DNA Methyltransferase 3 Alpha Research Articles

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected ‘hub’ cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.MethodsSingle-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. ‘Hub’ cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation.Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.Data availabilityThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.Graphical

Downregulation of UBB potentiates SP1/VEGFA-dependent angiogenesis in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC.

DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.

Abstract 15798: Clonal Hematopoiesis of Indeterminate Potential is Associated With Pro-Inflammatory Markers in Patients With Established Coronary Artery Disease

Introduction: Progression of atherosclerosis is a result of systemic inflammation. Clonal hematopoiesis of indeterminate potential (CHIP) has been shown to be associated with atherosclerosis, but pathways that lead to this are still not fully elucidated. Hypothesis: CHIP may contribute to atherosclerosis by facilitating low-grade inflammatory response. Methods: 130 patients younger than 80 years, who were hospitalized for acute coronary syndrome or for myocardial revascularization were included in this study and followed for 18 months post-intervention. Targeted next-generation sequencing (NGS) was performed on peripheral blood mononuclear cells to detect CHIP mutations with a variant allelic fraction > 2% in 92 genes. Subsequently, multiplexed proteomic analysis with extension proximity assays (EPA) was used to quantify circulating pro-inflammatory biomarkers potentially related to CHIP. Results: Some 17.6 % (23) of 130 patients were detected with CHIP mutations. Patients with mutations in either DNA methyltransferase 3alpha (DNMT3A) or Tet methylcytosine dioxygenase 2 (TET2) showed significantly higher body mass index (BMI) ( p =0.010), inflammatory markers (high sensitivity C-reactive protein p =0.015, interleukin- 6 p =0.002, chemerin p =0.034) as well as higher markers of myocardial overload (troponin T p =0.022 and NT-proBNP). In multivariable model adjusted for BMI, age, and gender we found that the inflammatory markers HGF, IL10RB, IL5RA, IL18, OSM, PD-L1, TNFRSF9, and TNFSF14 presented statistically significant association with CHIP (p<0.01). Interestingly, these differently expressed proteins form an overlapping network between TET2 and DNMT3A mutations pointing to cytokine receptor activity, inflammatory response, and leucocyte activation pathways. Conclusions: There is a significant association between CHIP and enhanced inflammatory response in patients with coronary artery disease, even after adjustment for age and BMI.

DNMT3A-mediated epigenetic silencing of SOX17 contributes to endothelial cell migration and fibroblast activation in wound healing.

Wound healing, especially impaired chronic wound healing, poses a tremendous challenge for modern medicine. Understanding the molecular mechanisms underlying wound healing is essential to the development of novel therapeutic strategies. A wound-healing mouse model was established to analyze histopathological alterations during wound healing, and the expression of SRY-box transcription factor 17 (SOX17), DNA methyltransferase 3 alpha (DNMT3A), and a specific fibroblast marker S100 calcium-binding protein A4 (S100A4) in wound skin tissues was tested by immunofluorescence (IF) assay. Cell proliferation and migration were evaluated using 5-ethynyl-2'-deoxyuridine (EdU) and Transwell migration assays. RT-qPCR and western blotting were used to measure RNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the secretion of transforming growth factor-beta (TGF-β). Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) and DNA pull-down assays were performed to confirm the interaction between DNMT3A and the CpG island of the SOX17 promoter. Promoter methylation was examined by pyrosequencing. SOX17 and DNMT3A expression were regularly regulated during the different phases of wound healing. SOX17 knockdown promoted HUVEC migration and the production and release of TGF-β. Through establishing an endothelial cells-fibroblasts co-culture model, we found that SOX17 knockdown in HUVECs activated HFF-1 fibroblasts, which expressed α-smooth muscle actin (α-SMA) and type I collagen (COL1). DNMT3A overexpression reduces SOX17 mRNA levels. ChIP-qPCR and DNA pull-down assays verified the interaction between DNMT3A and CpG island in the SOX17 promoter region. Pyrosequencing confirmed that DNMT3A overexpression increased the methylation level of the SOX17 promoter. DNMT3A-mediated downregulation of SOX17 facilitates wound healing by promoting endothelial cell migration and fibroblast activation.

Insights into the Inhibitory Mechanisms of the Covalent Drugs for DNMT3A.

