Search Results (52)

Colchicine is commonly prescribed for inflammation and gout, but its nephrotoxicity and underlying mechanisms remain incompletely understood. The objective of this research was to clarify the association between m6A methylation modifications and nephrotoxicity caused by colchicine. A significant decrease in HK2 cell viability was observed following colchicine treatment, and mRNA sequencing (mRNA-seq) revealed the differential expression of genes associated with DNA damage and autophagy. Further methylated RNA immunoprecipitation sequencing (MeRIP-seq) analysis revealed an association between N6-methyladenosine (m6A) modifications and the expression of genes involved in DNA damage and autophagy after colchicine exposure. Molecular docking and a molecular dynamics (MD) analysis identified ZC3H13 as a potential regulator of colchicine-induced cytotoxicity in HK2. Experimental validation confirmed that colchicine induces DNA damage and autophagy in HK2 cells, with ZC3H13 playing a significant role in these processes. In conclusion, the findings suggested that colchicine-induced damage in HK2 cells is associated with changes in m6A methylation levels in target genes and the altered expression of m6A regulator. Full article

Plants are constantly exposed to environmental stressors such as drought, salinity, and extreme temperatures, which threaten their growth and productivity. To counter these challenges, they employ complex molecular defense systems, including epigenetic modifications that regulate gene expression without altering the underlying DNA sequence. This review comprehensively examines the emerging roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as central signaling molecules orchestrating epigenetic changes in response to abiotic stress. In addition, biotic factors such as pathogen infection and microbial interactions are considered for their ability to trigger ROS/RNS generation and epigenetic remodeling. It explores how ROS and RNS influence DNA methylation, histone modifications, and small RNA pathways, thereby modulating chromatin structure and stress-responsive gene expression. Mechanistic insights into redox-mediated regulation of DNA methyltransferases, histone acetyltransferases, and microRNA expression are discussed in the context of plant stress resilience. The review also highlights cutting-edge epigenomic technologies such as whole-genome bisulfite sequencing (WGBS), chromatin immunoprecipitation sequencing (ChIP-seq), and small RNA sequencing, which are enabling precise mapping of stress-induced epigenetic landscapes. By integrating redox biology with epigenetics, this work provides a novel framework for engineering climate-resilient crops through the targeted manipulation of stress-responsive epigenomic signatures. Full article

Background: Ovarian cancer remains the most lethal gynecological cancer, primarily due to its asymptomatic nature in early stages and consequent late diagnosis. Early detection improves survival, but current biomarkers lack sensitivity and specificity. Cell-free DNA (cfDNA) released from tumor cells captures tumor-associated epigenetic alterations and represents a promising source for minimally invasive biomarkers. Among these, aberrant DNA methylation occurs early in tumorigenesis and may reflect underlying disease biology. This study aimed to investigate genome-wide cfDNA methylation profiles in patients with ovarian cancer, benign ovarian conditions, and healthy controls to identify cancer-associated methylation patterns that may inform future biomarker development. Results: We performed genome-wide cfDNA methylation profiling using cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) on plasma samples from 40 patients with high-grade serous ovarian carcinoma, 38 patients with benign ovarian conditions, and 38 healthy postmenopausal women. A total of 536 differentially methylated regions (DMRs) were identified between ovarian cancer and controls (n = 76), with 97% showing hypermethylation in ovarian cancer. DMRs were enriched in CpG islands and gene bodies and depleted in repetitive elements, consistent with known cancer-associated methylation patterns. Fifteen genes showed robust hypermethylation across analyses. These genes exhibited methylation across intronic, exonic, and upstream regulatory regions. Separate comparisons of ovarian cancer to each control group (benign and healthy) supported the reproducibility of these findings. Gene Ontology enrichment analysis revealed enrichment in gland development, embryonic morphogenesis, and endocrine regulation, suggesting biological relevance to ovarian tumorigenesis. Conclusions: This study identifies consistent cfDNA hypermethylation patterns in ovarian cancer, affecting genes involved in developmental regulation and hormone-related processes. Our findings underscore the potential of cfMeDIP-seq for detecting tumor-specific methylation signatures in plasma and highlight these 15 hypermethylated genes as biologically relevant targets for future studies on cfDNA methylation in ovarian cancer. Full article

