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The incidence of diabetic kidney disease (DKD) has increased rapidly worldwide in recent decades, and DKD is the leading cause of chronic kidney disease. The Wnt/β-catenin pathway is widely recognized as a critical contributor to DKD. However, how this pathway is activated in DKD is still unknown. Recent studies have revealed that epigenetic mechanisms play key roles in DKD. DNA methylation is an epigenetic mechanism that is essential for regulating gene transcription. Here, we demonstrated that reducing the expression of DNMT3B, a DNA methyltransferase, markedly decreased extracellular matrix (ECM) deposition and diabetic renal fibrosis (DRF). Furthermore, we found that DNMT3B activated the Wnt/β-catenin pathway by suppressing SFRP5 expression in HG-induced renal tubular epithelial cells. Mechanistically, we observed that DNMT3B increased the promoter methylation levels of sfrp5 , which contributed to a decrease in SFRP5 protein expression. Additionally, Pharmacological disruption of DNA methylation (via 5-Aza) and genetic knockdown of DNMT3B suppressed the Wnt/β-catenin pathway, leading to the attenuation of ECM deposition and DRF. Thus, our study provides a novel understanding of the epigenetic regulation of DKD pathogenesis and a new therapeutic strategy for DKD by disrupting the Wnt/β-catenin pathway.

Ovarian cancer (OC) remains the leading cause of death among gynecologic cancers. Most women diagnosed with OC at advanced stages eventually develop relapse and chemoresistance, leading to poor clinical outcomes. While piRNAs have emerged as critical regulators of gene expression and tumor biology, their specific roles in OC remain to be fully elucidated. This study integrated clinical and computational analyses to investigate the expression pattern and functional relevance of P-element-induced wimpy testis (PIWI)-interacting RNA-823 (piR-823) and its associated protein piwi-like RNA-mediated gene silencing 1 (PIWIL1)/DNA methyltransferase 3B (DNMT3B)/E-cadherin (CDH1) axis in OC tissues from 40 patients, with 20 non-cancer control samples. Expression profiling was performed using qPCR on OC and normal ovarian tissues, followed by correlation and regression analyses. Public databases, including GEPIA, TNM plot, and MethBank, were explored to validate gene expression, methylation status, and pathway enrichment. Our results revealed that piR-823, PIWIL1, and DNMT3B were significantly upregulated in OC tissues (p < 0.001, p = 0.009, and p < 0.001, respectively), and they correlated positively with each other and inversely with CDH1 expression. CDH2, OCT4, and NANOG were significantly upregulated (p = 0.011, p = 0.03, and p < 0.001, respectively), whereas CDH1 expression was significantly downregulated (p < 0.001) in OC tissues. In silico analyses supported DNMT3B-mediated CDH1 promoter methylation, epithelial–mesenchymal transition (EMT), and stemness pathway enrichment. Our integrated computational and clinical analyses indicate that the piR-823/PIWIL1/DNMT3B/CDH1 axis is a putative epigenetic regulator of EMT and cancer stemness in ovarian cancer. Additionally, piR-823 may serve as a promising prognostic biomarker and therapeutic target, offering novel insights into OC pathogenesis and treatment.

Stem cell proviral silencing Endogenous retroviruses are widely dispersed in mammalian genomes, and are silenced in somatic cells by DNA methylation. Here, an endogenous retroviruses silencing pathway independent of DNA methylation is shown to operate in embryonic stem cells. The pathway involves the histone H3K9 methyltransferase ESET/SETDB1 and might be important for endogenous retrovirus silencing during the stages in embryogenesis when DNA methylation is reprogrammed. Endogenous retroviruses (ERVs) are widely dispersed in mammalian genomes, and are silenced in somatic cells by DNA methylation. Here, an ERV silencing pathway independent of DNA methylation is shown to operate in embryonic stem cells. The pathway involves the histone H3K9 methyltransferase ESET and might be important for ERV silencing during the stages in embryogenesis when DNA methylation is reprogrammed. Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising ∼10% of the mouse genome 1 . These parasitic elements are responsible for >10% of spontaneous mutations 2 . Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells 3 , 4 , 5 , an additional DNA-methylation-independent pathway also functions in embryonal carcinoma and embryonic stem (ES) cells to inhibit transcription of the exogenous gammaretrovirus murine leukaemia virus (MLV) 6 , 7 , 8 . Notably, a recent genome-wide study revealed that ERVs are also marked by histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3 in ES cells but not in mouse embryonic fibroblasts 9 . However, the role that these marks have in proviral silencing remains unexplored. Here we show that the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and the Krüppel-associated box (KRAB)-associated protein 1 (KAP1, also called TRIM28) 10 , 11 are required for H3K9me3 and silencing of endogenous and introduced retroviruses specifically in mouse ES cells. Furthermore, whereas ESET enzymatic activity is crucial for HP1 binding and efficient proviral silencing, the H4K20 methyltransferases Suv420h1 and Suv420h2 are dispensable for silencing. Notably, in DNA methyltransferase triple knockout ( Dnmt1 -/- Dnmt3a -/- Dnmt3b -/- ) mouse ES cells, ESET and KAP1 binding and ESET-mediated H3K9me3 are maintained and ERVs are minimally derepressed. We propose that a DNA-methylation-independent pathway involving KAP1 and ESET/ESET-mediated H3K9me3 is required for proviral silencing during the period early in embryogenesis when DNA methylation is dynamically reprogrammed.