The perturbations of DNA methyltransferase 3 alpha (DNMT3A) may cause uncontrolled gene expression, resulting in cancers and tumors. The DNMT inhibitors Azacytidine (AZA) and Zebularine (ZEB) inhibit the DNMT family with no specificities, and consequently would bring side effects during the treatment. Therefore, it is vital to understand the inhibitory mechanisms in DNMT3A to inform the new inhibitor design for DNMTs. Herein, we carried out molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations to investigate the inhibitory mechanisms of the AZA and ZEB. The results were compared to the methyl transfer of cytosine. We showed how the AZA might stop the methyl transfer process, whereas the ZEB might be stuck in a methyl-transferred intermediate (IM3). The IM3 state then fails the elimination due to the unique protein dynamics that result in missing the catalytic water chain. Our results brought atomic-level insights into the mechanisms of the two drugs in DNMT3A, which could benefit the new generation of drug design for the DNMTs.

IRX1 is a novel gene, overexpressed in high-grade IDH-mutant astrocytomas.

IDH-mutant astrocytomas include CNS WHO grade 2 (A2), grade 3 (A3) and grade 4 (A4), of which A3 and A4 are high-grade. A3 has a heterogenous clinical outcome that cannot be explained entirely by the existing molecular biomarkers. We comprehensively studied the transcriptome profile of A3 to determine clinical significance. TCGA mRNA-sequencing data of A3 was analyzed to derive differentially expressed genes (DEG), which were short-listed using various approaches. mRNA expression of the short-listed genes was validated using NanoString platform on a uniformly treated and molecularly characterized A3 cohort. Protein expression of one prognostically significant gene, Iroquois-class homeodomain (IRX1) was assessed by immunohistochemistry and correlated with patient survival and tumor recurrence. IRX1 expression was also studied in different grades of astrocytoma. Since DNA methyltransferase 3 alpha (DNMT3A) influences IRX1 expression, its mutations were evaluated in a subset of tumors. TCGA analysis identified 96 DEG in A3 tumours. 57 genes were short-listed and finally narrowed down to 14 genes. mRNA values of 12/14 genes validated in our cohort. On multiple-variable analysis, IRX1 was the most prognostically relevant gene, with respect to progression free survival of patients. Further, IRX1 immunoexpression was significantly higher in A3 and A4 when compared to A2 and glioblastoma. Higher IRX1 immunoexpression correlated with poor prognosis in patients with A3 tumours. Also, a higher IRX1 expression was associated with DNMT3A mutation. Our study identifies IRX1 as a novel biomarker overexpressed in high-grade IDH-mutant astrocytomas with prognostic significance in A3. DNMT3A mutation probably modulates IRX1 expression.

Effects of in ovo feeding of chlorogenic acid on antioxidant capacity of postnatal broilers.

In this study, chlorogenic acid (CGA) was injected into the amniotic cavity of chicken embryos to study the effects of in ovo feeding of CGA on the antioxidant capacity of postnatal broilers. On the 17th day of embryonic age, a total of 300 healthy broiler fertile eggs with similar weights were randomly subjected to five groups as follows; in ovo injection with 0.5ml CGA at 4 mg/egg (4CGA) or 7 mg/egg (7CGA) or 10 mg/egg (10CGA), or sham-injection with saline (positive control, PC) or no injection (negative control, NC). Each group had six replicates of ten embryos. Six healthy chicks with similar body weights hatched from each replicate were selected and reared until heat stress treatment (35°C ± 1°C, 8h/d) at 28-42days of age. The results showed that there was no significant difference in the hatching rate between the groups (p > 0.05). After heat stress treatment, 4CGA group showed an improved intestinal morphology which was demonstrated by a higher villus height in the duodenum and a higher villus height/crypt depth ratio in the jejunum, compared with the NC group (p < 0.05). The antioxidant capacity of chickens was improved by in ovo feeding of CGA since 4CGA decreased the plasma content of malondialdehyde (MDA) (p < 0.05), whereas, it increased the superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities compared with NC group (p < 0.05). Also, the MDA content of the different injection groups had a quadratic effect, with the 4CGA group having the lowest MDA content (P quadratic < 0.05). In the duodenum, 4CGA injection significantly increased the mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (H O -1), glutathione synthetase (GSS), and SOD1 compared to the NC and PC groups (p < 0.05). The mRNA expressions of glutathione reductase (GSR) and GPX7 were significantly increased in all CGA-treated groups compared with the PC group (p < 0.05), while the mRNA expression of CAT was significantly increased by 4CGA group than the NC group (p < 0.05). The mRNA expressions of epigenetic-related genes, ten eleven translocation 1 and 2 (Tet1 and Tet2), and DNA-methyltransferase 3 alpha (DNMT3A) in the duodenum of 4CGA injected group was significantly increased compared with the NC and PC groups (p < 0.05). The mRNA expressions of Nrf2, SOD1, and Tet2 showed a significant quadratic effects with the 4CGA group having the highest expression (P quadratic < 0.05). In conclusion, in ovo feeding of CGA alleviated heat stress-induced intestinal oxidative damage. Injection with CGA of 4 mg/egg is considered most effective due to its actions in improving intestinal antioxidant capacity, especially in the duodenum. The antioxidant effects of in ovo CGA on postnatal heat-stressed broilers may be related to its regulation of epigenetic mechanisms. Thus, this study provides technical knowledge to support the in ovo feeding of CGA to alleviate oxidative stress in postnatal heat-stressed broilers.