MSTN has been used as a candidate gene in the genetics, breeding, and improvement of animal breeds. However, the possible mechanism by which the MSTN gene regulates muscle development through PSMA6 is not well understood. Previous methylome and transcriptome sequencing analyses of gluteal muscle tissues from MSTN+/−Luxi cattle and wild-type Luxi cattle identified that the PSMA6 gene exhibited a negative correlation between methylation levels and transcriptional activity. To investigate whether MSTN expression regulates PSMA6 gene expression, we examined the effects of MSTN on DNA methyltransferases (DNMT1, DNMT2, DNMT3A, and DNMT3B) and DNA demethylases (TET1, TET2, and TET3). Additionally, chromatin immunoprecipitation (ChIP) assays were performed to detect the binding interaction between PSMA6 and TET2. In this paper, we first established an MSTN knockdown cellular model to preliminarily validate its regulatory effect on PSMA6 expression. Subsequently, the developmental impact of PSMA6 on bovine skeletal muscle satellite cells was further investigated through both knockdown and overexpression of the PSMA6 gene. Furthermore, we examined changes in the expression of key components of the AKT/mTOR signaling pathway to elucidate the mechanisms underlying the PSMA6-mediated regulation of satellite cell development. The results demonstrate that myostatin (MSTN) inhibition significantly decreased proteasome 20S subunit alpha-6 (PSMA6) gene expression, while increasing demethylase expression, particularly ten-eleven translocation-2 (TET2), which exhibited the most pronounced changes. During the cell proliferation stage, the markers Paired Box 7 (PAX7) and Ki-67 exhibited no significant changes, whereas the PSMA6 gene was either overexpressed or disrupted. Conversely, PSMA6 overexpression altered the myogenic differentiation markers, causing the differential regulation of myosin heavy chain (MyHC) and myogenin (MyoG) expression, with MyHC upregulation and concurrent MyoG downregulation. PSMA6 gene overexpression led to the downregulation of AKT1 and Rac1, as well as the activation of the AKT/mTOR pathway, including key factors such as mTOR, p-mTOR, RPS6, p-RPS6, and RhoA. PSMA6 interference resulted in the downregulation of p-mTOR and the upregulation of p-RPS6. Gene expression profiling in our study revealed that the myostatin (MSTN) knockout model significantly reduced the transcriptional levels of the proteasome α6 subunit (PSMA6) (p < 0.05), with the regulatory intensity showing a significant negative correlation with MSTN expression. This molecular evidence substantiates a negative regulatory axis between MSTN and PSMA6. Functional experiments demonstrated that PSMA6 overexpression specifically enhanced myotube formation rates in bovine skeletal muscle satellite cells, whereas siRNA-mediated PSMA6 knockdown exhibited no significant effects on cellular proliferation, indicating the functional specificity of this gene in myogenic differentiation. Mechanistic investigations further revealed that PSMA6 activates the canonical AKT/mTOR signaling transduction cascade through the phosphorylation of AKT and its downstream effector mTOR, thereby mediating the expression of myogenic regulatory factors MyoD and myogenin. Collectively, these findings demonstrate that MSTN deficiency alleviates the transcriptional repression of PSMA6, remodels skeletal muscle differentiation-associated signaling networks, and ultimately drives the directional differentiation of satellite cells toward myofiber specification. Full article

Background: Breast cancer (BrCa) patients with tumors expressing high interleukin-6 (IL6) levels have poor clinical outcomes. In BrCa, altered occupancy of CCCTC-binding factor (CTCF) within its DNA binding sites deregulates the expression of its targeted genes. In this study, we investigated whether CTCF contributes to the altered IL6 expression in BrCa. Methods/Results: We performed CTCF gain- and loss-of-function assays in BrCa cell lines and observed an inverse correlation between CTCF and IL6 expression levels. To understand how CTCF negatively regulates IL6 gene expression, we performed luciferase gene reporter assays, site-directed mutagenesis assays, and chromatin immunoprecipitation assays. Our findings revealed that CTCF interacted with the IL6 promoter, a form of regulation disrupted in a CpG methylation-independent fashion in MDA-MB-231 and Tamoxifen-resistant MCF7 cells. Data from TCGA and GEO databases allowed us to explore the clinical implications of our results. An inverse correlation between CTCF and IL6 expression levels was seen in disease-free survival BrCa patients but not in patients who experienced cancer recurrence. Conclusions: Our findings provide evidence that the CTCF-mediated negative regulation of the IL6 gene is lost in highly tumorigenic BrCa cells. Full article

