figure 2A and 2B to establish how well the model systems targeting G9a are working. The G9a\ninhibitor seems to have a very mild effect on H3K9me2 levels based on these blots.\nResponse: I appreciate your carefulness in examining our immunoblotting images and your instruction\nto improve the quality of our data. Following your advice, we have repeated the immunoblotting\nexperiments with improved technical conditions to eliminate overexposure and band bleeding issues\npresented in the original Figure 2A; we also carried out quantitative analyses of the histone\nmodification changes across all experimental replicates in the original Figures 1, 2, and Supplementary\nFigure S2B. The revised Figure 2A and 2B (now Supplementary Fig.4e and 4f in the revised\nmanuscript) now clearly demonstrate the effectiveness of our G9a targeting approaches. Additionally,\nwe have provided corresponding uncropped western blot images to ensure full transparency and allow\nverification of our results. I think these modifications provide robust evidence for the effectiveness of\nour experimental approaches and strengthen the reliability of our conclusions regarding G9a-mediated\nH3K9me2 regulation.\n3. Fig S4C western blot has some important technical issues. The image shown as a ‘blot’ is missing\na membrane in both G9a blots. Lines above the G9a band are clearly visible showing where the\nmembrane has been cut. The box shapes around these should be readjusted accordingly to avoid\nshowing empty space where it seems like a blot should be.\nResponse: Thank you for pointing out this technical issue in original Figure S4C. Following your\nsuggestion, we have provided the original uncropped immunoblotting images and made corresponding\ncorrections to Fig. S4C in the revised manuscript. The membrane cutting lines and empty spaces have\nbeen properly addressed, and the presentation now accurately reflects the actual blot results without\nmisleading cropping or artificial boundaries.\n4. Figure 3 and 4 make the same broad conceptual point as figure 2 but without additional interesting\ndetails that aid mechanistic interpretations of the data. G9a is clearly important for proliferation and\nmaintenance of the stem cell compartment- and therefore intestinal epithelia. However, this same\npoint is made repetitively using different assays over there three figures. There I no apparent\nspecificity of this phenotype in any particular differentiated cells, it seems to arise from issues in\nthe stem cells. Much of this data could be moved to a supplementary figure supporting figure 2.\nResponse: I highly appreciate your insightful comment regarding the conceptual overlap between\nFigures 2, 3, and 4. Our original rationale was to present a logical progression from the pathological\nrole of G9a in intestinal injury and repair, to its effects on crypt structure, and finally to its impact on\ncellular proliferation - essentially moving from disease relevance to phenotypic manifestations, and\nthen to cellular function. Thanks to your criticism, I acknowledge that these three figures did contain\nredundancy without providing substantially new mechanistic insights. To address this issue and\nimprove the manuscript's clarity, I have made following revisions: First, I have retained Figure 2 as\nthe primary demonstration of the overall phenotypic effects of G9a deficiency on intestinal\nregeneration. Second, I have streamlined Figures 3 and 4 by moving the detailed villus-crypt\nmorphological analyses, cell type quantifications, and portions of the EdU pulse-chase experiments to\nsupplementary figures. Overall, I have only preserved the most mechanistically relevant panels that\ndirectly illustrate how H3K9me2 specifically regulates stem cell proliferation, such as key\nOLFM4/Ki67 staining and cell cycle protein expression data in the main figure (Fig. 3d, 3e and 3f in\nthe revised manuscript). Third, I have reorganized the figure presentation framwork to create a more\nlogical flow: Figure 2 (overall phenotype) → Figure 3 (streamlined, focusing on stem cell-specific\nmechanisms) → Figure 4 (the current Figures 5-6, emphasizing molecular mechanisms). I think this\nrestructuring has eliminated redundancy while maintaining the integrity of our mechanistic findings\nand better highlighting the specific role of H3K9me2 in suppressing cell cycle arrest genes to promote\nintestinal regeneration.\n5. The discussion states that H3K27me3 is present only in the villi and not the crypt. However, this\nis not true (doi: 10.15252/embj.201694550). The citations the authors provide for this section are\nalso incorrect and cite two studies (#30 and #31) that do not examine the intestinal epithelium at\nall.