A new risk factor associated with cardiovascular disease: clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis is a prevalent disease associated with all-cause death. Not only because it can be a precancerous lesion of blood system diseases but also has a strong association with cardiovascular disease. A narrow term, clonal hematopoiesis of indeterminate potential (CHIP), was proposed by Steensma et al. [1] to describe individuals with detectable somatic clonal mutations in their genes in blood or bone marrow but without a diagnosis of hematological disease or unexplained cytopenia. Recently, studies have suggested that CHIP is associated with adverse cardiovascular disease progression, particularly in patients with ten-eleven translocation 2 (TET2) mutations or DNA methyltransferase 3 alpha (DNMT3A) mutations. Age is the most crucial factor which is associated with increased CHIP prevalence. The underlying mechanisms appear to be related to inflammatory status. However, new evidence suggests that genetic factors, lifestyle and environmental factors such as smoking, obesity, and diet also play essential roles in developing CHIP. More research needs to be done on the potential genetic mechanisms driving CHIP and the environmental factors that modulate CHIP risk. This review summarizes the latest research on CHIP, discusses in detail the strong association between clonal hematopoiesis and accelerated cardiovascular disease, and rationalizes the intervention of CHIP in combination with existing evidence, which may be beneficial for future treatment.

Unveiling the methyl transfer mechanisms in the epigenetic machinery DNMT3A-3 L: A comprehensive study integrating assembly dynamics with catalytic reactions

In epigenetic mechanisms, DNA methyltransferase 3 alpha (DNMT3A) acts as an initiator for DNA methylation and prevents the downstream genes from expressing. Perturbations of DNMT3A functions may cause uncontrolled gene expression, resulting in pathogenic consequences such as cancers. It is, therefore, vitally important to understand the catalytic process of DNMT3A in its biological macromolecule assembly, viz., heterotetramer: (DNMT3A-3 L)dimer. In this study, we utilized molecular dynamics (MD) simulations, Markov State Models (MSM), and quantum mechanics/molecular mechanics simulations (QM/MM) to investigate the de novo methyl transfer process. We identified the dynamics of the key residues relevant to the insertion of the target cytosine (dC) into the catalytic domain of DNMT3A, and the detailed potential energy surface of the seven-step reaction referring to methyl transfer. Our calculated potential energy barrier (22.51 kcal/mol) approximates the former experimental data (23.12 kcal/mol). The conformational change of the 5-methyl-cytosine (5mC) intermediate was found necessary in forming a four-water chain for the elimination step, which is unique to the other DNMTs. The biological assembly facilitates the creation of such a water chain, and the elimination occurs in an asynchronized mechanism in the two catalytic pockets. We anticipate the findings can enable a better understanding of the general mechanisms of the de novo methyl transfer for fulfilling the key enzymatic functions in epigenetics. And the unique elimination of DNMT3A might ignite novel methods for designing anti-cancer and tumor inhibitors of DNMTs.