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies. Full article

Background: The current study’s objective is to evaluate the molecular genetic mechanisms influencing the biological behavior of hepatocellular carcinoma (HCC) by analyzing the transcriptomic and epigenetic signatures of the tumors. Methods: Transcriptomic data were downloaded from the NCBI GEO database. We investigated the expression differences between the GSE46444 (48 cirrhotic tissues versus 88 HCC tissues) and GSE63898 (168 cirrhotic tissues versus 228 HCC tissues) data sets using GEO2R. Differentially expressed genes were evaluated using GO and KEGG metabolic pathway analysis websites. Whole genome bisulfite sequencing (WGBS) and Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) data sets (26 HCC tissues versus 26 adjacent non-tumoral tissues) were also downloaded from the NCBI SRA database. These data sets were analyzed using Bismark and QSEA, respectively. The methylation differences between the groups were assessed using functional enrichment analysis. Results: In the GSE46444 data set, 80 genes were upregulated, and 315 genes were downregulated in the tumor tissue (HCC tissue) compared to the non-tumor cirrhotic tissue. In the GSE63898 data set, 1261 genes were upregulated, and 458 genes were downregulated in the cirrhotic tissue compared to the tumor tissues. WGBS revealed that 20 protein-coding loci were hypermethylated. while the hypomethylated regions were non-protein-coding. The methylated residues of the tumor tissue, non-tumorous cirrhotic tissue, and healthy tissue were comparable. MeDIP-Seq, conducted on tumoral and non-tumoral tissues, identified hypermethylated or hypomethylated areas as protein-coding regions. The functional enrichment analysis indicated that these genes were related to pathways including peroxisome, focal adhesion, mTOR, RAP1, Phospholipase D, Ras, and PI3K/AKT signal transduction. Conclusions: The investigation of transcriptomic and epigenetic mechanisms identified several genes significant in the biological behavior of HCC. These genes present potential targets for the development of targeted therapy. Full article

Retinoic acid receptor-related orphan receptor alpha (RORα), a candidate tumor suppressor, is prevalently downregulated or lost in malignant breast cancer cells. However, the mechanisms of how RORα expression is regulated in breast epithelial cells remain incompletely understood. Protein arginine N-methyltransferase 5 (PRMT5), a type II methyltransferase catalyzing the symmetric methylation of the amino acid arginine in target proteins, was reported to regulate protein stability. To study whether and how PRMT5 regulates RORα, we examined the direct interaction between RORα and PRMT5 by immunoprecipitation and GST pull-down assays. The results showed that PRMT5 directly bound to RORα, and PRMT5 mainly symmetrically dimethylated the DNA-binding domain (DBD) but not the ligand-binding domain (LBD) of RORα. To investigate whether RORα protein stability is regulated by PRMT5, we transfected HEK293FT cells with RORα and PRMT5-expressing or PRMT5-silencing (shPRMT5) vectors and then examined RORα protein stability by a cycloheximide chase assay. The results showed that PRMT5 increased RORα protein stability, while silencing PRMT5 accelerated RORα protein degradation. In PRMT5-silenced mammary epithelial cells, RORα protein expression was decreased, accompanied by an enhanced epithelial–mesenchymal transition morphology and cell invasion and migration abilities. In PRMT5-overexpressed mammary epithelial cells, RORα protein was accumulated, and cell invasion was suppressed. These findings revealed a novel mechanism by which PRMT5 regulates RORα protein stability. Full article