DNA methylation is involved in numerous biologic events and associates with transcriptional gene silencing, playing an important role in the pathogenesis of endometrial cancer. ESR1/PGR frequently undergoes de novo methylation and loss expression in a wide variety of tumors, including breast, colon, lung, and brain tumors. However, the mechanisms underlying estrogen and progesterone receptors (ER/PR) loss in endometrial cancer have not been studied extensively. The aims of this study were to determine the expression of DNA (cytosine-5)-methyltransferase 3A/3B (DNMT3A/3B) in endometrial cancer to investigate whether the methylation catalyzed by DNMT3A/3B contributes to low ER/PR expression. The clinicopathologic information and RNA-Seq expression data of DNMT3A/3B of 544 endometrial cancers were derived from The Cancer Genome Atlas (TCGA) uterine cancer cohort in May 2018. RNA-Seq level of DNMT3A/3B was compared between these clinicopathologic factors with t-test or one-way analysis of variance. DNMT3A/3B was overexpressed in endometrioid carcinoma (EEC) and was even higher in non-endometrioid carcinoma (NEEC) (DNMT3A, EEC vs. NEEC: 37.6% vs. 69.9%, t = -7.440, P < 0.001; DNMT3B, EEC vs. NEEC: 42.4% vs. 72.8%, t = -6.897, P < 0.001). In EEC, DNMT3A overexpression was significantly correlated with the hypermethylation and low expression of the ESR1 and PGR (P < 0.05). The same trend was observed in the DNMT3B overexpression subgroup. In the ESR1/PGR low-expression subgroups, as much as 83.1% of ESR1 and 59.5% of PGR were hypermethylated, which was significantly greater than the ESR1/PGR high-expression subgroups (31.3% and 11.9%, respectively). However, the above phenomena were absent in NEEC, while DNMT3A/3B overexpression, ESR1/PGR hypermethylation, and low ER/PR expression occurred much more often. In univariate analysis, DNMT3A/3B overexpressions were significantly correlated with worse prognosis. In multivariate analysis, only DNMT3A was an independent predictor of disease-free survival (P < 0.05). DNMT3A/3B expression increases progressively from EEC to NEEC and is correlated with poor survival. The mechanisms underlying low ER/PR expression might be distinct in EEC vs. NEEC. In EEC, methylation related to DNMT3A/3B overexpression might play a major role in ER/PR downregulation.

In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer. Intragenic DNA methylation, dependent on Dnmt3b, protects the gene body from spurious entry of RNA Polymerase II and aberrant transcription initiation events. The role of intragenic DNA methylation The DNA methylation of CpG sequences at gene promoters is associated with the silencing of gene expression, but the function of DNA methylation along the gene body has been less clear. Here, Salvatore Oliviero and colleagues show that this intragenic DNA methylation, dependent on the enzyme Dnmt3b, protects the gene body from the spurious entry of RNA polymerase II and the aberrant initiation of transcription. Elongating RNA polymerase triggers crosstalk between DNA methylation and histone H3 K36 methylation to ensure the fidelity of transcription.

Genome-wide localization and activity analysis of the de novo DNA methyltransferases DNMT3A and DNMT3B in mouse embryonic stem cells identifies overlapping and individual targeting preferences to the genome, including a role for DNMT3B in gene body methylation. Mechanism of de novo DNA methylation Genomic patterns of DNA methylation are established by the de novo DNA methyltransferases DNMT3A and DNMT3B. Dirk Schübeler and colleagues determine the genome-wide localization and activity of these two enzymes in mouse embryonic stem cells. Both localize to methylated CpG-rich regions and are excluded from active gene regulatory regions. DNMT3B also binds to the bodies of actively transcribed genes, dependent on its PWWP domain and methylation of Lys36 on histone H3. This leads to de novo methylation of active genes that scales with co-transcriptional deposition of H3K36me3. DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication 1 . Absence of these enzymes is lethal 2 , and somatic mutations in these genes have been associated with several human diseases 3 , 4 . How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development. The canonical DNA methylation maintenance enzyme Dnmt1 displays global de novo methylation activity with greater targeting towards IAP transposons, which may contribute to their stable repression during early development.

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery. Local activity of the DNA methylation machinery remains poorly understood. Here, the authors present a theoretical and experimental framework to infer methylation and demethylation rates at genome scale in mouse embryonic stem cells, finding that maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies.

Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, in vivo and in vitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

Mouse embryos acquire global DNA methylation of their genome during implantation. However the exact roles of DNA methyltransferases (DNMTs) in embryos have not been studied comprehensively. Here we systematically analyze the consequences of genetic inactivation of Dnmt1 , Dnmt3a and Dnmt3b on the methylome and transcriptome of mouse embryos. We find a strict division of function between DNMT1, responsible for maintenance methylation, and DNMT3A/B, solely responsible for methylation acquisition in development. By analyzing severely hypomethylated embryos, we uncover multiple functions of DNA methylation that is used as a mechanism of repression for a panel of genes including not only imprinted and germline genes, but also lineage-committed genes and 2-cell genes. DNA methylation also suppresses multiple retrotransposons and illegitimate transcripts from cryptic promoters in transposons and gene bodies. Our work provides a thorough analysis of the roles of DNA methyltransferases and the importance of DNA methylation for transcriptome integrity in mammalian embryos. DNA methyltrasferases play important role during mouse embryo development. Here the authors reveal the consequences of genetic inactivation of Dnmt1, Dnmt3a and Dnmt3b on the methylome and transcriptome of mouse embryos genome-wide.