Targeting TET2 as a Therapeutic Approach for Angioimmunoblastic T Cell Lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL), a type of malignant lymphoma with unique genomic aberrations, significant clinicopathological features, and poor prognosis, is characterized by immune system dysregulation. Recent sequencing studies have identified recurrent mutations and interactions in tet methylcytosine dioxygenase 2 (TET2), ras homology family member A (RHOA), DNA methyltransferase 3 alpha (DNMT3A), and mitochondrial isocitrate dehydrogenase II (IDH2). Notably, since B-cell lymphomas are frequently observed along with AITL, this review first summarizes its controversial mechanisms based on traditional and recent views. Epigenetic regulation represented by TET2 plays an increasingly important role in understanding the multi-step and multi-lineage tumorigenesis of AITL, providing new research directions and treatment strategies for patients with AITL. Here, we review the latest advances in our understanding of AITL and highlight relevant issues that have yet to be addressed in clinical practice.

DNMT3A-mediated high expression of circ_0057504 promotes benzo[a]pyrene-induced DNA damage via the NONO-SFPQ complex in human bronchial epithelial cells

Benzo[a]pyrene (B[a]P) is a class I carcinogen and hazardous environmental pollutant with genetic toxicity. Understanding the molecular mechanisms underlying genetic deterioration and epigenetic alterations induced by environmental contaminants may contribute to the early detection and prevention of cancer. However, the role and regulatory mechanisms of circular RNAs (circRNAs) in the B[a]P-induced DNA damage response (DDR) have not been elucidated. In this study, human bronchial epithelial cell lines (16HBE and BEAS-2B) were exposed to various concentrations of B[a]P, and BALB/c mice were treated with B[a]P intranasally. B[a]P exposure was found to induce DNA damage and upregulate circular RNA hsa_circ_0057504 (circ_0057504) expression in vitro and in vivo. In addition, B[a]P upregulated TMEM194B mRNA and circ_0057504 expression through inhibition of DNA methyltransferase 3 alpha (DNMT3A) expression in vitro. Modulation (overexpression or knockdown) of circ_0057504 expression levels using a lentiviral system in human bronchial epithelial cells revealed that circ_0057504 promoted B[a]P-induced DNA damage. RNA pull-down and western blot assays showed that circ_0057504 interacted with non-POU domain-containing octamer-binding (NONO) and splicing factor proline and glutamine rich (SFPQ) proteins and regulated formation of the NONO-SFPQ protein complex. Thus, our findings indicate that circ_0057504 acts as a novel regulator of DNA damage in human bronchial epithelial cells exposed to B[a]P. The current study reveals novel insights into the role of circRNAs in the regulation of genetic damage, and describes the effect and regulatory mechanisms of circ_0057504 on B[a]P genotoxicity.

DNA methyltransferase 3 alpha and TET methylcytosine dioxygenase 2 restrain mitochondrial DNA-mediated interferon signaling in macrophages

Deleterious somatic mutations in DNA methyltransferase 3 alpha (DNMT3A) and TET mehtylcytosine dioxygenase 2 (TET2) are associated with clonal expansion of hematopoietic cells and higher risk of cardiovascular disease (CVD). Here, we investigated roles of DNMT3A and TET2 in normal human monocyte-derived macrophages (MDM), in MDM isolated from individuals with DNMT3A or TET2 mutations, and in macrophages isolated from human atherosclerotic plaques. We found that loss of function of DNMT3A or TET2 resulted in a type I interferon response due to impaired mitochondrial DNA integrity and activation of cGAS signaling. DNMT3A and TET2 normally maintained mitochondrial DNA integrity by regulating the expression of transcription factor A mitochondria (TFAM) dependent on their interactions with RBPJ and ZNF143 at regulatory regions of the TFAM gene. These findings suggest that targeting the cGAS-type I IFN pathway may have therapeutic value in reducing risk of CVD in patients with DNMT3A or TET2 mutations.

Mesenchymal stem cell-derived extracellular vesicles alleviate cervical cancer by delivering microRNA-331-3p to reduce LIM zinc finger domain containing 2 methylation in tumor cells.