\nResponse: I sincerely apologize for the carelessness in drafting the manuscript and acknowledge that\nthis represents a significant oversight in academic rigor. Yes, the statement regarding H3K27me3\ndistribution in our original DISCUSSION is inaccurate! As a matter of fact, H3K27me3 is not\nexclusively present in villi but is found throughout both crypt and villus compartments, as\ndemonstrated by Chiacchiera et al. (EMBO J. 2016;35(21):2301). I have also removed the incorrect\ncitations (#30 and #31) that did not examine intestinal epithelium and the erroneous spatial distribution\nclaim.\nAccordingly, I have revised this section to accurately reflect the current understanding of histone\nmodifications in intestinal epithelium:\n“Previous studies have revealed that histone modifications control IECs self-renewal and\ndifferentiation processes. For example, MLL1-mediated H3K4me3 modification is essential for\nintestinal stem cell maintenance by promoting the expression of key transcription factors, with its loss\nresulting in ISC depletion and secretory lineage bias (PLoS Genet. 2021;17(12):e1009250). PRC2-\nmediated H3K27me3 modification controls progenitor cell proliferation and lineage balance, being\nparticularly critical for radiation-induced regeneration through Cdkn2a repression (Mol Cell.\n2020;78(1):141-151.e5; EMBO J. 2016;35(21):2301-2314.). SETDB1-mediated H3K9me3\nmodification contributes to genomic stability by silencing endogenous retroviral elements (Gut.\n2021;70(3):485; Nature. 2020;580(7803):386).”\nI hope our revision could now satisfy you\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: Thank you for participating as a co-reviewer in this peer review process. I hope our above\nresponses to the reviewer’s comments have adequately addressed your concerns."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have clearly addressed all my comments and concerns (both major and minor) with some substantial new data and changes to their manuscript text. Their new data is particularly impressive regarding 1) New histone mass spectrometry data highlighting the specific importance of G9a/H3K9me2 in the IEC context 2) Vulnerability of G9a mutant mice to irradiation highlighting a potent phenotype in tissue repair and 3) integrating new ChIP-seq data for H3K9me2 and G9a together with their RNA-seq and ATAC-seq to understand how G9a can influence the cell cycle.\nThis revised manuscript is technically sound and provides a substantial advance in our understanding of histone modifications, G9a and intestinal tissue homeostasis. I recommend this manuscript for publication in Nature Communications.", "reviewer_4": "The data presented in the manuscript demonstrate the role of G9a-mediated H3K9me2 in controlling the proliferation of intestinal crypt cells during the regenerative phase after 8 Gy irradiation or DSS treatment. However, even though the authors attempt to focus on intestinal regeneration in the text, the deletion of G9a-mediated H3K9me2 likely impacts cell cycle progression in general, given that G9IECKO mice exhibit decreased intestinal proliferation at homeostasis. In addition, the legends of several Figures remain unclear regarding whether the samples were collected from mice at homeostasis or at specific time points after injury. Together, the authors should more explicitly clarify the effect of G9a-mediated H3K9me2 on intestinal homeostasis versus the injury response, which remains elusive in the current manuscript."}, "author_response": "Point-to-point Response to the Reviewers’ Comments\nWe are grateful to all reviewers for their valuable feedback and the time invested in evaluating our\nmanuscript. We were pleased that Reviewer #2 found our revised manuscript technically sound and\nrecommended it for publication in Nature Communications. In response to Reviewer #4's concern, we\nhave now explicitly clarified the distinction between G9a-mediated H3K9me2 functions under\nhomeostatic versus regenerative conditions throughout the manuscript, and have revised all relevant\nfigure legends to clearly indicate whether samples were collected at steady-state or at specific time\npoints after injury. Our detailed point-by-point responses are provided below.\nReviewer #2:\nThe authors have clearly addressed all my comments and concerns (both major and minor) with some\nsubstantial new data and changes to their manuscript text. Their new data is particularly impressive\nregarding 1) New histone mass spectrometry data highlighting the specific importance of\nG9a/H3K9me2 in the IEC context 2) Vulnerability of G9a mutant mice to irradiation highlighting a\npotent phenotype in tissue repair and 3) integrating new ChIP-seq data for H3K9me2 and G9a together\nwith their RNA-seq and ATAC-seq to understand how G9a can influence the cell cycle.