Type 1 diabetes (T1D) affects gastrointestinal (GI) motility, favoring gastroparesis, constipation, and fecal incontinence, which are more prevalent in women. The mechanisms are unknown. Given the G-protein-coupled estrogen receptor’s (GPER) role in GI motility, we investigated sex-related diabetes-induced epigenetic changes in GPER. We assessed GPER mRNA and protein expression levels using qPCR and Western blot analyses, and quantified the changes in nuclear DNA methyltransferases and histone modifications (H3K4me3, H3Ac, and H3K27Ac) by ELISA kits. Targeted bisulfite and chromatin immunoprecipitation assays were used to evaluate DNA methylation and histone modifications around the GPER promoter by chromatin immunoprecipitation assays in gastric and colonic smooth muscle tissues of male and female control (CTR) and non-obese diabetic (NOD) mice. GPER expression was downregulated in NOD, with sex-dependent variations. In the gastric smooth muscle, not in colonic smooth muscle, downregulation coincided with differences in methylation ratios between regions 1 and 2 of the GPER promoter of NOD. DNA methylation was higher in NOD male colonic smooth muscle than in NOD females. H3K4me3 and H3ac enrichment decreased in NOD gastric smooth muscle. H3K4me3 levels diminished in the colonic smooth muscle of NOD. H3K27ac levels were unaffected, but enrichment decreased in NOD male gastric smooth muscle; however, it increased in the NOD male colonic smooth muscle and decreased in the female NOD colonic smooth muscle. Male NOD colonic smooth muscle exhibited decreased H3K27ac levels, not female, whereas female NOD colonic smooth muscle demonstrated diminished enrichment of H3ac at the GPER promoter, contrary to male NOD. Sex-specific epigenetic mechanisms contribute to T1D-mediated suppression of GPER expression in the GI tract. These insights advance our understanding of T1D complications and suggest promising avenues for targeted therapeutic interventions. Full article

Apobec-1 complementation factor (A1CF) functions as an RNA-binding cofactor for APO-BEC1-mediated C-to-U conversion during RNA editing and as a hepatocyte-specific regulator in the alternative pre-mRNA splicing of metabolic enzymes. Its role in RNA editing has not been clearly established. Western blot, co-immunoprecipitation (Co-IP), immunofluorescence (IF), methyl thiazolyl tetrazolium (MTT), and 5-ethynyl-2′-deoxyuridine (EdU) assays were used to examine the role of A1CF beyond RNA editing in renal carcinoma cells. We demonstrated that A1CF interacts with NKRF, independent of RNA and DNA, without affecting its expression or nuclear translocation; however, it modulates p65(Ser536) phosphorylation and IFN-β levels. Truncation of A1CF or deletion on NKRF revealed that the RRM1 domain of A1CF and the p65 binding motif of NKRF are required for their interaction. Deletion of RRM1 on A1CF abrogates NKRF binding, and the decrease in IFN-β expression and p65(Ser536) phosphorylation was induced by A1CF. Moreover, full-length A1CF, but not an RRM1 deletion mutant, promoted cell proliferation in renal carcinoma cells. Perturbation of A1CF levels in renal carcinoma cells altered anchorage-independent growth and tumor progression in nude mice. Moreover, p65(Ser536) phosphorylation and IFN-β expression were lower, but ki67 was higher in A1CF-overexpressing tumor tissues of a xenograft mouse model. Notably, primary and metastatic samples from renal cancer patients exhibited high A1CF expression, low p65(Ser536) phosphorylation, and decreased IFN-β levels in renal carcinoma tissues compared with the corresponding paracancerous tissues. Our results indicate that A1CF-decreased p65(Ser536) phosphorylation and IFN-β levels may be caused by A1CF competitive binding to the p65-combined site on NKRF and demonstrate the direct binding of A1CF independent of RNA or DNA in signal pathway regulation and tumor promotion in renal carcinoma cells. Full article

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy. Full article

Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O₂ environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention. Full article