The aim of this study is to investigate if extracellular vesicles (EVs) from bone marrow mesenchymal stem cells (BMSCs) deliver microRNA (miR)-331-3p to regulate LIM zinc finger domain containing 2 (LIMS2) methylation in cervical cancer cells. Cervical cancer cells were incubated with EVs from BMSCs with altered expression of miR-331-3p, DNA methyltransferase 3 alpha (DNMT3A) or/and LIMS2 and then subjected to 5-ethynyl-2'-deoxyuridine, Transwell, flow cytometry and western blotting analyses. Dual-luciferase reporter assay was conducted to verify the binding between miR-331-3p and DNMT3A. A xenograft model was established to evaluate the effect of BMSC-derived EV-miR-331-3p on cervical tumor growth. miR-331-3p was lowly and DNMT3A was highly expressed in cervical cancer. BMSC-derived EVs delivered miR-331-3p to control the behaviors of cervical cancer cells. miR-331-3p inhibited the expression of DNMT3A by binding DNMT3A mRNA. DNMT3A promoted LIMS2 methylation and reduced the expression of LIMS2. Overexpression of DNMT3A or silencing of LIMS2 in BMSCs counteracted the tumor suppressive effects of miR-331-3p. BMSC-derived EV-miR-331-3p also inhibited the growth of cervical tumors in vivo. BMSC-derived EVs alleviate cervical cancer partially by delivering miR-331-3p to reduce DNMT3A-dependent LIMS2 methylation in tumor cells.

Heterozygous Dnmt3a R878C induces expansion of quiescent hematopoietic stem cell pool

Somatic mutation of DNMT3A (DNA methyltransferase 3 alpha) is implicated in the development of a wide range of hematological disorders, including clonal hematopoiesis of indeterminate potential. To elucidate the functional roles of endogenous levels of a DNMT3A R882 mutant, we generated a novel Dnmt3a R878C conditional knock-in mouse model. In contrast to viable heterozygotes, mice homozygous for the Dnmt3a R878C mutation in the hematopoietic system were not viable (Dnmt3a R878C is homologous to human DNMT3A R882C). Hematopoietic cell-specific heterozygous expression of Dnmt3a R878C led to significant expansion of adult quiescent hematopoietic stem cells (HSCs); however, these mice had no hematological malignancies. The expanding HSC population in heterozygous Dnmt3a R878C knock-in mice had an accumulation of G0-phase cells. In contrast to aberrantly enhanced self-renewal capacity in vitro, heterozygous Dnmt3a R878C knock-in HSCs had no competitive repopulating advantage in vivo over wild-type HSCs. Considering the capacity of the heterozygous Dnmt3a R878C mutant for HSC pool expansion, our Dnmt3a R878C knock-in mouse line is a useful platform on which to dissect the pathophysiology of clonal hematopoiesis. This mouse line can also help to elucidate the biological and molecular actions of DNMT3A mutations in the malignant transformation of normal HSCs.

TDG suppresses the migration and invasion of human colon cancer cells via the DNMT3A/TIMP2 axis.

Background: Colorectal cancer (CRC) is one of the most common malignant tumors with high rates of recurrence and mortality. Thymine DNA glycosylase (TDG) is a key molecule in the base excision repair pathway. Recently, increasing attention has been paid to the role of TDG in tumor development. However, the specific functions of TDG in CRC remain unclear.Methods: The biological functions of TDG and DNA methyltransferase 3 alpha (DNMT3A) in CRC were evaluated using migration and invasion assays, respectively. A tumor metastasis assay was performed in nude mice to determine the in vivo role of TDG. The interaction between TDG and DNMT3A was determined via co-immunoprecipitation (Co-IP). Chromatin immunoprecipitation analysis (ChIP) was used to predict the DNA-binding site of DNMT3A. We also performed methylation-specific PCR (MSP) to detect changes in TIMP2 methylation.Results: TDG inhibited the migration and invasion of human colon cancer cells both in vitro and in vivo. TDG promoted the ubiquitination and degradation of DNMT3A by binding to it. Its interference with siDNMT3A also inhibits the migration and invasion of human colon cancer cells. Furthermore, the ChIP, MSP, and rescue experiments results confirmed that TDG accelerated the degradation of DNMT3A and significantly regulated the transcription and expression of TIMP2, thereby affecting the migration and invasion of human colon cancer cells.Conclusion: Our findings reveal that TDG inhibits the migration and invasion of human colon cancer cells through the DNMT3A-TIMP2 axis, which may be a potential therapeutic strategy for the development and treatment of CRC.

Exploration of the Effect on Genome-Wide DNA Methylation by miR-143 Knock-Out in Mice Liver.