\nThis revised manuscript is technically sound and provides a substantial advance in our understanding\nof histone modifications, G9a and intestinal tissue homeostasis. I recommend this manuscript for\npublication in Nature Communications.\nResponse: Thank you for your positive evaluation of our revised manuscript. We greatly appreciate\nyour recognition of our new experimental data, including the histone mass spectrometry, irradiation\nexperiments, and integrated ChIP-seq analyses. Your constructive feedback throughout the review\nprocess has been instrumental in strengthening our work. We are grateful for your recommendation for\npublication.\nReviewer #4:\nThe data presented in the manuscript demonstrate the role of G9a-mediated H3K9me2 in controlling\nthe proliferation of intestinal crypt cells during the regenerative phase after 8 Gy irradiation or DSS\ntreatment. However, even though the authors attempt to focus on intestinal regeneration in the text, the\ndeletion of G9a-mediated H3K9me2 likely impacts cell cycle progression in general, given that\nG9aIECKO mice exhibit decreased intestinal proliferation at homeostasis. In addition, the legends of\nseveral Figures remain unclear regarding whether the samples were collected from mice at homeostasis\nor at specific time points after injury. Together, the authors should more explicitly clarify the effect of\nG9a-mediated H3K9me2 on intestinal homeostasis versus the injury response, which remains elusive\nin the current manuscript.\nResponse: Thank you for this insightful comment regarding the distinction between G9a-mediated\nH3K9me2 functions in intestinal homeostasis versus injury-induced regeneration. We appreciate the\nopportunity to clarify this important aspect of our study.\nWe acknowledge that G9a-mediated H3K9me2 regulates intestinal crypt cell proliferation under\nboth homeostatic and regenerative conditions. As you correctly noted, G9aΔIEC mice exhibit reduced\nintestinal proliferation at homeostasis, as demonstrated in Figure 3 and Supplementary Figure 9, where\nsamples were collected from mice under steady-state conditions without any injury treatment. This\nindicates that G9a-mediated H3K9me2 is indeed required for maintaining baseline proliferative\ncapacity of intestinal crypt cells.\nHowever, our data also reveal that G9a-mediated H3K9me2 plays a particularly critical role during\nthe regenerative response. Several lines of evidence support this conclusion. First, the dynamic\nregulation of H3K9me2 and G9a levels during injury and repair, as shown in Figure 1 and\nSupplementary Figure 1-3, demonstrates a biphasic pattern with initial reduction following injury and\nsubsequent upregulation during regeneration. This temporal regulation suggests an active modulation\nof this epigenetic pathway in response to tissue damage. Second, ChIP-qPCR analysis in Figure 5e\nshows that H3K9me2 occupancy at cell cycle arrest gene promoters significantly increases during the\nrepair process, indicating enhanced epigenetic silencing of these genes specifically during regeneration.\nThird, the upstream IL-4-STAT6 signaling pathway, which we identified as the regulator of G9a\nexpression, is specifically activated during the regenerative phase, with IL-4 showing significant\nupregulation at 48-96 hours post-irradiation (Figure 6). Fourth, the survival experiments following 10\nGy irradiation (Figure 2m) demonstrate that the consequences of G9a deficiency become substantially\nmore severe under regenerative stress, with both G9aΔIEC mice and UNC0642-treated mice showing\nsignificantly reduced survival compared to controls.\nTherefore, we propose that G9a-mediated H3K9me2 serves dual functions: maintaining baseline\nproliferative capacity under homeostatic conditions and enabling enhanced proliferative responses\nduring tissue regeneration. The regeneration-specific upregulation of G9a through IL-4-STAT6\nsignaling represents an additional regulatory layer that amplifies this epigenetic mechanism when rapid\ntissue repair is required.\nRegarding the figure legends, we apologize for any ambiguity and have now clarified the\nexperimental conditions throughout. Specifically, Figures 3, 4, 5a-c and Supplementary Figures 4, 6,\n7, 8, 9, 10, 11, 14 present data from mice under homeostatic conditions without any injury treatment.\nFigures 2, 5d-f, 6 and Supplementary Figures 5, 12 show data from mice at specific time points after\ninjury, as indicated in the experimental schematics within each figure. Figure 1b-e illustrates the\ntemporal dynamics of H3K9me2 and G9a levels across the injury-regeneration timeline.\nWe have revised the manuscript text to more explicitly distinguish between the homeostatic and\nregenerative functions of G9a-mediated H3K9me2, and have clarified the figure legends to indicate\nthe experimental conditions for each dataset."}]

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