Hepatocellular carcinoma (HCC) is a highly lethal malignant neoplasm, and the involvement of bone morphogenetic protein 9 (BMP9) has been implicated in the pathogenesis of liver diseases and HCC. Our goal was to investigate the role of BMP9 signaling in regulating N6-methyladenosine (m⁶A) methylation and cell cycle progression, and evaluate the therapeutic potential of BMP receptor inhibitors for HCC treatment. We observed that elevated levels of BMP9 expression in tumor tissues or serum samples from HCC patients were associated with a poorer prognosis. Through in vitro experiments utilizing the m⁶A dot blotting assay, we ascertained that BMP9 reduced the global RNA m⁶A methylation level in Huh7 and Hep3B cells, thereby facilitating their cell cycle progression. This effect was mediated by an increase in the expression of the inhibitor of DNA-binding protein 1 (ID1). Additionally, using methylated RNA immunoprecipitation qPCR(MeRIP-qPCR), we showed that the BMP9-ID1 pathway promoted CyclinD1 expression by decreasing the m⁶A methylation level in the 5′ UTR of mRNA. This occurred through the upregulation of the fat mass and obesity-associated protein (FTO) in Huh7 and Hep3B cells. In our in vivo mouse xenograft models, we demonstrated that blocking the BMP receptor with LDN-212854 effectively suppressed HCC growth and induced global RNA m⁶A methylation. Overall, our findings indicate that the BMP9-ID1 pathway promotes HCC cell proliferation by down-regulating the m⁶A methylation level in the 5′ UTR of CyclinD1 mRNA. Targeting the BMP9-ID1 pathway holds promise as a potential therapeutic strategy for treating HCC. Full article

Genome-wide association studies have revealed an association between the genetic variant rs17514846 in the FURIN gene and coronary artery disease. We investigated the mechanism through which rs17514846 modulates FURIN expression. An analysis of isogenic monocytic cell lines showed that the cells of the rs17514846 A/A genotype expressed higher levels of FURIN than cells of the C/C genotype. Pyrosequencing showed that the cytosine (in a CpG motif) at the rs17514846 position on the C allele was methylated. Treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine increased FURIN expression. An electrophoretic mobility super-shift assay with a probe corresponding to the DNA sequence at and around the rs17514846 position of the C allele detected DNA-protein complex bands that were altered by an anti-MeCP2 antibody. A chromatin immunoprecipitation assay with the anti-MeCP2 antibody showed an enrichment of the DNA sequence containing the rs17514846 site. siRNA-mediated knockdown of MeCP2 caused an increase in FURIN expression. Furthermore, MeCP2 knockdown increased monocyte migration and proliferation, and this effect was diminished by a FURIN inhibitor. The results of our study suggest that DNA methylation inhibits FURIN expression and that the coronary artery disease-predisposing variant rs17514846 modulates FURIN expression and monocyte migration via an allele-specific effect on DNA methylation. Full article

Booroola fecundity (FecB) gene, a mutant of bone morphogenetic protein 1B (BMPR-1B) that was discovered in Booroola Merino, was the first prolificacy gene identified in sheep related to increased ovulation rate and litter size. The mechanism of FecB impact on reproduction is unclear. Methods: In this study, adult Han ewes with homozygous FecB(B)/FecB(B) mutations (Han BB group) and ewes with FecB(+)/FecB(+) wildtype (Han ++ group) were selected. Methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) was used to identify differences in methylated genes in ovary tissue. Results: We examined differences in DNA methylation patterns between HanBB and Han ++ sheep. In both sheep, methylated reads were mainly distributed at the gene body regions, CpG islands and introns. The differentially methylated genes were enriched in neurotrophy in signaling pathway, Gonadotropin Releasing Hormone (GnRH) signaling pathway, Wnt signaling pathway, oocyte meiosis, vascular endothelial growth factor (VEGF) signaling pathway, etc. Differentially-methylated genes were co-analyzed with differentially-expressed mRNAs. Several genes which could be associated with female reproduction were identified, such as FOXP3 (forkhead box P3), TMEFF2 (Transmembrane Protein with EGF Like and Two Follistatin Like Domains 2) and ADAT2 (Adenosine Deaminase TRNA Specific 2). Conclusions: We constructed a MeDIP-seq based methylomic study to investigate the ovarian DNA methylation differences between Small-Tail Han sheep with homozygous FecB mutant and wildtype, and successfully identified FecB gene-associated differentially-methylated genes. This study has provided information with which to understand the mechanisms of FecB gene-induced hyperprolificacy in sheep. Full article