MiR-143 play an important role in hepatocellular carcinoma and liver fibrosis via inhibiting hepatoma cell proliferation. DNA methyltransferase 3 alpha (DNMT3a), as a target of miR-143, regulates the development of primary organic solid tumors through DNA methylation mechanisms. However, the effect of miR-143 on DNA methylation profiles in liver is unclear. In this study, we used Whole-Genome Bisulfite Sequencing (WGBS) to detect the differentially methylated regions (DMRs), and investigated DMR-related genes and their enriched pathways by miR-143. We found that methylated cytosines increased 0.19% in the miR-143 knock-out (KO) liver fed with high-fat diet (HFD), compared with the wild type (WT). Furthermore, compared with the WT group, the CG methylation patterns of the KO group showed lower CG methylation levels in CG islands (CGIs), promoters and hypermethylation in CGI shores, 5′UTRs, exons, introns, 3′UTRs, and repeat regions. A total of 984 DMRs were identified between the WT and KO groups consisting of 559 hypermethylation and 425 hypomethylation DMRs. Furthermore, DMR-related genes were enriched in metabolism pathways such as carbon metabolism (serine hydroxymethyltransferase 2 (Shmt2), acyl-Coenzyme A dehydrogenase medium chain (Acadm)), arginine and proline metabolism (spermine synthase (Sms), proline dehydrogenase (Prodh2)) and purine metabolism (phosphoribosyl pyrophosphate synthetase 2 (Prps2)). In summary, we are the first to report the change in whole-genome methylation levels by miR-143-null through WGBS in mice liver, and provide an experimental basis for clinical diagnosis and treatment in liver diseases, indicating that miR-143 may be a potential therapeutic target and biomarker for liver damage-associated diseases and hepatocellular carcinoma.

Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies and is now being developed for solid tumors and chronic viral infections. Although clinical trials have demonstrated the curative potential of CAR T cell therapy, a substantial and well-established limitation is the heightened contraction and transient persistence of CAR T cells during prolonged antigen exposure. The underlying mechanism(s) for this dysfunctional state, often termed CAR T cell exhaustion, remains poorly defined. Here, we report that exhaustion of human CAR T cells occurs through an epigenetic repression of the T cell’s multipotent developmental potential. Deletion of the de novo DNA methyltransferase 3 alpha (DNMT3A) in T cells expressing first- or second-generation CARs universally preserved the cells’ ability to proliferate and mount an antitumor response during prolonged tumor exposure. The increased functionality of the exhaustion-resistant DNMT3A knockout CAR T cells was coupled to an up-regulation of interleukin-10, and genome-wide DNA methylation profiling defined an atlas of genes targeted for epigenetic silencing. This atlas provides a molecular definition of CAR T cell exhaustion, which includes many transcriptional regulators that limit the “stemness” of immune cells, including CD28, CCR7, TCF7, and LEF1. Last, we demonstrate that this epigenetically regulated multipotency program is firmly coupled to the clinical outcome of prior CAR T cell therapies. These data document the critical role epigenetic mechanisms play in limiting the fate potential of human T cells and provide a road map for leveraging this information for improving CAR T cell efficacy.

Mindfulness intervention improves cognitive function in older adults by enhancing the level of miRNA-29c in neuron-derived extracellular vesicles

Although mindfulness-based stress reduction (MBSR) improves cognitive function, the mechanism is not clear. In this study, people aged 65 years and older were recruited from elderly communities in Chitose City, Japan, and assigned to a non-MBSR group or a MBSR group. Before and after the intervention, the Japanese version of the Montreal Cognitive Assessment (MoCA-J) was administered, and blood samples were collected. Then, neuron-derived extracellular vesicles (NDEVs) were isolated from blood samples, and microRNAs, as well as the target mRNAs, were evaluated in NDEVs. A linear mixed model analysis showed significant effects of the MBSR x time interaction on the MoCA-J scores, the expression of miRNA(miR)-29c, DNA methyltransferase 3 alpha (DNMT3A), and DNMT3B in NDEVs. These results indicate that MBSR can improve cognitive function by increasing the expression of miR-29c and decreasing the expression of DNMT3A, as well as DNMT3B, in neurons. It was also found that intracerebroventricular injection of miR-29c mimic into 5xFAD mice prevented cognitive decline, as well as neuronal loss in the subiculum area, by down-regulating Dnmt3a and Dnmt3b in the hippocampus. The present study suggests that MBSR can prevent neuronal loss and cognitive impairment by increasing the neuronal expression of miR-29c.

DNMT3A Regulates Hematopoietic Stem Cell Function Via DNA Methylation-Independent Functions

The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is strictly regulated to sustain blood production throughout adult life. De novo DNA methyltransferase 3-alpha (DNMT3A) is one of the major epigenetic regulators that is essential for efficient HSC differentiation. DNMT3A mutations are prevalent in myeloid diseases that include acute myeloid leukemia (AML; ~22%) and myelodysplastic syndrome (MDS; ~10%) where they act as initiating events However, the precise molecular mechanisms of how DNMT3A regulates normal hematopoiesis and its mutations prime HSCs for leukemic formation are unclear. Although DNMT3A is described as a DNA methyltransferase enzyme, the lack of consistent correlation between changes in DNA methylation and differential gene expression in Dnmt3a-null HSCs in mouse models, and AML patients with DNMT3A mutations undermine the conventional understanding of DNMT3A's canonical role in hematopoietic cells. Hence, we hypothesized that DNMT3A may have novel functions outside of DNA methylation that regulate HSC fate decisions.To answer this question, we first ectopically expressed GFP-labeled Dnmt3a constructs (wild-type Dnmt3a, Dnmt3aE752A; complete DNA methylation dead, and Dnmt3aR832A; reduced DNA methylation target recognition) and empty vector (negative control) in Dnmt3a-null (Vav-Cre: Dnmt3afl/fl = Dnmt3a-/- in hematopoiesis) bone marrow (BM) cells. The result showed that similar to restoring wild-type Dnmt3a, ectopic expression of Dnmt3aE752A as well as Dnmt3aR832A showed a rescue effect of decreased engraftment of transduced cells in the peripheral blood as well as reduced HSC numbers in the BM. Analysis of DNA methylation by whole-genome bisulfite sequencing (WGBS) in transduced cells showed this phenotypic and functional rescue of the Dnmt3a-/- phenotype occurred in the absence of restored DNA methylation patterns. To study the importance of Dnmt3a-mediated DNA methyltransferase activity in a more physiological system, we generated knock-in mice that have one copy of either wild-type Dnmt3a, Dnmt3aE752A, or Dnmt3aR832A (CAGG-Cre-ER T2 = ER T2-Cre: Dnmt3afl/+, Dnmt3afl/E752A, and Dnmt3afl/R832A) to be compared to the Dnmt3a-null group (ER T2-Cre: Dnmt3afl/-). These mice contain one allele with loxP-flanked Dnmt3a that is deleted by tamoxifen-inducible Cre-mediated recombination and one allele of either wild-type Dnmt3a, Dnmt3aE752A, Dnmt3aR832A, or germline knockout Dnmt3anull. 5-weeks post-tamoxifen (~93% floxed allele recombination), competitive transplantation of 250 phenotypically defined test HSCs against with 2.5x10 5 congenic competitor BM cells was performed.Dnmt3afl/R832A recipients had higher engraftment (35.6 % +/- 6.1) than Dnmt3afl/+ (28.5% +/- 7.2) and Dnmt3afl/- (10.7% +/- 2.79), while Dnmt3afl/E752A had slightly higherengraftment (12.5% +/- 3) than Dnmt3afl/-. Analysis of the BM 18 weeks post-transplant showed that Dnmt3afl/E752A and Dnmt3afl/R832A HSCs phenocopied the HSC self-renewal potential phenotype of heterozygous Dnmt3a fl/+ HSCs (Fig. 1). The absolute count of donor-derived HSCs per mouse after the transplant were: ER T2-Cre control (675.7 +/- 299.3), Dnmt3afl/+ (1870 +/- 961.4), Dnmt3afl/- (3546 +/- 1019), Dnmt3afl/E752A (1130 +/- 362.7), and Dnmt3afl/R832A (1184 +/- 344.5) (mean +/- S.E.M.). While the described clonal expansion of Dnmt3a-null HSCs was observed, HSCs with one copy of full-length Dnmt3a but devoid of its methyltransferase capacity mimicked the heterozygous state rather than the homozygous loss-of-function. This is the first evidence to suggest that DNMT3A potentially regulates HSCs by non-canonical (DNA methylation independent) mechanisms. DNA methylation analysis by WGBS is ongoing to determine if Dnmt3afl/E752A and Dnmt3afl/R832A HSCs show a methylome comparable to Dnmt3a-null HSCs whilst having the functional potential of Dnmt3a-heterozygous HSCs, which will be complemented with other molecular analyses including gene expression. Our study opens new avenues for investigations into the molecular mechanisms of DNMT3A function in HSC biology, which could ultimately benefit clinical practice by identifying new therapeutic approaches for the patients with DNMT3A mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.